bluuebunny/crossref_metadata_2025 huggingface dataset:
An Analysis of the Effects of Oestrogen Treatment in vivo on the Protein‐Synthetic Activity of Male Xenopus Liver Cell‐Free Systems
Authors: Clemens Michael J. , Tata Jamshed R.
Published: March 2005

A cell‐free protein‐synthesising system has been prepared from the liver of adult male Xenopus laevis which shows several adaptive changes following induction of egg‐yolk protein synthesis by administration of oestradiol‐17βin vivo.

The product of the cell‐free system from oestrogen‐treated animals is characterised by elevated incorporation of radioactive serine relative to phenylalanine. This is taken as an indication of the synthesis of phosvitin, which has a high serine content, in vitro.

Increased protein‐synthetic activity of Xenopus liver ribosomes following oestrogen treatment can be demonstrated and is maintained after the particles are washed in 0.5 M KCl. This effect of the hormone is also apparent when the ribosomes are first stripped of endogenous mRNA by preincubation and then added to a fresh cell‐free system containing poly (U). It is concluded that oestrogen treatment produces an intrinsic activation of synthetic ability of ribosomes in the liver.

Increased ability of ribosomes to translate poly(U) does not result from changes in their ability to bind the polynucleotide, either before or after preincubation.

Cell sap from liver of oestrogen‐treated animals shows increased ability to support amino acid incorporation by ribosomes from control or treated animals in vitro. This is not due to enhanced initiation of peptide synthesis or to increased availability of endogenous aminoacyl‐tRNA in vitro.

The effects of oestradiol on Xenopus liver are considered in relation to the translational control of protein synthesis by hormones and other agents in a variety of systems.
Yeast Chromatin
Authors: Wintersberger Ulrike , Letnansky Karl , Smith Phillip
Published: March 2005

A chromatin‐like material was prepared from isolated nuclei of Saccharomyces cerevisiae. This chromatin fraction contains a full complement of yeast histones as revealed by acrylamide‐gel electrophoresis. Yeast histones were found to resemble those of rat liver cells in electrophoretic properties except for the almost complete absence of histone F1. Yeast chromatin is only poorly transcribed by the isolated yeast RNA polymerases A and B and was found to contain endogenous RNA polymerases which are activated by the addition of salts. Salt causes a removal of DNA‐associated proteins from the chromatin, allowing bound RNA polymerase molecules to extend already initiated RNA chains. Some evidence was obtained indicating that different proteins have to be removed to allow the different endogenous polymerases to proceed with transcription. It is concluded that the properties of yeast chromatin studied, in particular the histone composition and the transcription capacity, are very similar to those of chromatin preparations from animal cells.
Phosphorylation of Very‐Lysine‐Rich Histone in Physarum polycephalum
Authors: Bradbury E. Morton , Inglis Robert J. , Matthews Harry R. , Sarner Nitza
Published: March 2005

Physarum polycephalum histones have been analysed by acrylamide gel electrophoresis. Two of the five major bands had electrophoretic mobilities identical with the mobilities of two bands from calf thymus histone. The P. polycephalum pattern is qualitatively the same at all stages of the synchronous mitotic cycle. Quantitative changes in the relative proportion and relative mobility of the very‐lysine‐rich histone are reported. In particular, a dramatic increase in phosphate content of this histone occurred in late G2 phase with a peak where chromosome condensation is seen to be occurring in the phase contrast microscope. Phosphate content is low during S phase and the peaks of RNA synthesis.
A Protein Factor from Rat‐Liver Tissue Enhancing the Transcription of Native Templates by Homologous RNA Polymerase B
Authors: Seifart Klaus H. , Juhasz Peter P. , Benecke Bernd J.
Published: March 2005

A protein factor which stimulates the transcription of native templates catalyzed by enzyme B from rat liver has been isolated from the cytoplasm of this tissue. It has been purified extensively by chromatography through DEAE‐cellulose and CM‐cellulose, hydroxylapatite, gel‐filtration, rechromatography on DEAE‐cellulose and isoelectrofocussing. The protein is of low molecular weight (±30000) with an isoelectric point of 9.6. This factor predominantly stimulates enzyme B (±500%) without markedly effecting other RNA polymerase species from this tissue. Experiments have been adressed to the question concerning the mechanism through which this phenomenon operates. The stimulation manifests itself without noticeable time‐lag, immaterial of whether the factor is added before or after initiation of RNA synthesis and approximate estimation of the initiation complexes formed with or without factor, measured by direct filtration on millipore filters, lends no proof to the assumption that initiation is elevated. The degree of stimulation, measured on a template blocked by actinomycin D, thus allowing initiation but limiting propagation, is markedly diminished. This observation, taken together with the pronounced sensitivity of the factor‐induced RNA synthesis toward pyrophosphate, an inhibitor of phosphodiester bond formation, suggests that the observed effect operates through the chain‐elongation step. This assumption is supported by the fact that Ks, determinations for CTP are not altered by the factor in the face of a greatly enhanced value for V, suggesting that the rate of chain propagation is raised. Experiments in which the 5′ termini of the RNA were labelled with purine nucleoside [γ‐32P] triphosphates show that the average chain length of the product is increased through the action of the stimulatory protein. The factor‐induced stimulation is most pronounced on native templates of high molecular weight and it is possible that the protein studied aids in the unwinding process of such a DNA or enhances the rate of chain propagation in some other way.
Purification and Further Properties of Single‐Strand‐Specific Nuclease from Aspergillus oryzae
Authors: Vogt Volker M.
Published: March 2005

A simple scheme has been devised for the purification of Aspergillus nuclease S1 from commercially available crude amylase powder. Five purification steps yield 90% pure nuclease with an overall yield of 27%. The enzyme is shown to be absolutely specific for single‐stranded nucleic acids. It degrades both DNA and RNA. The nuclease exhibits a sharp pH optimum at pH 4.2 and is active in low concentrations of dodecylsulfate. Aspergillus nuclease S1 is a metalloprotein of molecular weight 32000. The utility of this nuclease for measuring annealing of labeled DNA is discussed.
κ (Kappa), a Novel Factor for the Arrest of Transcription in vitro by DNA‐Dependent RNA Polymerase from Escherichia coli at Specific Sites of Natural Templates
Authors: Schäfer Rolf , Zillig Wolfram
Published: March 2005

A procedure for the isolation of a novel factor, named x, effecting termination or block of transcription of DNAs of bacteriophages T4, T5 and T7 by Escherichia coli RNA polymerase is described.

This protein is a dimer of two apparently identical peptide chains, the latter with a molecular weight of about 17000.

x is bound to double‐stranded DNA in almost tight packing. It does not influence initiation of transcription. The net rate of RNA synthesis is reduced. The kinetics of transcription in vitro of phage DNAs in the presence of a saturating amount of x lead to a plateau over a wide range of ionic strength. The average chain length of the product at this plateau is 40–50% of that of the product obtained without the factor.

The RNA remains bound to the DNA in the blocked end state.

Hybridization‐saturation and competition and sedimentation on analysis sucrose gradients yield evidence that the blocking action occurs at a few specific sites of the many x‐binding sites on the DNA.

The possible role of this factor is discussed.
Complexes of Messenger RNA and 40‐S Subunit in Rabbit Reticulocytes
Authors: Zehavi‐Willner Tova , Danon David
Published: March 2005

Native 40‐S subunits isolated from reticulocytes of anemic rabbits exposed for 2 h to [32P]‐phosphate are labelled to a much higher extent than are 60‐S subunits, monosomes and polysomes. Sucrose gradient analysis of the RNA content of these 40‐S subunits shows that most of the radioactivity appears in the 9‐S RNA peak. Analysis of native 40‐S subunits 40 h after [32P]‐phosphate‐administration indicates, that in addition to 18‐S RNA as a major component, they also contain mRNA and tRNA. EDTA treatment releases from the native 40‐S subunits 9‐S RNA in the form of a 15‐S mRNA ṁ protein particle. CsCl gradient profiles of the subunits followed by base analysis of the fractions obtained on the gradient, revealed the presence of two forms of 40‐S subunits with buoyant densities of 1.436 (major peak) and 1.512 g/cm3 (minor peak), respectively. It is proposed that the subunit particle with density of 1.436 g/cm3 is a 40‐S mRNA ṁ protein ṁ tRNA initiation complex, in view of its buoyant density characteristics, the absence of nucleoprotein particles other than the forms of 40‐S subunits and the molar ratio of the three components in the complex.
Slow Conformational Changes of the Inorganic Pyrophosphatase from Bakers' Yeast Induced by Divalent Metal Ions
Authors: Höhne Wolsfgang E. , Rapoport Tom A.
Published: March 2005

A study of the role of divalent metal ions, in particular of Mg2+ and Mn2+ in the reaction of the inorganic pyrophosphatase of bakers' yeast has been carried out. It is shown that the free metal ions compete with each other for the same metal binding site. Binding to this site causes reversible conformational changes of the enzyme as indicated by the kinetics. This assumption is in agreement with significant alterations in the circular dichroism spectrum. In the presence of inorganic pyrophosphate the changes of the enzyme are slow enough to be followed kinetically over a period of minutes. Other possibilities for a slow transition of the enzyme velocity which might exist according to the hysteretic enzyme concept of Frieden could be rejected. To explain the experimental data a kinetic scheme is proposed.

The results show that, the metal ions affect, the hydrolysis at the substrate site through a conformational change of the protein.
Further Studies with Labelled Polypeptide‐Chain‐Initiation Factor 3 (IF‐3)
Authors: Sabol Steven , Meier Dankwart , Ochoa Severo
Published: March 2005

The preparation of 35S‐labelled initiation factor 3 (IF‐3) from Escherichia coli is described. As reported earlier, labelled IF‐3 binds to 30‐S, but not to 50‐S ribosomal subunits or 70‐S ribosomes, and is released upon formation of a 70‐S couple. In continuation of this work we find that the 30‐S binding of IF‐3 is rather sensitive to aurin‐tricarboxylic acid, a powerful inhibitor of polypeptide chain initiation, but relatively insensitive to streptomycin. IF‐3 binds weakly to both 23‐S and 16‐S ribosomal RNA and to 23‐S but not 43‐S ribosomal cores, Free IF‐3 readily exchanges with the 30‐S‐bound factor even at low temperature. There is also an exchange of 50‐S or 30‐S ribosomal subunits labelled with [3H]uracil into 70‐S ribosomes in the absence of the reactions of protein synthesis. This exchange is markedly temperature‐dependent and strongly inhibited at high (20 mM) Mg2+ concentrations.
Molecular Morphology of Ribosomes
Authors: Michalski Chester J. , Sells Bruce H. , Morison Martin
Published: March 2005

Using the lactoperoxidase‐catalyzed iodination system as a probe for exposed proteins, the location of ribosomal proteins on 50‐S subunits of Escherichia coli was examined. These studies demonstrated that at least three of the proteins in the 50‐S ribosome are iodinated and may be termed exposed proteins. Most of the proteins, on the other hand, are not iodinated and can be classified as buried or partially buried proteins. Unfolding of the 50‐S ribosome by the removal of Mg2+ resulted in a decreased organization of the ribosome structure and allowed all the proteins except one to be iodinated.
Tryptophan Hydroxylase in Nucleated Thrombocytes of the Domestic Fowl
Authors: Sorimachi Masaru , Kataoka Kiyoshi , Hori Seiki , Hori Shinichiro , Fujisawa Hitoshi
Published: March 2005

Using the labeled CO2 trapping method, tryptophan hydroxylase activity was detected in the nucleated thrombocytes of the domestic fowl, but not in the mammalian platelets. This enzyme activity was around a 5‐hydroxytryptophan production of 60 pmol × mg protein−1× h−1. Certain properties such as optimal pH (7.0), Km value (12 μM), requirement for cofactor (2‐amino‐4‐hydroxy‐6,7‐dimethyl‐5,6,7,8‐tetrahydropteridine) and inhibition by p‐chlorophenylalanine indicated a close resemblance to tryptophan hydroxylase from brain tissue. Solubilization of this enzyme was to some extent successful by digitonin treatment of the cells.
The Interaction of Bilirubin with Human Serum Albumin
Authors: Beaven Gilbert H. , D'Albis Anne , Gratzer Walter B.
Published: March 2005

The interaction of bilirubin with de‐fatted human serum albumin has been studied by circular dichroism and spectrofluorimetry. From the dependence of the induced circular dichroism of the bilirubin on the ratio of protein to bilirubin, it is shown that in the presence of salt (0.5 M sodium chloride) there are two binding sites with markedly different optical characteristics. From an analysis of the profiles of ellipticity as a function of protein: bilirubin ratio, association constants of 7 and 0.3 μM−1 at pH 8.5 were derived. At low salt concentrations qualitatively similar binding profiles are obtained with a marked maximum of ellipticity at a protein: bilirubin ratio of about 1. These cannot however be adequately accounted for with only two binding sites. The addition of a third site allows the data to be fitted over the entire concentration range at all wavelengths tested, with association constants falling in the range 10–0.01 μM−1. When bilirubin is bound to albumin, the fluorescence of the protein is quenched and that of the bilirubin is enhanced. The Fluorescence characteristics of the binding sites are shown to be different: the weaker sites, which have lower fluorescence, but higher circular dichroism than the stronger, are relatively much more labile to salt. The pH‐dependence of the optical properties reveals the presence of a state defined by a plateau at between pH 8 and 9, another by a plateau centred at about pH 6.5, and another, the appearance of which is associated with the N–F transition of the protein, and which exists only within an extremely narrow pH range, at about pH 4. The pH at which the minimum occurs undergoes a large shift with salt concentration. This state is characterised by an extremely large circular dichroism (effective molar ellipticity ‐5 × 105 deg. cm2· dmol−1), opposite in sign to the system of Cotton effects observed at higher pH. This complex shows no fluorescence enhancement. The system shows large hysteresis, satisfactory results being obtained only when the complex is first formed by equilibration at pH 8–9 at low ionic strength. Under other conditions the self‐association of the bilirubin evidently prevents the attainment of equilibrium, and vitiates any attempts at analysis of the binding equilibria. The competition of oleate ions with the bilirubin binding sites has been studied. Circular dichroism, under physiological conditions, of human serum containing bilirubin shows that the binding of the bilirubin by albumin alone at the corresponding ionic strength and pH defines the state of the ligand in the plasma.
Biosynthesis of Melittin, a Toxic Peptide from Bee Venom
Authors: Kreil Günther
Published: March 2005

From the venom glands of honey bees fed with different radioactive amino acids, labelled promelittin could be isolated. Extensive analysis of this precursor peptide has led to the conclusion that it contains the entire sequence of melittin, including the C‐terminal glutamic acid diamide. At the amino end, promelittin was found to be heterogeneous. As compared to melittin, the main species contains eight additional amino acids. Other species of different chain length are present in varying amounts. The structure of the main component can be formulated as Glu‐Pro‐Glu‐Pro‐Asp‐Pro‐Glu‐Ala‐melittin(1–26). The methodology used for determining the sequence of radioactive peptides is discussed.
The Induction of Translocators for Di‐ and Tricarboxylic‐Acid Anions inAzotobacter vinelandii
Authors: Reuser Arnold J. J. , Postma Pieter W.
Published: March 2005

The conditions under which translocators for Krebs‐cycle intermediates can be induced inAzotobacter vinelandiiwere determined. With the exception of mesotartrate andd‐malate, only Krebs‐cycle intermediates themselves are good inducers. Glucose is needed for the induction of a translocator for C4‐dicarboxylic acid anions. Glucose at higher concentrations represses the translocator for C4‐tricarboxylic acid anions. This repression can be released by 3′: 5′‐AMP.

Induction of the translocator for C4‐dicarboxylic acid anions probably occurs on the outside of the cell, because intracellularly generated dicarboxylic acid anions do not lead to synthesis of their translocator. Furthermore, contact between the cell and the inducer is necessary only during the first 20–30 min of induction, synthesis of the translaocator going on for another 60 min at least.

Induction of a malonate translocator leads to the simultaneous induction of two malonate‐metabolizing enzymes: malonyl‐CoA synthetase and malonyl‐CoA decarboxylase.
Bovine‐Lens Leucine Aminopeptidase
Authors: Lasch Jürgen , Kudernatsch Walter , Hanson Horst
Published: March 2005

An attempt has been made, by means of kinetic experiments, to get some insight into the molecular mechanism of the binding and catalytic steps taking place during low‐molecular‐weight substrate hydrolysis by leucine aminopeptidase from bovine eye lenses. To this end the variation of the kinetic parameters with concentration and type of well‐known activators (anions and metal cations of valency two) was studied systematically.

Additional, we searched for substrate‐like competitive inhibitors. It was found that the end product l‐leucine shows no competition but the compounds N‐p‐tosyl‐N′‐butylcarbamide and N‐sulfanilyl‐N′‐butylcarbamide turned out to be rather potent competitive inhibitors with Ki values comparable to the Km of l‐leucine‐p‐nitroanilide. The variation of the ki values with temperature was studied so that the thermodynamic parameters of inhibitor binding could be calculated.

In order to be able to make a meaningful comparison between the kinetic parameters all data were subjected to a computer‐aided (CDC 1604‐A) statistical analysis.
Evidence for the Compartmentation of Cholesterol in Rat‐Liver Microsomes
Authors: Balasubramaniam Santhirasegaram , Mitropoulos Konstantinos A. , Myant Nicolas B.
Published: March 2005

The formation of 7α‐hydroxycholesterol from cholesterol by membrane‐bound cholesterol 7α‐hydroxylase in rat‐liver microsomes was investigated in vitro. When [14C]cholesterol of known specific radioactivity was incubated in suspensions of rat‐liver microsomes, the specific radioactivity of the total cholesterol in the suspension compared with that of the 7α‐hydroxychol‐esterol formed during the incubation showed that [14C]cholesterol entered a pool of cholesterol in the microsomes (substrate pool) accessible to the enzyme. Estimates of the maximal size of the substrate pool suggested that not more than 70% of the cholesterol in rat‐liver microsomes is immediately accessible to cholesterol 7α‐hydroxylase. The extent to which [14C]cholesterol entered the substrate pool was influenced by the form in which the [14C]cholesterol was added to the suspension. The specific radioactivity of 7α‐hydroxycholesterol reached a maximum value early during the incubation and remained constant thereafter. When nonradioactive cholesterol was added to a microsomal suspension pre‐incubated with a trace of [14C]cholesterol, the proportion of the nonradioactive cholesterol that entered the substrate pool was smaller than that of the [14C]cholesterol. When [14C]cholesterol was injected intravenously into rats, the [14C]cholesterol that entered the liver microsomes was found to be predominantly in the portion of microsomal cholesterol that is not accessible to cholesterol 7α‐hydroxylase.
Diacetyl (Acetoin) Reductase from Aerobacter aerogenes
Authors: Johansen Liv , Larsen Sigrun H. , StØRMER Fredrik C.
Published: March 2005

A kinetic study of diacetyl (acetoin) reductase in the reaction from diacetyl to acetoin has been performed.

Product inhibition plots, similar to those obtained for the reduction of acetoin and the reverse reaction, were obtained. The kinetic mechanisms for the reactions catalyzed by the enzyme are proposed.
Diacetyl (Acetoin) Reductase from Aerobacter aerogenes
Authors: Larsen Sigrun H. , Størmer Fredrik C.
Published: March 2005

The kinetics of the reversible reduction of acetoin and NADH to 2,3‐butanediol and NAD by diacetyl (acetoin) reductase from Aerobacter aerogenes, have been investigated at pH 7.0, 5.8, and 4.8. The reaction follows an ordered bi‐bi mechanism.

Acetate acts as an inhibitor of the enzyme with respect to the substrate 2,3‐butanediol. This inhibition increases for decreasing pH, indicating that it is acetic acid that is the effector.

These results, in combination with earlier observations, show that high acetate concentration allows maximal 2,3‐butanediol and NAD production at 5.8 from pyruvate and NADH.
Cyanide Binding to Carbonic Anhydrase
Authors: Feeney James , Burgen Arnold S. V. , Grell Ernst
Published: March 2005

From the similar 13C chemical shifts measured for the cyanide complexes of carbonic anhydrase B enzymes and inorganic zinc cyanide complexes, it is concluded that cyanide binds in its unprotonated form directly to the zinc ion in the enzyme. The cyanide 13C chemical shift of the enzyme · cyanide complex is pH independent indicating that neither the cyanide nor the atom to which it is bonded can undergo protolytic reactions.

The 13C spectrum of HCN is reported and the C‐H directly‐bonded coupling constant measured as 275 ± 10 Hz.
The Isolation and Properties of a Tyrosine‐Rich Protein from Wool: Component 0.62
Authors: Frenkel Maurice J. , Gillespie J. Morton , Woods E. Frank
Published: March 2005

An apparently homogeneous protein (component 0.62), of mol. wt 6900, has been isolated from reduced and carboxymethylated wool proteins by chromatography on QAE‐cellulose at pH 10.5, followed by chromatography on DEAE‐cellulose at pH 8.26% of the amino acid residues of this protein are glycine, another 31% are aromatic amino acids, these together with serine account for 70% of the molecule. The component is unusual in containing no lysine, histidine, glutamic acid, methionine or isoleucine.

Electrophoresis in starch gels at pH 8.6 and pH 2.4 and isoelectric focusing in polyacrylamide, indicate that the preparation contains a single component with an isoelectric point of 3.6. Chromatography on calibrated columns of Sephadex G‐75 and Biogel P‐10 gives single symmetrical peaks corresponding in elution volume with an apparent molecular weight of about 4700, whereas ultracentrifugation and sequence give a value close to 7000. It is probable that this discrepancy is due to an extreme retardation of the protein on Sephadex or Biogel due to the large number of aromatic amino acid residues present in the protein. Component 0.62 is almost completely insoluble below pH 9 but increases in solubility with increasing pH and is soluble to the extent of about 0.20% at pH 10.0. This protein component constitutes about 30% of the high‐tyrosine type I protein group and accounts for at least 2% of the mass of fine Merino wools.
The Primary Structure of an Acidic Protein from 50‐S Ribosomes of Escherichia coli which is Involved in GTP Hydrolysis Dependent on Elongation Factors G and T
Authors: Terhorst Cox , Möller Wim , Laursen Richard , Wittmann‐Liebold Brigitte
Published: March 2005

The primary structures of the ribosomal proteins A1 (= L7) and A2 (= L12) have been elucidated. Comparison of the two amino acid sequences confirms earlier studies by us (1972) which indicated that the two proteins are identical except that A1 possesses an N‐terminal acetyl group.

Sequencing of tryptic and chymotryptic peptides was accomplished primarily by automatic solid‐phase Edman degradation of 50‐ to 70‐nanomole peptide samples. A large tryptic peptide T1Phe, which could not be sequenced by this method, was fragmented with elastase and its sequence mainly derived by conventional methods. The sequence of the first 51 residues of the nonacetylated A2‐protein was obtained by Edman degradation in the Beckman sequencer.

A‐protein comprises three distinct regions: I (residues 1—55), which is negatively‐charged and hydrophobic, II (56—81), which is positively charged, and III (82—120), which is negatively charged and hydrophilic. α‐Helix promoting residues are located primarily in regions I and III. ɛ‐N‐Monomethyllysine, which occurs in 50% of the A1 and A2 chains is located in the more flexible region II at position 81.

The distinctive clustering of hydrophobic and charged residues is quite remarkable and suggests that in situ certain of these regions may contain a binding site for components involved in peptide chain elongation.
Spectrophotometric Studies on the Interaction of Sarcoplasmic‐Reticulum Fragments with Adenosine Triphosphate and Calcium
Authors: Nakamaru Yoshiaki , Schwartz Arnold
Published: March 2005

An interaction of sarcoplasmic reticulum of skeletal and cardiac muscle with bromeresol purple was studied in the absence and presence of ATP and/or calcium using dual‐wavelength spectrophotometry.

In the absence of added calcium, addition of ATP induced four specific phases that were measured as successive changes in absorption intensity of bromeresol purple at the wavelength pair 590—600 nm: phase 1, rapid decrease; phase 2, rapid increase; phase 3, gradual decrease; and phase 4, gradual increase.

The absorption intensity of the phases was entirely reversed in direction at the wavelength pair 615—640 nm. The wavelength of maximum absorption by bromeresol purple was 587 nm; the isosbestic point with respect to pH was 488 nm.

At the wavelength pair 488–540 nm and in the presence of ATP, phase 2 and phase 4 were not observed. The intensity‐increasing phase was completely calcium‐independent and represented a blue shift of the absorption spectrum of bromeresol purple.

The intensity‐decreasing phase was calcium‐dependent and included both a red shift and some pH changes of the medium. The blue shift (phase 2) accompanied the interaction of sarcoplasmic reticulum, ATP and magnesium.

Addition of calcium caused a shift back to longer wavelengths (phase 3). The initiation of the blue shift (phase 2) coincided with removal of calcium in the medium by active accumulation. Calcium had no effect on the shift in the absence of ATP.

The results suggest that the blue shift (shift to shorter wavelengths, phase 2) might reflect the formation of a sarcoplamic reticulum · ATP complex; the red shift (to longer wavelengths, phases 1 and 3) might reflect conversion of this complex to another intermediary form.
Adaptive Changes in Satellite Systems Related to Lipogenesis in Rat and Sheep Mammary Gland and in Adipose Tissue
Authors: Gumaa Khalid A. , Greenbaum A. Leslie , Mclean Patricia
Published: March 2005

The activities of a range of enzymes from the glycolytic and pentose phosphate pathways, tricarboxylic acid cycle and lipogenic pathways have been measured in the mammary glands of rats at different stages of the lactation cycle and in the lactating sheep mammary gland.

Changes of the enzyme activities are considered in the light of constant and specific proportion enzymes; in particular, in relation to the alternative systems for the generation of NADPH in the cytosol.

Specifically considered are the metabolic transitions in the rat at parturition and weaning; comparison between lactating sheep and rat mammary gland (which manufacture a similar spectrum of fatty acids from different carbon sources) and a comparison between lactating rat mammary gland and rat adipose tissue.

Evidence is adduced from enzyme profiles and flux data that the major source and pathway of generation of NADPH differed in the three tissues, an adaptation dependent upon carbon source and over‐all energy requirement of the tissue. Specifically, rat mammary gland has a greater emphasis upon NADPH formation via the pentose phosphate pathway, sheep mammary gland from cytosolic isocitrate dehydrogenase (combined with glutamate dehydrogenase acting as a transhydrogenase system) and adipose tissue showing participation of both pentose phosphate pathway and ‘malic’ enzyme.

The adaptation of mammary tissue to the use of the pentose phosphate pathway or of isocitrate dehydrogenase confers considerable advantage in the ATP yields relative to adipose tissue, where partial generation of NADPH via the ‘malic’ enzyme necessitates an ATP‐consuming reaction.

The modification in other satellite systems of lipid synthesis, such as pyruvate carboxylase, citrate cleavage enzyme and glycerol‐1‐phosphate dehydrogenase, is linked to the relative emphasis on different hydrogen‐generating systems, to the source of carbon and to the function of the tissue as a secretory or storage and release tissue.
Genetic Variants of Human Erythrocyte Glucose‐6‐Phosphate Dehydrogenase
Authors: Cancedda Ranieri , Ogunmola Gabriel , Luzzatto Lucio
Published: March 2005

Analysis of thermal inactivation profiles of human erythrocyte glucose‐6‐phosphate dehydrogenase, type B, reveals three distinct forms of the enzyme. The form with the lowest stability is observed at very low NADP+ concentration, and probably consists of the monomeric molecular species. As the NADP+ concentration is increased, the enzyme undergoes two successive transitions to forms of higher stability, which probably consist of two different conformational states of the dimer, in equilibrium with tetramer. Enzyme type A behaves similarly, but may have less tendency to monomer formation.

NADPH, like NADP+, is competent to stabilize the enzyme under suitable conditions and to induce dimer formation, but probably not to induce formation of the dimer with the highest thermostability. NADPH nay be integral part of the enzyme structure.

Stopped‐flow experiments suggest that the monomer of glucose‐6‐phosphate dehydrogenase is catalytically inactive, but that it recovers activity within 100 ms when exposed to a high concentration of NADPH+. By contrast, with the enzyme in dimeric form, the reaction starts immediately upon addition of glucose‐6‐phosphate.
Translation of Duck‐Globin Messenger RNA in a Partially Purified Mammalian Cell‐Free System
Authors: Schreier Max H. , Staehelin Theophil , Stewart Andrew , Gander Eugen , Scherrer Klaus
Published: March 2005

Duck globin messenger RNA purified by three different procedures from duck immature erythrocyte polyribosomes was translated in a mammalian cell‐free system which is strongly dependent upon exogenous mRNA. The system consists of purified mouse liver ribosome sub‐units, partially purified rabbit reticulocyte initiation factors, and rat liver pH‐5 enzymes (containing elongation and termination factors, aminoacyl‐tRNA synthetases and tRNA). If offered alone, duck globin mRNA directed the synthesis of all known duck globin chains. The efficiency of duck globin synthesis was similar to that of rabbit globin if the two types were translated separately. If duck and rabbit globin mRNA were offered together each in saturating amounts in this system, rabbit globin synthesis occurred almost exclusively. Differences in the secondary structure between the two types of globin mRNA must be involved in this strongly discriminating translation in the competition experiments. Furthermore, the quantitative requirement of at least one initiation factor was found to be different for duck and rabbit globin mRNA translation. While there is no evidence that this factor is directly involved in mRNA binding, it seems likely that the difference in factor requirement is related to the preferential translation of rabbit globin mRNA in the competition experiments.
Protonated Polynucleotide Structures
Authors: Sarocchi Marie‐Thérèse , Guschlbauer Wilhelm
Published: March 2005

Circular dichroism and absorbance‐difference spectra of polydeoxynucleotides and DNAs of various base composition have been measured at different submelting temperatures. The following results were obtained.

The changes observed by both techniques correlate poorly with (G + C) content, but follow the normalized YpY, i.e. (fCpC+fTpT)/xG+C, and not the mixed RpY, i.e. (fApT+fTpA)/yA+T, nearest‐neighbour frequencies.

Small pH changes have considerable effect on the difference spectra.

The metastable protonated form of poly(dG) · poly(dC) Correlates better with the circular dichroism spectra than the non‐protonated form.

It is concluded that dC clusters may be at the origin of DNA premelting, especially if they are in the metastable protonated form.
The Problem of Cell Heterogeneity of Liver Tissue in the Study of Fructose Metabolism
Authors: Zehner Jürgen , Loy Eberhard , Müllhofer Gerd , Bücher Theodor
Published: March 2005

Livers of rats fasted for 48 h were perfused with [2‐14C]fructose, [6‐14C]fructose or [U‐14C]‐fructose in a non‐recirculating perfusion system.

From the 14C‐labeling patterns of the metabolites glucose and lactate, which were isolated from the effluent perfusate, as well as from the labeling patterns of lactate, phosphoglycerate and phosphoenolpyruvate, which were separated from the tissue extract, the following conclusions were drawn:

The 14C activity of lactate in the perfusate leaving the liver is almost independent of the position of 14C label in the fructose, which was infused. This indicates that this lactate originates from a metabolic pathway in which both triose units of fructose are converted to lactate. On the other hand the 14C activity of cellular lactate depends on the position of the 14C label in the infused fructose. This lactate seems to be formed only from the C4‐C5‐C6 unit of the fructose. These two pathways of fructose metabolism are postulated to be located in different cell compartments of liver tissue and are initiated by phosphorylation of fructose at carbon position 6 and 1, respectively. We refer to them as the “glycolytic” and “gluconeogenic” compartments.

In experiments with [2‐14C]fructose there is no correlation between the specific 14C activities of phosphoglycerate, phosphoenolpyruvate and intracellular lactate indicating compartmentation of these metabolites.

By comparing the specific 14C activities of the C4‐C5‐C6 triose unit of newly formed glucose (taken as representative of the specific 14C activity of glyceraldehyde‐3‐phosphate in the “gluconeogenic” compartment) with the specific 14C activity of cellular lactate, the following can be postulated. Glyceraldehyde is transformed into lactate either by a pathway not involving glyceraldehyde‐3‐phosphate as an intermediate or a glyceraldehyde‐3‐phosphate which is spatially separated from the intermediate glyceraldehyde‐3‐phosphate pool of gluconeogenesis. The measured high tissue levels of 2‐phosphoglycerate in our experiments support the first concept.

In experiments with [2‐14C]fructose, the specific 14C activity of newly formed glucose corresponds to the 14C activity of fructose infused, while in experiments with [6‐14C]fructose a high dilution of the 14C activity of glucose was observed. This dilution must originate from a crossing over of some intermediate metabolite in the conversion of fructose to glucose with the inactive intermediate metabolite of some other metabolic pathway. An interaction with the citric acid cycle as possible source of this dilution was excluded.
Studies on the Conformation of Yeast Pyruvate Decarboxylase in Solution by Small‐Angle X‐Ray Measurements
Authors: Pilz Ingrid , Ullrich Johannes
Published: March 2005

Pyruvate decarboxylase, holo‐ and apo‐enzyme, was studied by X‐ray small‐angle scattering.

A concentration series of the holo‐enzyme in 50 mM sodium phosphate buffer pH 6.8 was measured. The solutions were homodisperse and the following data could be determined: radius of gyration = 4.13 nm, molecular weight = 185000 (partial specific volume 0.751), volume = 400 nm3, hydration = 0.55 g H2O/g protein. The overall shape of the holo‐enzyme can be described by an elongated body (ellipsoid of revolution or double sphere) with an axial ratio of about 1:1:2.2.

From the apo‐enzyme no homodisperse solutions could be obtained. There was always a mixture of particles, which had nearly the same radius of gyration as the holo‐enzyme, and larger aggregates. Therefore no detailed evaluation was possible. The partial specific volume was found to be identical with that of the holo‐enzyme within the limits of error.
The Solution Conformation and Some Spectroscopic Properties of 1,N6‐Ethenoadenosine Monophosphate, a Fluorescent Analogue of AMP
Authors: Penzer Geoffrey R.
Published: March 2005

The conformation of ethenoadenosine monophosphate in aqueous solution at pH 2.0 has been shown to be very similar to that of AMP through use of the lanthanide‐shift proton‐magnetic‐resonance technique.

The pKa, for protonation of the 9‐position of etheno‐AMP is 4.3 at 298 K, but at all pH values in the range 1.0–9.6, fluorescence is emitted from the protonated form of the heterocyclic ring system. This conclusion is based on the pH, temperature and KI dependence of the emission spectrum.

When dissolved in ethanol the quantum yield of etheno‐AMP fluorescence falls to about 40% of its value in water.

There are unlikely to be large changes in the energies of the emissions of ethenoadenosine derivatives when bound to biological structures, but their quantum yields may well be reduced, especially if binding affects the ease with which the rings can be protonated.
Functional Behaviour of Transferrin
Authors: Hahn Dietrich
Published: March 2005

Radioactive iron was preferentially bound in vitro to the two functionally different binding sites of transferrin, referred to as hemoglobin‐orientated and nonhemoglobin‐orientated binding site, respectively. Its availability from these binding sites for different organs was tested in vivo in rats.

To distinguish different iron uptake characteristics by different organs from the two binding sites of the injected transferrin, the normally high percentage of the erythropoiesis‐related iron clearance was cut down to a level similar to the degree of radioactive iron clearance to the storing organs liver and spleen by a hypertransfusion‐induced polycythemia.

It was observed that the iron from the hemoglobin‐orientated binding site was taken up predominantly by bone marrow and intestine, while liver and spleen had a higher affinity to iron bound to the nonhemoglobin‐orientated binding site, thus indeed pointing out a different function of the two binding sites.
The Kinetics of the Redox Reactions of Ubiquinone Related to the Electron‐Transport Activity in the Respiratory Chain
Authors: KrÖGER Achim , Klingenberg Martin
Published: March 2005

The role of ubiquinone (Q) in the respiratory chain is quantitatively analyzed by correlating both the steady‐state redox level of Q and the kinetics of oxidation and reduction with the flux of the overall electron transport in uncoupled submitochondrial particles. This is achieved by experimentally defining donor and acceptor activities for Q.

The degree of reduction of Q in the steady state is proportional to the respiratory activity with NADH and succinate, if the respiratory activity is varied by titrating the donor side for Q with rotenone and malonate, respectively. The proportionality constant (acceptor activity, Vox) is independent of the substrate used.

The degree of oxidation of Q in the steady state is proportional to the respiratory activity as varied by titration of the acceptor side for Q with antimycin. The proportionality constant (donor activity, Vred) is independent of the acceptor activity and depends on the dehydrogenase activity for NADH and succinate.

From these experimental relations, the redox state of Q in the steady state and the respiratory activity can be described as functions of the donor and the acceptor activity only. The equations are valid with both NADH and succinate as the substrates.

The kinetics of oxidation of Q on the addition of oxygen as measured by the quench‐flow method are in agreement with that measured by direct absorption recording in a mixing chamber. The reaction is first order with a rate constant equal to the acceptor activity divided by the amount of redox‐active Q (Vox/Qa). The final steady‐state level (in the present case 15% reduction) is a result also of the reduction reaction with the first‐order rate constant, VredQa.

The acceptor activity can also be measured as maximum respiratory activity with duro‐hydroquinone. This activity is independent of the presence of Q but sensitive to antimycin. Thus, the acceptor activity for ubiquinone can be measured by three independent methods.

It is concluded, that also in the steady state the reduction and oxidation of the active Q‐pool follow pseudo‐first‐order reactions, the rates of which are equal to the respiratory rate. The total amount of redox‐active Q is kinetically and functionally homogeneous and is not divided into substrate‐specific compartments.
The Steric Course with Respect to the Reduced Nicotinamide‐Adenine Dinucleotide of the Reduction of 3‐Oxo Steroids Catalysed by Cortisone Reductase
Authors: Gibb William , Jeffery Jonathan
Published: March 2005

The stereochemistry of hydrogen transfer from NADH during the reduction of the 3‐oxo groups of various steroids by cortisone reductase has been investigated for a series of substrates showing interesting structural features or reaction characteristics. It was found that the 4B‐hydrogen of the NADH was transferred to the substrate in all cases whether the resulting hydroxyl group was 3α or 3β. The 4B‐hydrogen was also transferred when the same enzyme catalysed the reduction of the 20‐oxo group of pregn‐4‐ene‐3,20‐dione, so, although the enzyme can bind steroids allowing hydrogen transfer in various ways, it binds NADH in such a way that only the 4B‐hydrogen is available for catalytic transfer to any considerable extent. No reaction was detected with NADPH.

The reaction rate with NADH was lowered by the presence of NAD+ in the two cases tested and, in the one case in which the reaction was studied in the reverse direction, NADH lowered the rate of reaction with NAD+. In all three cases, the apparent V values did not change significantly, whereas the apparent Km values for the reacting nucleotide changed considerably (“competitive inhibition”). This was consistent with an ordered reaction sequence in which the nucleotide bound before and left after the steroid, but these findings did not eliminate other possibilities.
Amino‐Acid Sequence of a Peptide from Human‐Serum Albumin
Authors: Babin Donald R. , Goos Susan M.
Published: March 2005

A peptide obtained by cleavage of human serum albumin with cyanogen bromide was isolated by gel filtration and ion‐exchange chromatography. The peptide contains 31 amino acids and has proline as its amino terminal residue. The amino acid sequence of this peptide has been determined by conventional methods.
Rat‐Liver‐Sialidase Activity Utilizing a Tritium‐Labeled Sialic‐Acid Derivative of Glycoprotein Substrates
Authors: Bernacki Ralph J. , Bosmann H. Bruce
Published: March 2005

Plasma glycoproteins were oxidized with periodic acid and reduced with tritiated KBH4, resulting in macromolecules specifically labeled with tritium on their sialic acid residues. The resulting tritiated seven‐carbon analog of N‐acetylneuraminic acid, abbreviated [3H]AcNeu, covalently attached as part of the fetuin molecule, [3H‐AcNeu]fetuin, had a specific activity of 3.67 counts × min−1× ng−1. It served as a very suitable substrate for the measurement of bacterial and mammalian neuraminidase. Clostridium perfringens neuraminidase had a Km of 9 μM and a V of 50 counts × min−1× h−1× ng−1. [3H‐AcNeu]Fetuin was homogeneous on polyacrylamide gel electrophoresis and migrated exactly as native fetuin following treatment by sodium dodecyl‐sulfate, mercaptoethanol, and boiling. Mild acid hydrolysis removed 70% of the radioactivity, which chromatographed as a homogeneous product utilizing butylacetate, acetic acid and water (3:2:1, v/v/v) as the solvent system.

Mammalian neuraminidase was located in a particulate fraction of rat liver. The enzyme had an acid pH optimum and may be lysosomal in origin. The use of [3H‐AcNeu7]glycoprotein substrates increased the sensitivity of measurement of the released sialic acid and provided a more reliable estimate of neuraminidase levels in mammalian tissue sources.

Livers from rats fed a vitamin‐K‐deficient diet were also assayed for activity. The results indicated that vitamin‐K‐deficient animals had a slightly elevated amount of neuraminidase activity with [3H‐AcNeu7]prothrombin substrate. Prothrombin, unlabeled or labeled, was not extensively degraded by Cl. perfringens neuraminidase. Therefore, either differences exist between bacterial enzyme and mammalian enzyme in the substrate specificities, or the mammalian enzyme source may contain more than one neuraminidase, i.e. one capable of the degradation of the sialic acid residues of fetuin and another capable of the degradation of the sialic acid residues of prothombin.
The Effects of Electrolytes on the Interaction of Cationic Dyes with Energized Mitochondrial Fragments
Authors: Dell'ANTONE Paolo , Frigeri Luciano , Azzone Giovanni Felice , Ziche Bruno , Pregnolato Luciano
Published: March 2005

The binding of cationic dyes to energized submitochondrial particles and the extent and rate of absorbance changes accompanying the binding are enhanced by the presence of inorganic anions. The anion effect follows the sequence: SCN‐ > ClO4− > I‐ > NO3− > Br‐ > Cl− > BrO3−= H2PO4− > CH3COO− > F−. With the more lipophilic anions the phase of enhancement is followed by one of inhibition: this effect is more marked with more lipophilic dyes. The rate and extent of absorbance changes is also affected by cations according to the sequence: Ba2+ > Sr2+ > K++ valinomycin > Li+ > Mg2+ > Ca2+ > Cs+∼ Rb+∼ K+∼ Na+. Under conditions where the anions markedly enhance the binding of the dyes by the energized fragments there is no parallel increase of anion binding. It is suggested that the membrane conformation and therefore the accessibility of the sites for various ligands, is modified during the interaction of electrolytes with energized fragments.
Assay of Yeast Enzymes in situ
Authors: Serrano Ramón , Gancedo Juana M. , Gancedo Carlos
Published: March 2005

A permeabilization method which allows the assay of several intracellular enzymes within the boundaries of the yeast cell wall is described. The kinetic properties of hexokinase and pyruvate kinase examined in the permeabilized cells, including the allosteric activation of the latter by fructose bisphosphate, are essentially the same as in cell‐free extracts.