bluuebunny/crossref_metadata_2025 huggingface dataset:
Identification of Neurophysin‐Related Proteins in Bovine Neurosecretory Granules
Authors: MASSE Marie J. O. , DESBOIS‐PERICHON Pierette , COHEN Paul
Published: July 2010

A highly purified fraction of neurosecretory granules isolated from the bovine posterior hypophysis was obtained using a continuous isotonic Percoll gradient. The analysis of their protein content indicates that the main products consist of 10000‐Mr neurophysin (95%) and about 5% of high‐molecular‐weight forms of this protein. Species ranging from Mr 80000–14000 were detected by radioimmunoassay and immunoprecipitated by anti‐neurophysin antibodies after 125I labeling of the neurosecretory granule lysates. The chemical relationship with neurophysin, of one of the immunoreactive species (Mr 19000), was ascertained by tryptic map analysis of the radioiodinated material. It is concluded that species both immunologically and chemically related to neurophysin represent a large majority of the protein content of the granules. They compare with the higher molecular weight forms of these neurosecretory components detected in the hypothalamo‐neurohypophyseal tract and proposed as putative precursors of both neurophysin and vasopressin.
Excision of Apurinic Sites from DNA with Enzymes Isolated from Rat‐Liver Chromatin
Authors: GOFFIN Colette , VERLY Walter G.
Published: July 2010

Apurinic sites were excised from φX174 RF DNA with two enzymes isolated from rat liver chromatin: an apurinic/apyrimidinic endodeoxyribonuclease and a 5′‐3′‐exonuclease; the resulting gap was filled with DNA polymerase β also prepared from rat liver chromatin and the repair was fully terminated with T4 ligase.
A Pulse‐Radiolysis Study of Cytochrome c3
Authors: VAN LEEUWEN Johan W. , VAN DIJK Cees , GRANDE Hans J. , VEEGER Cees
Published: July 2010

Pulse‐radiolysis experiments were performed in the presence of methyl viologen and cytochrome c3. After the pulse, methyl viologen radicals are formed and the kinetics of these radicals with cytochrome c3 are studied. The reaction between cytochrome c3 and methyl viologen radicals (MV+) is diffusion controlled. The ionic strength dependence and the pH‐dependence of this reaction were studied. From the ionic strength dependence (at pH 7.8) we found that the net charge of the fully oxidized cytochrome c3 molecule was Z =+ 4.7 ± 0.7.

After the pulse an equilibrium is reached for the reaction of MV+ with cytochrome c3. From this equilibrium an apparent midpoint potential can be obtained. The apparent midpoint potential of this multihaem molecule was found to depend on the degree of reduction, α. With the help of the Nernst equation an empirical equation is obtained to describe this dependence of the midpoint potential: E0=− 0.250–0.088α (in V).

An estimation is made of the energy of interaction between the haems due to electrostatic interactions (Δɛ < 32 mV) and due to ionic strength effects (−12 mV < Δɛ < 26 mV). The results suggest that the redox properties of the individual haems in the cytochrome c3 molecule are dependent on the degree of reduction of the other haems in the molecule.

The reaction of cytochrome c3 with MV+ or with ethanol radicals (EtOH) has been compared with the reactions of horse‐heart cytochrome c and of metmyoglobin with the same radicals. The reaction of MV+ or EtOH with horse‐heart cytochrome c is found to be diffusion controlled; the reactions with metmyoglobin on the other hand are most probably controlled by an activation energy.
Short‐Term Regulation of the Nitrogenase Activity in Rhodopseudomonas sphaeroides
Authors: HAAKER Huub , LAANE Colja , HELLINGWERF Klaas , HOUWER Berend , KONINGS Wil N. , VEEGER Cees
Published: July 2010

The nitrogenase activity in whole cells of Rhodopseudomonas sphaeroides could be inhibited by lowering the electrical potential across the cytoplasmic membrane. The membrane potential was partly dissipated either by lowering the light intensity or by the addition of a lipophilic cation, tetraphenylphosphonium. Under these circumstances, it was shown that the intracellular ATP/ADP ratio was not affected and that the inhibition of the whole cell nitrogenase activity was not due to an inactivation of the nitrogenase enzyme. From these results it is concluded that electron transport to nitrogenase in Rps. sphaeroides is dependent on a high membrane potential.

The nitrogenase enzyme in whole cells could be inactivated by lowering the membrane potential across the cytoplasmic membrane by incubating the cells in the dark or in the light in the presence of uncouplers. Nitrogenase could be reactivated in the light in the absence of uncouplers.

Some possible mechanisms of action of NH+4 inhibition of whole cell nitrogenase activity could be excluded. Inhibition by NH4Cl of whole cell nitrogenase activity in Rps. sphaeroides could neither be explained by a rapid inactivation of the nitrogenase enzyme, nor by an effect on the intracellular ATP/ADP ratio or the membrane potential. NH+4 inhibits whole cell nitrogenase activity not directly but probably after being assimilated by glutamine synthetase. The role of glutamine, glutamate and 2‐oxoglutarate on the regulation of electron transport to nitrogenase will be discussed.
Luteinizing Hormone‐Releasing Hormone and Thyrotropin‐Releasing Hormone in Human and Bovine Milk
Authors: AMARANT Tanchum , FRIDKIN Mati , KOCH Yitzhak
Published: July 2010

Two hypothalamic peptide hormones, luteinizing hormone‐releasing hormone (LHRH) and thyrotropin‐releasing hormone (TRH), have been isolated from human milk and bovine colostrum. Acidified methanolic extracts, prepared from human milk, bovine colostrum and rat hypothalami, as well as synthetic LHRH and TRH markers were subjected to high‐pressure liquid chromatography (HPLC). The eluates were tested for the presence of LHRH and TRH by specific radioimmunoassays. It was found that milk extracts contain significant amounts of LHRH (3.9–11.8 ng/ml) and TRH (0.16–0.34 ng/ml), which comigrate with the corresponding marker hormones and with those of hypothalamic origin. The HPLC‐purified LHRH from both human and bovine milk was bioactive in a dose‐response manner similar to synthetic LHRH.
Synthesis of the Proteins of Complex III of the Mitochondrial Respiratory Chain in Heme‐Deficient Cells
Authors: LIN Ching‐I P. , GOLLUB Edith G. , BEATTIE Diana S.
Published: July 2010

The presence of several proteins of complex III of the respiratory chain has been demonstrated in mitochondria from a mutant of Saccharomyces cerevisiae lacking 5‐aminolevulinic acid synthase and, hence, devoid of heme. The two ‘core’ proteins, apocytochrome b and the iron‐sulfur protein, were observed in equal amounts in the heme‐deficient and heme‐sufficient cells with antiserum against complex III and the sensitive immunotransfer technique. In addition, three other bands were detected with the complex III antiserum in the mitochondria from the cells lacking heme. One of these has a molecular weight similar to that reported for a precursor form of cytochrome c1.

By contrast, when mitochondria from the heme‐deficient cells were solubilized with mild detergents and treated with the complex III antiserum, almost no immunoprecipitation was obtained above that obtained with control serum. The presence of only one major labeled band with a molecular weight similar to subunit I was observed after gel electrophoresis. These results suggest that heme may be necessary for proper processing of the apoprotein of cytochrome c1 and for the assembly into the membrane of the subunits of complex III, rather than for the synthesis of the proteins.
Isoleucyl‐tRNA Synthetase from Escherichia coli MRE 600
Authors: FREIST Wolfgang , STERNBACH Hans , CRAMER Friedrich
Published: July 2010

The substrate specificity of isoleucyl‐tRNA synthetase from Escherichia coli MRE 600 with regard to ATP analogs has been compared with the results obtained with isoleucyl‐tRNA synthetase from yeast. The enzyme from E. coli is less specific, the two enzymes exhibit different topographies of their active centres.

The order of substrate addition to isoleucyl‐tRNA synthetase from E. coli MRE 600 has been investigated by bisubstrate kinetics, product inhibition and inhibition by substrate analogs. The inhibition studies were done in the aminoacylation and in the pyrophosphate exchange reaction, the aminoacylation was investigated in the absence and presence of inorganic pyrophosphatase. As found for isoleucyl‐tRNA synthetase from yeast, the results of the pyrophosphate exchange studies indicate the possibility of formation of E · Ile‐AMP · ATP complexes by random addition of one ATP and one isoleucine molecule, followed by adenylate formation, release of pyrophosphate and subsequent addition of a second molecule of ATP. For the aminoacylation in the absence of pyrophosphatase, a rapid‐equilibrium random ter addition of the substrates is found whereas the enzyme from yeast exhibits a steady‐state ordered ter‐ter mechanism; in the presence of pyrophosphatase the mechanism is bi‐uni uni‐bi ping‐pong similarly as observed for the yeast enzyme.

A comparison of inhibition patterns obtained with N(6)‐benzyladenosine 5′‐triphosphate under different assay conditions (spermine or magnesium ions, addition of pyrophosphatase) indicates that even more than two pathways of the aminoacylation may exist.

The catalytic cycles of the two mechanisms derived from the observed orders of substrate addition and product release include the same enzyme substrate complex (E · tRNA · Ile‐AMP) for the aminoacyl transfer reaction. The kcat values, however, are considerably different: kcat of the sequential pathway is about 40% lower than kcat of the ping‐pong mechanism.
Porcine Pancreatic Lipase
Authors: BENKOUKA Fatima , GUIDONI Andrea A. , DE CARO Josiane D. , BONICEL Jacques J. , DESNUELLE Pierre A. , ROVERY Mireille
Published: July 2010

Following complete sequence analysis of the 449 amino acids in porcine pancreatic lipase [J. De Caro et al. (1981) Biochim. Biophys. Acta, 671, 129–138], the position of the six disulfide bridges and of the two free thiols of the protein was investigated using a variety of techniques. Three bridges (Cys‐4–Cys‐10, Cys‐237–Cys‐261 and Cys‐433–Cys‐449) were easily identified in the peptic digest of lipase at pH 2.0. In the latter digest, two other bridges (Cys‐285–Cys‐296 and Cys‐299–Cys‐304) were also identified by means of the cystine peptide constituted by two peptide segments: Ala281‐Gly‐Phe‐Pro‐Cys‐Asp‐Ser287 and Thr292‐Ala‐Asn‐Lys‐Cys‐Phe‐Pro‐Cys‐Pro‐Ser‐Glu‐Gly‐Cys‐Pro‐Gln‐Met307. A disulfide bridge, formed by Cys‐285 and one of the three half‐cystines of the other segment, connected the two peptide moieties. A second disulfide bridge linked the two remaining half‐cystines. It was not possible to split any peptide bond between Cys‐296 and Cys‐304 with proteolytic enzymes. The determination of the pairing of the four half‐cystines was resolved as follows. Bond Lys‐295–Cys‐296 was cleaved with trypsin. A single cycle of Edman degradation was then performed on the peptide compound, thus freeing, in particular, Cys‐296 of the peptide bond (296–297). Cys‐296 only retained its S‐S connection with the half‐cystine partner. At the completion of the above operations, the two peptide segments (282–287) and (297–307) could be separated. Consequently it was concluded that Cys‐285 is linked to Cys‐296. Therefore Cys‐299 and Cys‐304 are paired.

The most reactive SHI group of the enzyme was localized on Cys‐181 by condensation of the native protein with radioactive N‐ethylmaleimide. The alkylation of the SHII group required previous denaturation of the molecule at alkaline pH. The results obtained for the characterization of the SHII group suggested the existence of two isomeric forms, the SHII being either on Cys‐101 or Cys‐103 and the bridge alternately between Cys‐90–Cys‐103 or Cys‐90–Cys‐101.

It is not yet known whether these two forms pre‐exist in native lipase or result from an exchange reaction. The bridge Cys‐90–Cys‐101 was characterized in a thermolytic digest of a cyanogen bromide fragment (CN II) of the protein. However, another bridge involving Cys‐181 was also found in the digest. This bridge is considered as being an artefact. It is possible that considering the treatments undergone by the large peptide (CN II), the SH groups of lipase were oxidized and transformed in an S‐S bridge.

The disulfide bridges of lipase form relatively small loops along the main chain. This arrangement is consistent with a high flexibility of the molecule. As reported earlier [R. Verger et al. (1971) Biochim. Biophys. Acta, 242, 580–592], the SHI group is not essential for lipase activity. The role of the SHII group should be more precisely investigated.
Kinetic Anticooperativity in Pre‐Steady‐State Formation of Tryptophanyl Adenylate by Tryptophanyl‐tRNA Synthetase from Beef Pancreas
Authors: MAZAT Jean‐Pierre , MERLE Michel , GRAVES Pierre‐Vincent , MERAULT Guy , GANDAR Jean‐Claude , LABOUESSE Bernard
Published: July 2010

The kinetics of formation of tryptophanyl adenylate by tryptophanyl‐tRNA synthetase from beef pancreas has been followed by stopped‐flow, using the quenching of fluorescence of the enzyme linked to the amino acid activation reaction. Both subunits of this α2 enzyme catalyze the adenylate formation. At saturation with substrates the rate constant of the activation reaction is the same for both subunits. The same behaviour is observed for the pyrophosphorolysis reaction. Both subunits exhibit the same affinity for ATP‐Mg in the forward reaction and the same affinity for magnesium pyrophosphate in the backward reaction. On the contrary the formation of tryptophanyl adenylate follows biphasic kinetics when tryptophan concentration is much below saturation. This is independent of ATP‐Mg concentration and is the consequence of different affinities of the two subunits for tryptophan as already observed by Graves et al. (1979, Eur. J. Biochem. 96, 509–518) in equilibrium dialysis experiments. A monoadenylate‐enzyme complex on one subunit has been prepared. This complex made possible the study of the formation of the second adenylate on the other subunit. The formation of this second adenylate followed first‐order kinetics at all ATP‐Mg and tryptophan concentrations. The tryptophan concentration dependence of the rate of formation of this second adenylate leads to a Michaelis constant close to the dissociation constant of the low affinity tryptophan site of the enzyme. No isomerization step could be evidenced. The experiments were carried out under two conditions corresponding to those used by Merault et al. (1978, Eur. J. Biochem. 87, 541–550) in the steady state of the tRNATrp aminoacylation reaction (10 mM total magnesium in 100 mM KCl and 1 mM free magnesium ions, both at pH 8.0, 25°C). No great difference either in the mechanism or in the dissociation and rate constants was observed but an inhibitory effect of KCl. It is concluded that the enzyme is symmetrical as far as the ATP‐Mg and the magnesium pyrophosphate sites are concerned and that the rate of the activation reaction reflects the anticooperative occupancy of the tryptophan sites carried by the two subunits.
A Coupled Diffusion and Barrier Model for the Recombination Kinetics of Myoglobin with Carbon Monoxide
Authors: MARDEN Michael C.
Published: July 2010

The rebinding kinetics of myoglobin (Mb) with carbon monoxide (CO) after photodissociation have been analyzed using a single energy barrier coupled by a two‐dimensional channel to the solvent. The total binding rate is written as the product of the barrier rate coefficient and the probability of the ligand being at the barrier. Assuming that the photodissociated ligands are initially localized near the barrier, the probability then decays as 1/tas the ligands migrate away toward the solvent. The time‐dependent rate coefficient yields power‐law kinetics as observed in the MbCO recombination kinetics.

At a given temperature two parameters are sufficient to simulate power‐law kinetics of arbitrary slope. Using a model of diffusion from a region of lower to higher diffusivity, four parameters can simulate an initial power law followed by a final slow exponential process. Estimates of the diffusion coefficients inside the protein are two to three orders of magnitude slower than the ligand diffusivity in the external solvent. A single Arrhenius rate coefficient for the barrier can be used for the entire range from 60 K to 320 K. The barrier/diffusion model implies a certain shape of the kinetics which is in good agreement with the observed recombination kinetics of many heme protein/ligand systems.
NMN Adenylyltransferase: Its Association with Chromatin and with Poly(ADP‐ribose) Polymerase
Authors: UHR Marie Louise , SMULSON Mark
Published: July 2010

The nuclear location of NMN adenylyltransferase, which catalyses the formation of NAD and pyrophosphate from ATP and NMN, has been examined to ascertain if the enzyme is bound to the domains of chromatin which undergo poly(ADP‐ribos)ylation. This latter reaction utilizes much of the cellular NAD.

A radioisotope assay using [α‐32P]ATP was developed to enable precise measurement of picomole amounts of NAD. With this assay, it appeared that the reaction catalysed by NMN adenylyltransferase proceeded with a rapid, early ‘burst’ of NAD before steady‐state velocities were established. From this it was calculated that there could be 107 active sites of NMN adenylyltransferase per HeLa nucleus in asynchronously growing cells; that is, approximately one per 10–20 nucleosomes.

Very little enzyme activity was liberated by digesting HeLa nuclei with micrococcal nuclease in 80 mM NaCl, and the enzyme which was solubilized was not bound to oligonucleosomes separated by electrophoresis on polyacrylamide gels. In contrast, poly(ADP‐ribose) polymerase activity was clearly demonstrated on these particles. The enzyme was readily liberated by DNase I digestion, especially when the digestion was carried out in low‐ionic‐strength buffer.

The results demonstrated that the enzyme was neither bound to oligonucleosomes nor part of the nuclear envelope or matrix. Preliminary results suggested that there could be some direct channelling of NAD between the two enzymes in intact nuclei. It appears that NMN adenylyltransferase is bound within rather than to chromatin.
An Improved Purification Method and a Physical Characterization of Phage T7 DNA Polymerase
Authors: RANDAHL Hakan , SLABÝ Ivan , HOLMGREN Arne
Published: July 2010

The immunoadsorbent used to purify T7 DNA polymerase contains antibodies directed towards thioredoxin. Elution of the enzyme is made by a pulse of buffer at pH 12.0. This decreases the binding capacity of the column. Binding experiments with [3H]thioredoxin showed that the effect was caused by reduction of the antibodies by thiols in alkaline buffers. T7 DNA polymerase aggregated and irreversibly lost activity in buffers of low ionic strength. Experiments with gel chromatography and glycerol density gradient centrifugation showed that 0.2 M sodium chloride was required to keep the enzyme in its monomeric form. The sedimentation coefficient and the Stokes' radius are 5.3 S and 4.6 nm respectively, evaluated by gel chromatography and glycerol density gradient centrifugation techniques. The frictional ratio of 1.49 indicates that the T7 DNA polymerase is an asymmetrical protein.
Role of Gangliosides in Reception of Influenza Virus
Authors: BERGELSON Lev D. , BUKRINSKAYA Alla G. , PROKAZOVA Nina V. , SHAPOSHNIKOVA Galina I. , KOCHAROV Sergey L. , SHEVCHENKO Valeriy P. , KORNILAEVA Galina V. , FOMINA‐AGEEVA Elena V.
Published: July 2010

The ganglioside composition of Ehrlich ascites carcinoma (EAC) cells and the role of the individual gangliosides in binding and penetration into the cell of influenza virus were determined. EAC gangliosides identical with or close to GM3, GM2, GM1, GT1a and GT1b were characterized by thin‐layer chromarography, compositional analyses, methylation analysis and mass‐spectrometry. The ganglioside uptake capacity of native and neuraminidase‐treated EAC cells was studied with tritium‐labeled gangliosides of definite structure and the binding of influenza virus to cells was determinated by using [3H]uridine‐labeled virus and by hemagglutination studies. Treatment of the cells with Vibrio cholerae neuraminidase largely decreased binding of the virus. Exogenous gangliosides with a terminal galactose unit or a penultimate galactose masked by neuraminic acid were able to restore the virus‐binding capacity of neuraminidase‐treated cells, however, the main ganglioside of EAC cells, GM2, which carbohydrate chain is terminated by N‐acetylgalactosamine, was completely ineffective. The common carbohydrate sequence of the ganghosides showing binding activity

image
is proposed to be the main recognition structure of the influenza virus receptor on the surface of EAC cells.

Penetration of labeled influenza virus into the nuclei of EAC cells was evaluated by measuring the radioactivity of the nuclei of neuraminidase‐treated ganglioside‐loaded cells after exposition to the labeled virus. Of all gangliosides tested only trisialogangliosides of the GT1b type were able to induce increased entry of the virus into the cells and accumulation of its radioactive component into the nuclei. It is suggested that GT1b gangliosides react specifically with the virus protein responsible for membrane fusion (apparently the hemagglutinin HA2 subunit) and thus are involved in virus penetration and delivery of the virus genome to the nuclei.
Detection and Localization of a Class of Proteins Immunologically Related to a 100‐kDa Nucleolar Protein
Authors: BUGLER Beatrix , CAIZERGUES‐FERRER Michèle , BOUCHE Gérard , BOURBON Henri , AMALRIC François
Published: July 2010

A high‐molecular‐mass nucleolar protein (100‐kDa protein) is associated with nascent pre‐rRNA and preribosomes in Chinese hamster ovary cells. We have prepared antiserum against the 100‐kDa protein and we have used it to study the intracellular localization and the possible processing of this protein.

Serologically related proteins were detected in the nucleolus and in ribosomes. Proteins of various subcellular fractions were separated by sodium dodecyl sulfate/polyacrylamide gel electrophoresis, transferred to nitrocellulose filters, reacted with serum and the protein‐immunoglobulin complexes were revealed by 125I‐labeled protein A. In the nucleolus, four proteins with molecular masses of 100 kDa, 95 kDa, 76 kDa and 70 kDa were thus visualized. In the ribosomes, two proteins (of 100 kDa and 76 kDa) gave a strong reaction while six others (of 70 kDa, 60 kDa, 50 kDa, 30 kDa, 21 kDa, 18 kDa) reacted slightly. By immunological precipitation of total cell extracts, we have shown that the 100‐kDa protein antiserum cross‐reacts with five proteins with molecular masses of 120 kDa, 100 kDa, 95 kDa, 70 kDa and 60 kDa.

Specific degradation of the 100‐kDa protein into similar peptides with molecular masses of 95 kDa, 76 kDa, 70 kDa, 60 kDa and 50 kDa can be achieved by incubation of isolated nucleoli or of purified 100‐kDa protein in vitro. Cleavage of the protein is due to a thiol endoprotease which is tightly bound to the 100‐kDa protein. Possible relations between the maturation of this preribosomal protein into ribosomal proteins and the processing of preribosomal RNA into mature ribosomal RNA are discussed.
Desensitisation of β‐Adrenergic Responsivenessin vivo
Authors: SVARTENGREN Jan , SVOBODA Petr , CANNON Barbara
Published: July 2010

Chronic catecholamine stimulationin vivoof brown adipose tissue during acclimation of hamsters to cold does not result in any alteration of p‐adrenergic receptor number or affinity when determined in isolated adipocytes by (–)‐[3H]dihydroalprenolol binding. Norepinephrine displacement of (–)‐[3H]dihydroalprenolol showed the sameKifor both groups. However, the slope of the displacement curve was shallower for cells from cold‐acclimated animals than for controls. Cyclic AMP accumulation was stimulated by norepinephrine in cells from both groups of animals, although the dose‐response curve for cells from cold‐acclimated animals was shifted to the right and the maximum value obtained was less than half that found in cells from control animals. The slope of the curve was again lower. Other catecholamines stimulated CAMP accumulation with an order of potency in agreement with a response mediated through β1‐adrenergic receptors. The dose‐response curve for norepinephrine‐stimulated oxygen consumption was also shifted to the right for cells from cold‐acclimated animals, although the maximal respiration was only slightly reduced. The slope factor was again decreased. The results are interpreted in terms of a reduced coupling between the β‐receptor and the metabolic response in isolated brown adipocytes from cold‐acclimated animals as a result of chronic catecholamine stimulationin vivo.
Transient Kinetics of the One‐Electron Transfer Reaction between Reduced Flavocytochrome b2 and Oxidized Cytochrome c
Authors: CAPEILLERE‐BLANDIN Chantal
Published: July 2010

The one‐electron transfer reaction from reduced flavocytochrome b2 (fully reduced by three electron equivalents) to ferricytochrome c, both purified from the yeast Hunsenula anomala, has been studied using stopped‐flow spectrophotometry in the course of a single turnover, for reactants initially mixed in a heme molar ratio equal to one. The cytochrome c reduction proceeded to completion through an apparently first‐order process. Depending on the experimental conditions (concentrations and/or ionic strength), the reduction is of second‐order or first‐order character.

To interpret these kinetic results computer simulation studies have been performed based on a kinetic scheme involving, besides the formation of a complex before the electron transfer step, intramolecular electron transfer steps within flavocytochrome b2 to maintain the concentration of the specific electron donor center, the reduced cytochrome b2. As far as the cytochrome c reduction rate constant, ka, and its variations were concerned the simulated data showed that this complicated scheme could approximate a mechanism which is by far the simplest, involving only the two former steps. Such a scheme accounts firstly for the hyperbolic dependence of the rate of reduction of cytochrome c, ka, upon reductant concentrations which had provided clear evidence for the kinetic existence of a complex in the reaction pathway. At 5°C the rate constant for the electron transfer is 380 s−1 with an activation energy of 13.8 kJ mol,−1 (3.3 kcal mol−1). Secondly it predicts the observed variations of ka with ionic strength and provides estimates of the rate constants of the binding step.
Kinetic and Thermodynamic Properties of the Ternary Complex between F‐actin, Myosin Subfragment 1 and Adenosine 5′‐[β,γ‐imido]triphosphate
Authors: KONRAD Manfred , GOODY Roger S.
Published: July 2010

Equilibrium constants for the formation of a ternary complex between actin, myosin subfragment 1 (S1) and the non‐hydrolyzable ATP analog adenosine 5′‐[β,γ‐imido]triphosphate (AdoPP[NH]P) were determined from light‐scattering titrations under a variety of conditions. The affinities of S1 (binding constant K1) and acto · S1 (K4) for AdoPP[NH]P have relatively low dependencies on temperature (ΔH°∼ ‐15–30 kJ mol−1) and ionic strength, in contrast to the affinities of S1 (K2 and S1 · AdoPP[NH]P (K3) for actin which are influenced quite strongly by temperature (ΔH°∼ 50–65 kJ mo1−1) and ionic strength, K2 decreasing by a factor of 10–15 between I= 0.05 M and I= 0.2 M and K3 decreasing by a factor of 5. K1, and by detailed balance K2 as well, were found to be about 10‐times higher than hitherto reported values (K1= 3.4 × 107 M−1, K2= 6 × 108 M−1, at 24°C, I= 0.09 M, pH 8.0). The binding of ADP to S1 is about 10‐fold weaker than that of AdoPP[NH]P, being however much more exothermic (ΔH°= ‐70 kJ mol−1 at I= 0.1 M) and having a negative standard entropy change (ΔS°= ‐125 J mol−1 K−1), in contrast to AdoPP[NH]P binding for which the calculated ΔS° had positive values.

The observed rate constant of dissociation of acto · S1 by AdoPP[NH]P showed an almost hyperbolic dependence on the nucleotide concentration, reaching a maximum of 15 s −1 at I= 0.055 M and 5 s −1 at I= 0.275 M, pH 8.0, 23°C; at 5°C this value was somewhat higher. The rate constant of dissociation of AdoPP[NH]P from its complex with acto · S1 was estimated to exceed 400 s−1 at 23°C, and to be of the order of 150 s−1 at 4°C. The observed rate constant for the association of the S1 middot; nucleotide complex and actin was proportional to actin concentrations up to 60 μM, thus defining an apparent second‐order rate constant of 2 × 104 M−1 s−1 at I= 0.125 M and 23°C. A reaction scheme is proposed in which isomerizations of the acto · S1 and acto · S1 · nucleotide complexes can occur.
The Pressure‐Induced, Reversible Inactivation of Mouse Brain Enolases
Authors: KORNBLATT J. , KORNBLATT J. , HUI BON HOA Gaston
Published: July 2010

The three isozymes of mouse brain enolase (αα, αγ and γγ) were subjected to pressures between 160 MPa and 380 MPa. Enzymatic activity 2 min after the pressure exposure was used to monitor pressure‐induced changes in the physical state of the protein. Pressure caused a reversible inactivation of all three forms; αα showed a continual inactivation with increasing pressure whereas the inactivation curves for αγ and γγ were biphasic. P1/2 for αα was 190 MPa, for αγ 210 MPa and for γγ 280 MPa. Volume changes associated with this inactivation were calculated for all three forms of the enzyme; ΔV0 varied from – 150 ml mol−1 to – 170 ml mol−1, assuming a model in which there was no change in the aggregation state of the enzyme, or from – 230 ml mol−1 to – 250 ml mol−1 assuming that the enzyme dissociated on exposure to pressure. The half‐time for reactivation was measured as a function of the concentration of enzyme; the results are consistent with an inactivation that is due to dissociation of the dimeric enzyme into monomers.
Purification of Twelve Cyanogen Bromide Fragments from Bovine Plasma Fibronectin and the Amino Acid Sequence of Eight of Them
Authors: SKORSTENGAARD Karnd , THØGERSEN Hans C. , VIBE‐PEDERSEN Karen , PETERSEN Torben E. , MAGNUSSON Staffn
Published: July 2010

Twelve cyanogen bromide fragments (CB1 – 12) from bovine plasma fibronectin have been isolated and eight of these completely sequenced. Altogether they account for 502 of the total expected 1880 residues in each of the two chains of fibronectin. Four of these fragments (CB1 – 4) constitute residues 1–289 in fibronectin with CB4 overlapping the N‐terminal 29‐kDa plasmic fragment to the second plasmic fragment, of 170‐kDa in fibronectin. Fragments CB 5 – 9 are all contained within a 45‐kDa gelatin‐binding region, which is N‐terminal in the 170‐kDa fragment. The sequence of two of these five fragments in the 45‐kDa fragment (CB7 – 8) contains two mutually homologous stretches with 57 sequence identity. Another two fragments (CB10 – 11) are derived from the heparin‐binding region of the 170‐kDa fragment. CB12 constitutes the C‐terminal 13‐residue stretch in fibronectin and contains a partly phosphorylated serine residue in the C‐terminal sequence: ‐Arg‐Glu‐Asp‐Ser(P)‐Arg‐Glu.
Preferential Transcription and Nuclear Transport of Globin Gene Sequences, as Control Steps Leading to Final Differentiation of Murine Erythroleukemic Cells
Authors: SHAUL Yosef , GINZBURG Irith , AVIV Haim
Published: July 2010

Murine erythroleukemic (MEL) cells undergo a specific program of differentation in vitro, which is mainly characterized by accumulation of globin mRNA. These cells serve as a model system to study in detail the expression of a specific gene product at the transcriptional and post‐transcriptional level. In this report we describe experiments in which the transcription rate of globin and non‐globin genes, as well as their cytoplasmic appearance, was measured during the differentiation process. Two independent steps for regulating the abundance of globin mRNA were observed. On the transcriptional level we have observed that, in contrast to the transcription of globin genes, the transcription rate of non‐globin genes is dramatically reduced throughout the period of induction. When the rate of cytoplasmic appearance was measured newly synthesized globin RNA molecules were found to be preferentially transported into the cytoplasm. It was shown that the reduction in cytoplasmic appearance of non‐globin genes is not a result of a shut‐off in their transcriptional activity. In cells treated with 12‐O‐tetradecanoylphorbol 13‐acetate the transcription rate remains constant while a continuous reduction in the cytoplasmic appearance is observed. These two independent phenomena which affect the non‐globin genes, i.e. the suppression of their transcription and reduced cytoplasmic appearance, lead to the reduction in the relative amounts of the stable poly(A)‐rich mRNA population and to the accumulation of globin sequences in the cytoplasm of the differentiated erythroid cells. These observations are in agreement with our previous model, which claimed that disappearance of the stable poly(A)‐rich mRNA population is an obligatory process leading to the final differentiation of MEL cells.
Synthesis in vitro of Novel Nucleic‐Acid‐Like Polymers of Ehrlich Ascites Tumor Cells
Authors: KAGAWA Kazuko , KAGAWA Haruo
Published: July 2010

When cytoplasmic particles isolated from Ehrlich ascites tumor cells were incubated with [14C]ribose, es and inorganic phosphate in the presence of MgCl2 and Tris/HCl (pH 7.0), radioactivity was incorporated into trichloroacetic‐acid‐insoluble materials.

Radioactive products, extracted and purified by DEAE‐cellulose column chromatography, were hydrolyzed in i M HCl at 100°C for 1 h and the resultant radioactive neutral sugar components were purified and identified as ribose, glucose and rhamnose by gas chromatography and mass spectrometry.

When [14C]glucose was incubated in the same reaction system, radioactivity was also incorporated into trichloroacetic‐acid‐insoluble materials. Purified radioactive products were hydrolyzed in 1 M HCl at 100°C for 1 h and the resultant radioactive neutral sugar components were also shown to be ribose, glucose and a little rhamnose, as was the case with the products synthesized from [14C]ribose.

When [14C]deoxyribose was incubated in the same reaction system, little incorporation was observed.

The radioactive products were incubated with venom phosphodiesterase and the digests were analyzed by Dowex lX4 column chromatography. The results suggest that the radioactive products contained ribo‐nuclcotides and ‘nucleotides’ containing glucose (glucose ‘nucleotides’).

When the radioactive products were incubated with both venom phosphodiesterase and alkaline phosphatase, ribonucleosides and glucose ‘nucleosides’ were detected. In Dowex 1X4 (borate form) chromatography, tibonuclcosides were eluted coincidentalhy with ribonucleoside markers (Cyd Ado Urd and Guo), whereas the elution profiles of glucose ‘nucleosides’ were totallydifferent fromfacto of ribonucleosides.
The Phosphorylation of Ribosomal Protein S6 from Progesterone‐Stimulated Xenopus laevis Oocytes
Authors: KRUPPA Joachim , DARMER Dorothea , KALTHOFF Holger , RICHTER Dietmar
Published: July 2010

Xenopus laevis oocytes were prelabeled with [32P]orthophosphate overnight before maturation was induced by progesterone stimulation. The phosphorylation status of ribosomal protein S6 from control oocytes and the temporal changes in S6 phosphorylation after progesterone treatment were analyzed by two‐dimensional gel electrophoresis. S6 protein was separated in up to five distinct S6 species, which differed in their degree of phosphorylation. 32P labeling of S6, as judged from the shift of radioactivity into more highly phosphorylated S6 derivatives, continuously increased in progesterone‐stimulated oocytes even at later times when germinal vesicle breakdown was completed. S6 protein of unstimulated oocytes was labeled to a lower degree. Trypsin cleavage of total S6 protein, isolated from control and maturing oocytes, gave rise to different complex phosphopeptide patterns reflecting the existence of various multiply phosphorylated S6 derivatives in both samples. Two of the more highly phosphorylated S6 derivatives showed considerable differences between the phosphopeptide elution profiles of control and stimulated oocytes indicating that dissimilar sites had been modified under both physiological conditions. Only phosphoserine was detected in the phosphoamino acid analysis of individual S6 derivatives.
Structural and Functional Studies on Protein S20 from the 30‐S Subunit of the Escherichia coli Ribosome
Authors: PATERAKIS Konstantinos , LITTLECHILD Jennifer , WOOLLEY Paul
Published: July 2010

Fragments resistant to proteolysis have been obtained from the ribosomal protein S20. They provide evidence for a structural domain stretching from the middle of the protein to its C terminus. With the exception of a large fragment of this protein lacking only 14 residues at the N terminus, all fragments had lost their ability to bind to 16‐S rRNA. The protein in the S20. 16‐S‐RNA complex was highly protected against enzymic digestion, indicating that the entire protein is involved in interaction with the nucleic acid. Circular dichroism showed a high α helix content (36%) for the intact protein and a low α helix content (2%) for the large fragment. Intrinsic fluorescence studies demonstrated that the single tyrosine residue in protein S20 is exposed to the solvent in the intact protein and is not exposed in the S20. 16‐S‐RNA complex. Irreversible thermal denaturation of the protein was followed by fluorescence of the tyrosine and was found between 50°C and 70°C.
Activation and Deactivation of the Glucocorticoid Hormone‐Receptor Complex
Authors: ARÁNYI Péter
Published: July 2010

Cytosol glucocorticoid receptors acquired the ability to bind to DNA‐cellulose on incubation at 25–37°C and/or in media of high ionic strength (0.3 M K Cl). However, this activation was transient only. It was followed by deactivation whose rate was also dependent on incubation temperature and on the presence of potassium chloride. Deactivation resulted in a decreased but non‐zero binding to DNA‐cellulose. Specific triamcinolone acetonide binding to the receptor protein was not lost under the same conditions.

Deactivation commenced before it became apparent, probably together with activation. Preactivated complex underwent deactivation even in conditions that would not allow significant decrease in DNA‐cellulose binding without previous incubation at 37°C.

In contrast to previous reports it was found that fast activation/deactivation took place in the presence of 4 mM Ca2+.

Molybdate ions slow down both activation and deactivation, but do not prevent activation by heat.

Heat activated/deactivated complex differed in size from both non‐activated and Ca2+‐deactivated complexes. This finding suggests that heat and Ca2+‐induced deactivation follow different mechanisms.
The Catalytic Mechanism of Tryptophan Synthase from Escherichia coli
Authors: LANE Andrew N. , KIRSCHNER Kasper
Published: July 2010

The mechanism by which indole condenses with L‐serine in the active site of tryptophan synthase was studied by the stopped‐flow technique. The single turnover occurs by rapid binding of indole to the pre‐formed enzyme—L‐serine complex, followed by C—C bond formation, reprotonation of the α carbon carbanion of L‐tryptophan, and its final release. The effects of isotopic substitution at C‐3 of indole, of pH, and of the presence of indolepropanol phosphate on these processes were also studied. The mechanism of binding of indole complements the known mechanisms of binding of L‐serine and L‐tryptophan to give a detailed picture of the mechanism of catalysis. It invokes two competent species of enzyme—L‐serine complexes, leading to a branched pathway for the central condensation process. The rates of dehydration of L‐serine and reprotonation of the carbanion of L‐tryptophan are probably limited by rearrangements at the active site. Analysis of absorption, fluorescence and circular dichroic spectra, as well as of published data on the stereoisomers obtained by reduction with borohydride, suggests that the rearrangement includes a reorientation of the pyridoxal phosphate C‐4′ atom. The mechanism provides a detailed framework for explaining all available information, including the activating effect of the α subunit on the reaction catalyzed by the β2 subunit.
The Role of the Proton‐Motive Force and Electron Flow in Light‐Driven Solute Transport in Rhodopseudomonas sphaeroides
Authors: ELFERINK Marieke G. L. , FRIEDBERG Ilan , HELLINGWERF Klaas J. , KONINGS Wil N.
Published: July 2010

The initial rate of uptake of alanine was studied in whole cells of Rhodopseudomonas sphaeroides under conditions such that the proton‐motive force generated by light‐induced cyclic electron transfer, was composed of the membrane potential only. The proton‐motive force was varied by varying the light intensity or by pretreatment of the cells with the uncoupler carbonylcyanide p‐trifluoromethoxyphenylhydrazone. At constant light intensity a threshold ΔΨ is required before alanine uptake occurs. Above this threshold value the rate of alanine uptake increases with the ΔΨ. The threshold value of the ΔΨ is higher the lower the light intensity. Under conditions of constant ΔΨ the rate of alanine uptake increases linearly with the light intensity. At low ΔΨ values a threshold light intensity is required which is higher the lower the ΔΨ. These results demonstrate that both a proton‐motive force and electron transfer are required for alanine uptake.
Rapid Induction of the Synthesis of Phenylalanine Ammonia‐Lyase and of Chalcone Synthase in Elicitor‐Treated Plant Cells
Authors: LAWTON Michael A. , DIXON Richard A. , HAHLBROCK Klaus , LAMB Chris
Published: July 2010

Changes in the rate of synthesis of phenylalanine ammonia‐lyase and chalcone synthase, two characteristic enzymes of phenylpropanoid biosynthesis, have been investigated by direct immunoprecipitation of in vivo [35S]methionine‐labelled enzyme subunits in elicitor‐treated cells of dwarf French bean (Phaseolus vulgaris). Elicitor, heat‐released from cell walls of Colletotrichum lindemuthianum, the causal agent of anthracnose disease of bean, causes marked but transient increases in the rates of synthesis of both enzymes concomitant with the phase of rapid increase in enzyme activity at the onset of phaseollin accumulation during the phytoalexin defence response.

Increased rates of synthesis of both enzymes can be observed 20 min after elicitor treatment and the pattern of induction of synthesis of phenylalanine ammonia‐lyase and chalcone synthase are broadly similar with respect to elicitor concentration and time, maximum rates of synthesis being attained between 2.5 h and 3.0 h after elicitor treatment. Within this overall co‐ordination small but distinct differences between the enzymes were observed in: (a) the elicitor concentrations giving maximum enzyme synthesis, and (b) the precise timing of maximum enzyme synthesis, with that for chalcone synthase occurring 20–30 min earlier than that for phenylalanine ammonia‐lyase. However, for a given rate of enzyme synthesis, induction of the activities of phenyl‐alanine ammonia‐lyase and chalcone synthase is more efficient at high elicitor concentrations. This may reflect the operation under certain circumstances of post‐translational control of the activity levels of these enzymes as implicated for phenylalanine ammonia‐lyase by previous density‐labelling experiments [Lawton et al. (1980) Biochim. Biophys. Acta, 633, 162–175]. The same pattern of induction of enzyme synthesis is observed with elicitor preparations from a variety of sources.
The Complete Amino‐Acid Sequence of the Low‐Spin Class II Cytochrome c‐556 from Agrobacterium tumefaciens Strain B2a
Authors: TEMPST Paul , VAN BEEUMEN Jozef
Published: July 2010

The amino acid sequence of the soluble monohaem cytochrome c‐556 from Agrobacterium tumefaciens, strain B2a, has been determined. The sequence was derived from peptides obtained by digestion of the apoprotein with trypsin and chymotrypsin, and by subdigestion of some of the peptides with Staphylococcus aureus protease and thermolysin. Sequencing of the various peptides was achieved by a combination of manual dansyl‐Edman degradation and automatic liquid‐phase sequence analysis.

The main characteristic of this cytochrome is that the haem‐binding sequence Cys‐Xaa‐Yaa‐Cys‐His occurs in the C‐terminal region of the polypeptide chain, the first cysteine being located 11 residues ahead of the C‐terminal lysine‐122. As such, the protein belongs to cytochrome c sequence class II (sensu Ambler). The cytochrome c‐556 is the first example known of a class II cytochrome of the low‐spin type isolated from an obligate aerobic organism.
The Quarternary Structure of Tryptophan Synthase from Escherichia coli
Authors: LANE Andrew N. , KIRSCHNER Kasper
Published: July 2010

Single cysteine residues on the separated α and β2 subunits of tryptophan synthase were labelled with various fluorescent ligands suitable for fluorescence energy transfer measurements. Labelled α subunits, reconstituted into an αL2β2 complex, retained more than 75% activity. Labelled β2 subunits, either free or reconstituted into an α2βL2′ complex, retained 10–30% of their activity, depending on the label. The coenzyme pyridoxal 5′‐phosphate served as a natural fluorescent label of the active site of the β2 subunit. Different combinations of labelled subunits resulted in doubly labelled αL2βL2′ complexes that allowed five independent distances to be estimated by fluorescence energy transfer: within one β protomer, CysL–pyridoxal 5′‐phosphate = 1.4–3.2 nm; between two β protomers, CysL–CysL= 0.5–2.3 nm; between one α protomer and a β protomer, CysL–pyridoxal 5′‐phosphate = 1.9–3.2 nm, CysL–CysL= 3.1–4.4 nm; between two α protomers, CysL–CysL= 3.1–3.9 nm. Translational frictional ratios obtained from measurements of sedimentation and diffusion constants and partial specific volumes provided independent information on the shapes of the different particles in solution. Both the α and the β protomer are known to consist of two globular domains of known molecular weight. These were assumed to be approximately spherical and used to construct models first of the α and β2 subunits and then of the α2β2 complex. The data on specific distances and shape factors, together with considerations of symmetry and compactness allow many possible spatial arrangements of the domains to be eliminated. The radii of gyration, maximum dimensions and hydrated volumes calculated for the best models of the α and β2 subunits and the α2β2 complex agree reasonably well with the results of independent small‐angle X‐ray scattering studies [Wilhelm et al. (1982) Eur. J. Biochem. 129, 51–56. The active sites responsible for cleavage of indoleglycerol phosphate to indole and for condensation of indole with L‐serine are approximately 2 nm apart, ruling out the composite active sites hypothesis.