bluuebunny/crossref_metadata_2025 huggingface dataset:
The evolution of lysozyme and α‐lactalbumin
Authors: NITTA Katsutoshi , SUGAI Shintaro
Published: March 2005

From the analysis of phylogenetic trees constructed from the amino acid sequences and metal‐binding properties of various lysozymes c and α‐lactalbumins, it was found that before the divergence of the lineages of birds and mammals, calcium‐binding lysozyme diverged from non‐calcium‐binding lysozyme. α‐Lactalbumin evolved from the calcium‐binding lysozyme along the mammalian lineage after the divergence of birds and mammals. Rapid evolution took place, not in the process of acquisition of the activity of α‐lactalbumin, but after the loss of lysozyme activity, due to the change in the distribution of selective pressure on each amino acid site.

A general process for the change in function of a protein during evolution is suggested to be as follows: after duplication of the gene, one of their protein products acquires a new function, besides that already present; the old function is eventually lost.
Covalent cofactor binding to flavoenzymes requires specific effectors
Authors: BRANDSCH Roderich , BICHLER Veronika
Published: March 2005

Modification by covalent FAD attachment to a histidine residue via an 8α‐(N3‐histidyl)‐riboflavin linkage occurs in several flavoenzymes. Among them is 6‐hydroxy‐D‐nicotine oxidase (6‐HDNO) of Arthrobacter oxidans and the flavoprotein subunits of the fumarate reductase and succinate dehydrogenase complex of Escherichia coli and other bacterial and eukaryotic cells. We found that 6‐HDNO holoenzyme formation from apo‐6‐HDNO, monitored by [14C]FAD incorporation and increase in enzyme activity, can be mediated not only by phosphoenolpyruvate [Nagursky, H., Bichler, V. and Brandsch, R. (1988) Eur. J. Biochem. 177, 319–325], but also by one of the glycolytic intermediates glyceraldehyde‐3‐P, glycerate‐3‐P, or the intermediate in glycerol utilization by bacteria, glycerol‐3‐P. Apoflavoprotein of fumarate reductase and succinate dehydrogenase was obtained in an E. coli riboflavin‐requiring strain (E. coli RR28rf) overexpressing the frdABCD or the sdhCDAB operon from the recombinant plasmids pGS39 and pGS141, respectively. In extracts obtained from these cells, flavoprotein flavinylation, analyzed as covalent [14C]FAD incorporation into the apoflavoprotein polypeptide by polyacrylamide gel electrophoresis and fluorography, was stimulated severalfold by the citric acid cycle intermediates citrate, isocitrate, succinate and fumarate. Our results suggest that covalent modification and thus activation of these enzymes is dependent on specific metabolic intermediates which may act as allosteric effectors in the reaction.
Purification and characterisation of bovine brain protein kinase C isotypes α, β and γ
Authors: MARAIS Richard M. , PARKER Peter J.
Published: March 2005

Several polyclonal antisera specific for each of the protein kinase C isotypes α, β1, β2 and γ have been generated and used to monitor the purification and subseqenct separation of these polypeptides. A simple protocol has been developed for the efficient co‐purification of these isotypes from bovine brain. The separation of the α, β1, β2 and γ isotypes has been monitored using the antibodies and pools containing pure α, β1, and γ forms have been produced. These isotypes have been characterised for activator dependence and substrate specificity. The results. indicate that while the isotypes have similar requirements for magnesium, calcium, ATP and phosphatidylserine, they differ in their dependence on phorbol esters and diacylglycerols. The isotypes also differ in their range of substrate specificities. The implications of these results are discussed.
The peptide antibiotic subtilin acts by formation of voltage‐dependent multi‐state pores in bacterial and artificial membranes
Authors: SCHÜLLER Friederike , BENZ Roland , SAHL Hans‐Georg
Published: March 2005

The peptide antibiotic subtilin was shown to induce a rapid efflux of amino acids from intact bacterial cells and cytoplasmic membrane vesicles, and to prevent amino acid uptake by cells preincubated with the peptide. Upon addition of subtilin the trans‐membrane potential (ΔΦ) was greatly reduced. Starved bacterial cells were less sensitive to subtilin than energized cells. Depolarization of cells by carbonyl cyanide m‐chlorophenylhydrazone prevented subtilin action, but its activity could be restored by a valinomycin‐induced potassium diffusion potential. Using this technique, we deduced a threshold potential of about −90 to −100 mV to be essential for subtilin action on intact cells. A similar value was obtained in macroscopic conductance measurements with black lipid membranes. The current‐voltage characteristic was symmetric, i.e. subtilin induced membrane currents with trans‐negative and trans‐positive voltages. Single‐channel experiments revealed short‐lived multi‐state pores of the alamethicin type. The pores had lifetimes of several hundred milliseconds and pore diameters of up to approximately 2 nm.
Analysis of the biotin‐binding site on acetyl‐CoA carboxylase from rat
Authors: BAI Dong‐Hoon , MOON Tae‐Wha , LÓPEZ‐CASILLAS Fernando , ANDREWS P. C. , KIM Ki‐Han
Published: March 2005

The biotin‐binding site of acetyl‐CoA carboxylase from rat was characterized as to its amino acid sequence and relative position in the enzyme molecule. Biotin binds to the lysyl residue in the tetrapeptide Val‐Met‐Lys‐Met; this tetrapeptide is located in close proximity to the NH2 terminus. In all other biotin‐containing enzymes, the conserved tetrapeptide Ala‐Met‐Lys‐Met is the counterpart to that of rat acetyl‐CoA carboxylase; and the lysyl residue is 35 residues from the COOH terminus. To examine the significance of these unusual features of the biotinylation site of animal acetyl‐CoA carboxylase, cDNA fragments were expressed in a bacterial system and the effects of specific site‐directed mutagenesis were examined. Replacement of Val by Ala in the conserved tetrapeptide abolished biotinylation of the expressed protein. However, introduction of a termination codon at residue 36, in such a way that the distance between the lysine on which biotin binds and the COOH‐terminal amino acid was 35 residues and the penultimate amino acid was the hydrophobic residue leucine, increased the efficiency of biotinylation, provided a substantial portion of the NH2‐terminal peptide was removed.
Structure‐function relationships of elongation factor Tu
Authors: JENSEN Michael , COOL Robbert H. , MORTENSEN Kim K. , CLARK Brian F. C. , PARMEGGIANI Andrea
Published: March 2005

The guanine‐nucleotide‐binding domain (G domain) of elongation factor Tu(EF‐Tu) consisting of 203 amino acid residues, corresponding to the N‐terminal half of the molecule, has been recently engineered by deleting part of the tufA gene and partially characterized [Parmeggiani, A., Swart, G. W. M., Mortensen, K. K., Jensen, M., Clark, B. F. C., Dente, L. and Cortese, R. (1987) Proc. Natl Acad. Sci. USA 84, 3141–3145]. In an extension of this project we describe here the purification steps leading to the isolation of highly purified G domain in preparative amounts and a number of functional properties. The G domain is a relatively stable protein, though less stable than EF‐Tu towards thermal denaturation (t50%= 41.3°C vs. 46°C, respectively). Unlike EF‐Tu, its affinity for GDP and GTP, as well as the association and dissociation rates of the relative complexes are similar, as determined under a number of different experimental conditions. Like EF‐Tu, the GTPase of the G domain is strongly enhanced by increasing concentrations of Li+, K+, Na+ or NH4+, up to the molar range. The effects of the specific cations shows similarities and diversities when compared to the effects on EF‐Tu. K+ and Na+ are the most active followed by NH4+ and Li+ whilst Cs+ is inactive. In the presence of divalent cations, optimum stimulation occurs in the range 3–5 mM, Mg2+ being more effective than Mn2+ and Ca2+. Monovalent and divalent cations are both necessary components for expressing the intrinsic GTPase activity of the G domain. The pH curve of the G domain GTPase displays an optimum at pH 7–8, similar to that of EF‐Tu.

The 70‐S ribosome is the only EF‐Tu ligand affecting the G domain in the same manner as that observed with the intact molecule, although the extent of the stimulatory effect is lower. The rate of dissociation of the G domain complexes with GTP and GDP as well as the GTPase activity are also influenced by EF‐Ts and kirromycin, but the effects evoked are small and in most cases different from those exerted on EF‐Tu. The inability of the G domain to sustain poly(Phe) synthesis is in agreement with the apparent lack of formation of a ternary complex between the G domain GTP complex and aa‐tRNA. Our results indicate that the isolated G domain of EF‐Tu is a suitable model for studying the basic properties of the guanine nucleotide interaction and the catalytic activity of a guanine‐nucleotide‐binding protein.
The complex of actin and deoxyribonuclease I as a model system to study the interactions of nucleotides, cations and cytochalasin D with monomeric actin
Authors: POLZAR Bernhard , NOWAK Ewa , GOODY Roger S. , MANNHERZ Hans Georg
Published: March 2005

The stoichiometric actin – DNase‐I complex was used to study the actin – nucleotide and actin – divalentcation interactions and its ATPase activity in the presence of MgCl2 and cytochalasin D. Treatment of actin – DNase‐I complex with 1 mM EDTA results in almost complete restoration of its otherwise inhibited DNase I activity, although the complex does not dissociate, as verified by size‐exclusion chromatography. This effect is due to a loss of actin‐bound nucleotide but is prevented by the presence of 0.1 – 0.5 mM ATP, ADP and certain ATP analogues. In this case no increase in DNase I activity occurs, even in the presence of EDTA. At high salt concentrations and in the presence of Mg2+ (‘physiological conditions’) the association rate constants for ATP, ADP and ɛATP (1,N6‐ethenoadenosine 5′‐triphosphate) and the dissociation rate constant for ɛATP were determined. Both the on and off rates were found to be reduced by a factor about 10 when compared to uncomplexed actin. Thus the binding constant of ɛATP to actin is almost unaltered after complexing to DNase I (2.16 × 108 M−1). Titrating the increase in DNase I activity of the actin – DNase I complex against nucleotide concentration in the presence of EDTA, the association constant of ATP to the cation‐free form of actin – DNase I complex was found to be 5 × 103 M−1, which is many orders of magnitude lower than in the presence of divalent metal ions. The binding constant of Ca2+ to the high‐affinity metal‐binding site of actin was found not to be altered when complexed to DNase I, although the rate of Ca2+ release decreases by a factor of 8 after actin binding to DNase I. The rate of denaturation of nucleotide‐free and metal‐ion‐free actin – DNase I complex was found to be reduced by a factor of about 15. The ATPase activity of the complex is stimulated by addition of Mg2+ and even more effectively by cytochalasin D, proving that this drug is able to interact with monomeric actin.
The effect of light on the biosynthesis of leaf‐specific thionins in barley, Hordeum vulgare
Authors: REIMANN‐PHILIPP Ulrich , BEHNKE Susanna , BATSCHAUER Alfred , SCHÄFER Eberhard , APEL Klaus
Published: March 2005

In barley seedlings grown in the dark large amounts of thionin‐specific mRNAs are present, the concentration of which rapidly declines once the seedling is exposed to light. This rapid light effect is mediated by a complex interaction of possibly two photoreceptors, phytochrome and a blue‐light‐absorbing photoreceptor. Parallel to the decline in mRNA content, the de novo synthesis of leaf‐specific thionins ceases rapidly upon illumination of etiolated seedlings. However, thionins which have accumulated before the onset of illumination remain stable within the seedling at high concentrations. In younger leaves of mature, nonstressed barley plants grown under a 16‐h‐light/8‐h‐dark cycle thionins are still present, although at much lower concentrations. In these plants, synthesis and accumulation of thionins occur predominantly in the meristematic zone at the leaf basis, which is shielded from light through the sheath of the preceding leaf. In mature light‐adapted barley plants, mRNA encoding leaf‐specific thionins may reaccumulate if these plants are exposed to pathogens or other stresses. Thus, the inhibitory effect of light on the biosynthesis of thionins may be overruled by stress‐ and pathogen‐induced signals.
Primary structure of spinach‐chloroplast thioredoxin f
Authors: KAMO Masaharu , TSUGITA Akira , WIESSNER Christoph , WEDEL Norbert , BARTLING Dieter , HERRMANN Reinhold G. , AGUILAR Fernando , GARDET‐SALVI Laura , SCHÜRMANN Peter
Published: March 2005

The primary structure of thioredoxin f from spinach chloroplasts was determined by standard amino acid sequencing and furthermore by sequencing the corresponding nuclear genome region. The protein, with a calculated molecular mass of 12564 Da and a molar absorption coefficient at 280 nm of 17700 M−1 cm−1, consists of 113 residues and exhibits 24% residue identities with spinach chloroplast thioredoxin mb or Escherichia coli thioredoxin.

A monospecific antibody elicited against thioredoxin f has been used to select recombinant phage from spinach cDNA libraries in λgt11. The inserts of positive clones were sequenced. They code for a polypeptide of 190 amino acids, composed of the thioredoxin f sequence (113 residues) and an upstream element (77 residues) which most probably consitutes the N‐terminal transit peptide that directs the polypeptide into chloroplasts. In vitro transcription and translation of this construct generates a polypeptide of approximately 21 kDa, which is imported by isolated spinach chloroplasts and processed to the mature 12.5‐kDa protein.
Characterization of crystalline formate dehydrogenase from Candida methanolica
Authors: IZUMI Yoshikazu , KANZAKI Hiroshi , MORITA Shigeru , FUTAZUKA Hideaki , YAMADA Hideaki
Published: March 2005

The crystalline formate dehydrogenase from Candida methanolica, which showed the highest specific activity (7.52 U/mg) so far reported, was characterized in detail. The enzyme is a dimer composed of identifical subunits, each containing one SH group related to the catalytic activity. The molecular mass of the enzyme is about 82–86 kDa. The Km values were found to be 3.0 mM for formate and 0.11 mM for NAD+. Even if the enzyme was incubated at pH 6.5–9.5 or at 55°C, the activity remained at 100%. Hg2+, Ni2+, NaCN, NaN3 and p‐chloromercuribenzoate strongly inhibited the enzyme activity, while the enzyme showed relatively high resistance to various chelating agents. The amino acid composition and some other physicochemical properties of the enzyme were studied. Immunological studies revealed that formate dehydrogenases of methanol‐utilizing yeasts immunologically more or less resemble each other, but differ from those of methanol‐utilizing bacteria. Furthermore, yeast formate dehydrogenases can be immunologically classified into three types: (a) the Candida type, (b) the Torulopsis/Hansenula/Pichia type and (c) the formaldehyde‐resistant yeast type. For simple and large‐scale preparation of the enzyme for practical use, treatment of cells of C. methanolica with the commercial cationic detergent, ‘Benzalkonium’ cation, is useful: the total and specific activities of the enzyme are 1.17‐fold and 3.10‐fold higher than those of the crude cell‐free extract, respectively.
Antibodies directed against phosphothreonine residues as potent tools for studying protein phosphorylation
Authors: HEFFETZ Daphna , FRIDKIN Mati , ZICK Yehiel
Published: March 2005

Here we report the development of novel antibodies which specifically react with phosphothreonine residues [anti‐(P‐Thr)antibodies]. The specificity of the antibodies was assessed in radioimmunoassays where we could demonstrate that half‐maximal and maximal binding of the antibodies to plates coated with BSA –P‐Thr occurred at serum dilutions of 1:4000 and 1:1000, respectively. P‐Thr inhibited antibody binding with a half‐maximal effect at 40 μM. P‐Ser was 200‐fold less potent while P‐Tyr was essentially ineffective. Anti‐(P‐Thr) antibodies could specifically bind to phosphothreonine‐containing proteins on Western blots. Using such a procedure we could demonstrate enhanced threonine phosphorylation of the EGF receptor upon treatment of intact unlabeled A431 cells with EGF. We could further demonstrate antibodies binding to proteins present in extracts of rat hepatoma cells (Fao). P‐Thr at 10 μM completely inhibited antibody binding while P‐Ser, P‐Tyr, Thr or Ser, each present at tenfold higher concentrations, had no such inhibitory effect. Anti‐(P‐Thr) antibodies were also capable of specifically immunoprecipitating 32P‐labeled phosphoproteins present in Triton extracts of Fao cells. Immunoprecipitation of proteins of 38 kDa, 55 kDa, 85 kDa, 100 kDa and 155 kDa was inhibited by 1 mM P‐Thr but not by P‐Tyr. These findings suggest that anti‐(P‐Thr) antibodies could be powerful tools in studies aimed at monitoring alterations in threonine phosphorylation of specific proteins as they occur under physiological conditions in response to various extracellular stimuli. Identification of such proteins can be conveniently monitored by immunoblotting.
Spectroscopic characterization of the nickel and iron‐sulphur clusters of hydrogenase from the purple photosynthetic bacterium Thiocapasa roseopersicina
Authors: CAMMACK Richard , KOVACS Kornel L. , McCRACKEN John , PEISACH Jack
Published: March 2005

Pulsed electron‐spin‐resonance techniques were applied to the hydrogenase of the purple photosynthetic bacterium Thiocapsa roseopersicina, an enzyme which contains nickel and iron‐sulphur clusters but no flavin. The linear electric field effect profile of the spectrum in the region of g= 2.01 indicated that the strong ESR signal in the oxidized protein is due to a [3Fe–4S] cluster. The electron spin‐echo envelope of this spectrum was modulated by hyperfine interactions with 1H and 14N nuclei, probably from the polypeptide chain. The ESR spectrum of this species shows a complex pattern arising from spin‐spin interaction with another paramagnet. When the protein was partially reduced by ascorbate plus phenazine methosulphate, the complexity of the spectrum was abolished but the form of the electron spin‐echo envelope modulation (ESEEM) pattern was unchanged. This indicates that the reversible disappearance of the spin‐spin interaction pattern on partial reduction is not due to cluster interconversion to a [4Fe–4S] cluster. In the ESR spectrum of nickel(III), weak hyperfine interactions with 1H and 14N were also observed by ESEEM. The nature of the interacting nuclei is discussed.
On the regulatory significance of inhibitors acting on non‐equilibrium enzymes in the Calvin photosynthesis cycle
Authors: PETTERSSON Gösta , RYDE‐PETTERSSON Ulf
Published: March 2005

Control analyses and kinetic model studies have been performed in order to obtain quantitative information on the regulatory significance of 12 experimentally well‐documented inhibitory interactions of Calvin cycle intermediates with the four non‐equilibrium cycle enzymes. Evidence is presented to show that none of these interactions contributes significantly to the cycle flux control over the range of external orthophosphate concentrations where the reaction cycle shows close to optimal activity. Contrary to what has been generally supposed, the examined inhibitions appear to be of little interest for our understanding of the biological regulation of the Calvin photosynthesis cycle under conditions of light and carbon dioxide saturation.
Triacylglycerol synthesis in cultured rat hepatocytes
Authors: MAYOREK Nina , GRINSTEIN Irene , BAR‐TANA Jacob
Published: March 2005

The limiting role of diacylglycerol acyltransferase with respect to triacylglycerol synthesis in cultured rat hepatocytes was evaluated by following the inhibition of the overall synthetic flux by 2‐bromooctanoate acting as an inhibitor of the diacylglycerol acyltransferase step. The flux‐control coefficient of diacylglycerol acyltransferase in intact cultured hepatocytes amounted to 0.76 in the presence of saturating glycerol and either palmitate or oleate as the fatty acyl substrates. The flux‐control coefficient of diacylglycerol acyltransferase in lysolecithin‐permeabilized cultured hepatocytes amounted to 0.80 and 0.99 in the presence of saturating glycerol 3‐phosphate and either palmitate or oleate as the fatty acyl substrate, respectively. Hence, triacylglycerol synthesis in liver cells under the experimental conditions employed is rate‐limited by the diacylglycerol acyltransferase.
Insect immunity. Isolation of cDNA clones corresponding to diptericin, an inducible antibacterial peptide from Phormia terranovae (Diptera)
Authors: REICHHART Jean‐Marc , ESSRICH Michael , DIMARCQ Jean‐Luc , HOFFMANN Danièle , HOFFMANN Jules A. , LAGUEUX Marie
Published: March 2005

We have previously isolated and characterized a family of novel 8‐kDa cationic antibacterial peptides synthesized by larvae of Phormia terranovae (Diptera) in response to various injuries. These molecules have been named diptericins. The peptide sequence of diptericin A was used to prepare oligonucleotides for screening cDNA libraries and we report in the present paper the isolation of several cDNA clones encoding diptericin. The analysis of the nucleotide sequences indicates that diptericin is synthesized as a prepeptide which is matured in two steps: (a)cleavage of a signal peptide and (b) amidation of the C‐terminal residue. Interestingly, the 3′ untranslated region of the mRNA contains a consensus sequence TTATTTAT which is also observed in the mRNA of another insect antibacterial peptide (attacin‐related sarcotoxin IIA) and in mRNAs encoding proteins related to the inflammatory response in mammals. Our data illustrate that diptericins form a polymorphic family of immune peptides. The transcription of the diptericin genes is rapidly induced in the fat body after inoculation of bacteria as evidenced by the transcriptional profile.
Photofootprinting of drug‐binding sites‐on DNA using diazo‐ and azido‐9‐aminoacridine derivatives
Authors: JEPPESEN Claus , NIELSEN Peter E.
Published: March 2005

It is demonstrated that DNA photofootprinting analysis of the intercalating depsipeptide echinomycin, and the minor groove‐binders distamicyn, 4′,6‐diamidino‐2‐phenylindole (DAPI) and Hoechst 33258 can be performed using 9‐[6‐(2‐diazocyclopentadienylcarbonyloxy)hexylamino]acridine (DHA) [Nielsen et al. (1988) Nucleic Acids Res. 16, 3877–3888] or 2‐methoxy‐6‐azido‐9‐aminoacridine (MAA) [Jeppesen et al. (1988) Nucleic Acids Res. 16, 5755–5770]. Both the extent of the drug‐binding sites and their relative strength can be determined with either reagent. DNA has the advantage of giving virtually sequence‐uniform DNA photocleavage. On the other hand, structural changes in the DNA are detected by MAA. Using the 232‐base‐pair EcoRI–PvuII pUC19 restriction fragment, it is found that cleavage protection by distamycin, DAPI and Hoechst 33258 all require an (A · T)4 sequence, whereas protection by echinomycin was confined to a G+C‐rich 8‐base‐pair region.
The expression and purification of human rhinovirus protease 3C
Authors: KNOTT Jacqueline A. , ORR David C. , MONTGOMERY Douglas S. , SULLIVAN Carole A. , WESTON Anthony
Published: March 2005

Human rhinovirus type 14 protease 3C was expressed as a soluble and active protein in Escherichia coli. The protease was purified by a cationic‐exchange step followed by gel filtration on a TSK 3000 column. The final yield of purified protease was in the range 0.5–1.0 mg/l culture grown to A550= 1.0. Sequence analysis revealed that greater than 90% of the N‐terminal residues were methionine. The enzyme activity of the purified protease was measured by cleavage of a synthetic peptide representing a predicted Gln/Gly viral polyprotein cleavage site. A mutant protease (Cys146→Ser) was produced and purified in the same way. The yield of mutant protease 3C was approximately 150 μg/l from a culture grown to A550= 1.0. This mutant protease 3C did not cleave the synthetic peptide substrate.
Reaction of fluorescein isothiocyanate with an ATP‐binding site on the phosphorylase kinase α subunit
Authors: ZAMAN Nadia , VARSÁNYI Magdolna , HEILMEYERM Ludwig M. G. , SOTIROUDIS Theodore G. , JOHNSON Charles M. , CRABB John W.
Published: March 2005

Phosphorylase kinase can be labeled specifically on the α subunit with fluorescein 5′‐isothiocyanate (FITC) which concomitantly incactivates the enzyme (T. G. Sotiroudis and S. Nikolaropoulus (1984) FEBS Lett. 176, 421–425] Labeled peptides have been purified and their primary structure has been determined. The amino acid sequence of the fluorescein‐labeled tryptic peptide is Lys‐Met‐Gln‐Asp‐Gly‐Tyr‐Phe‐Gly‐Gly‐Ala‐Arg. The environment of this fluorescein‐labeled lysine has been detemined by sequencing peptides isolated from a Staphylococcus aureus V8 digest and two further cyanogen bromide fragments of the purifed [14C]carboxy‐methylated α subunit. The partial sequences obtained have then been localized in the primary structure of the α subunit [Zander et al. (1988) Proc. Natl Acad. Sci. USA 85, 2929–2933]. Both the incorporation of the fluorescent label and enzymatic inactivation are inhibited by ATP only at pH 7.0; ADP and AMP do not protect. Kinetic analysis reveals a competition between ATP and FITC; a Ki for ATP of 728±100 μM has been determined.
The coupling between protonmotive force and the NAD(P)+transhydrogenase in chromatophores from photosynthetic bacteria
Authors: COTTON Nicholas P. J. , LEVER Timothy M. , NORE Beston F. , JONES Michael R. , JACKSON J. Baz
Published: March 2005

The activity of NAD(P)+transhydrogenase in chromatophores ofRhodobacter capsulatusrelaxed from a high rate during illumination to a lower rate after darkening with a half‐time of approximately 100 ms.

The dissipative ionic current flowing across the chromatophore membrane was increased in the presence of transhydrogenase substrates. This is attributed to proton current through the transhydrogenase enzyme. Subject to the assumption that transhydrogenase does not conduct in the absence of nucleotide substrates, the ratio of protons translocated across the membrane per hydride ion transferred was 0.4±0.5. Within the error and uncertainities in the calibration procedure, this ratio may be consistent with a stoichiometry of one but higher values seem unlikely. The ratio of hydride ion transferred in the transhydrogenase to electrons transferred through the cyclic electron transport system was approximately 0.2.

TheKmappvalues for the transhydrogenase substrates were determined for chromatophores in illuminated and darkened suspensions over a range of pH. These values are discussed in relation to the equivalent parameters reported for mitochondria transhydrogenase [Rydstrom. J. (1977)Biochim. Biophys. Acta 255, 9641–9646] and were used to calculate the concentrations of substrates which effectively saturate the enzyme.

At substrate concentrations which were in excess of 8 xKmappthe dependence of transhydrogenase rate on the value of the membrane potential (zero pH gradient) was determined at pH 6.3, 6.9, 7.6 and 9.0. The relation was similar at pH 6.9 and 7.6. At alkaline pH the apparent threshold in the relation became more prominent as it was shifted to slightly higher values of membrane potential. At acid pH a shift in the opposite direction diminished the apparent threshold and saturation at high membrane potential became more dominant. We use these data in an attempt to discriminate between two models of energy transduction: (a) the driving force exerted by the membrane potential is mediated by a pH gradient formed through the operation of a proton well in the transhydrogenase; (b) the membrane potential increases a rate constant for charge translocation through transhydrogenase by decreasing the effective height of the Eyring barrier for charge transfer across the membrane through the enzyme. The second model leads to a more simple description than the first of the pH dependence of transhydrogenase rate on membrane potential.

Divalent cations at very low concentrations (∼ 0.1 mM) or monovalent cations at higher concentrations (∼ 50 mM) stimulate considerably the transhydrogenase activity in chromatophores. The effect of monovalent and divalent cations on the relationship between the rate of transhydrogenase and membrane potential suggests that they do not influence the reaction by altering the membrane‐potential‐dependent rate constant.
Characterization of trehalose‐6‐phosphate synthase and trehalose‐6‐phosphate phosphatase of Saccharomyces cerevisiae
Authors: VANDERCAMMEN Annick , FRANÇOIS Jean , HERS Henri‐Géry
Published: March 2005

The properties of yeast trehalose‐6‐phosphate synthase were reinvestigated in relation with the recent claim made by Panek et al. [Panek, A. C., de Araujo, P. S., Moura‐Neto, V. and Panek, A. D. (1987) Curr. Genet. 11, 459–465] that the enzyme would be stimulated by ATP and partially inactivated by cAMP‐dependent protein kinase. Trehalose‐6‐phosphate synthase activity was measured by the sum of [14C]trehalose 6‐phosphate and [14C]trehalose formed from UDP‐[14C]glucose and glucose 6‐phosphate. The activity measured in an extract of Saccharomyces cerevisiae was not affected by any treatment of the cells, such as incubation in the presence of glucose or of dinitrophenol, which are known to greatly increase the intracellular concentration of cAMP, nor by preincubation of the extract in the presence of ATP‐Mg, cAMP and bovine heart cAMP‐dependent protein kinase. The activity was also not significantly different in several mutants affected in the cAMP system. The kinetic properties of the partially purified enzyme were investigated; no effect of ATP could be detected but Pi acted as a potent noncompetitive inhibitor (Ki= 2 mM). The activity of trehalose‐6‐phosphate phosphatae was measured by the amount of [14C]trehalose formed from [14C]trehalose 6‐phosphate. The enzyme could be separated from other phosphatases and appeared to be highly specific for trehalose 6‐phosphate. It was Mg dependent and its kinetics for trehalose 6‐phosphate was hyperbolic. Studies performed with intact cells, crude extracts or the purified enzyme did not reveal any cAMP‐dependent change in its activity.

Remarkably, trehalose‐6‐phosphate synthase and trehalose‐6‐phosphate phosphatase copurified in the course of different chromatographic procedures, suggesting that they are part of a single bifunctional protein. A 50‐fold purification of the two enzymes could be achieved with a yeild of only 2% by chromatography on Mono S followed by gel filtration on Superose 6B.
Chains and fragments of tetanus toxin
Authors: WELLER Ulrich , DAUZENROTH Marie‐Elisabeth , HERINGDORF Dagmar MEYER ZU , HABERMANN Ernst
Published: March 2005

Tetanus toxin, as obtained from bacterial culture filtrates, consists of two chains. Since their roles in poisoning are unknown, we have made a detailed study of their preparation, reassociation and pharmacological activity.

Two‐chain tetanus toxin (pI 6.0) was subjected to isoelectric focussing under reducing conditions in 2 M urea. Both light (pI 4.8) and heavy (pI 7.2) chains separated as nearly homogeneous proteins of low toxicities. Upon removal of urea and reoxidation, partial homodimerization by formation of disulfide bonds took place in the purified fractions. The toxin was reconstituted nearly quantitatively by covalent heterodimerization of the complementary chains, as shown by SDS/gel electrophoresis, toxicity studies, inhibition of evoked [3H]‐noradrenaline release and binding to rat brain membranes.

Accordingly, fragment B (pI 5.6) resulting from papain hydrolysis, was separated into a light chain and the N‐terminal moiety of the heavy chain, called fragment β2 (pI 7.1 and 6.8, two maxima). Removal of urea and reoxidation led to reconstitution of fragment B. Covalent linkage did not occur between the two parts of the heavy chain, or between the light chain and the C‐terminal part of the heavy chain.

The heavy chain alone inhibited K+‐evoked [3H]noradrenaline release from a rat brain homogenate. However, the concentration‐response ratio was flat and 10–100‐fold higher concentrations were required than with native or reconstituted two‐chain toxin. The light chain was inactive. Purified heavy chain but not light chain decreased the [3H]noradrenaline content, whereas the two‐chain toxin increased it.

Binding to rat brain membranes was assessed by competition with 125I‐labelled two‐chain toxin. In hypotonic buffer, the heavy chain, the papain fragment C and native and reconstituted two‐chain toxin had comparable affinities to membranes. In isotonic buffer the heavy chain displayed an about 1000‐fold lower affinity than native or reconstituted two‐chain toxin. The light chain did not bind to membranes in either test.

Our data indicate that (a) the light chain and the N‐terminal part of the heavy chain are held together not only by one disulfide bond but also by hydrogen bonds and ionic forces to yield a two‐chain toxin or fragment B and (b) both chains contribute to the actions of the toxin in vivo and in vitro, and to its binding.
A novel inositol‐phospholipid‐specific phospholipase C
Authors: MELDRUM Eric , KATAN Matilda , PARKER Peter
Published: March 2005

A novel bovine brain inositol‐phospholipid‐specific phospholipase C has been identified on the basis of chromatographic behaviour and purified to apparent homogeneity by a rapid three‐step procedure. The purified enzyme has a molecular mass of 85 kDa on SDS/polyacrylamide gel electrophoresis and a specific activity of 24 μmol · min–1· mg–1. The enzyme is dependent on Ca2+ and shows a marked preference for inositol phospholipid substrates. The unique nature of this polypeptide was confirmed through partial protein sequence analysis.
Stability and activity of a thermostable malic enzyme in denaturants and water‐miscible organic solvents
Authors: GUAGLIARDI Annamaria , MANCO Giuseppe , ROSSI Mossé , BARTOLUCCI Simonetta
Published: March 2005

A study was made of the effects of common protein denaturants and water‐miscible organic solvents on both the stability and activity of the malic enzyme [(S)‐malate: NADP+ oxidoreductase (oxaloacetate‐decarboxylating); EC 1.1.1.40] from the extreme thermoacidophilic archaebacterium Sulfolobus solfataricus. At 25°C, the enzyme was not inactivated in 4 M urea or 0.05% SDS over 24 h, while the half‐life was 30 min in 6 M guanidine hydrochloride and 5 h in 0.075% SDS. The enzyme stability in water‐miscible organic solvents at 25°C is somewhat surprising: after a 24‐h incubation, the enzyme was completely active in 50% dimethylformamide; it lost 15% of its initial activity in 50% methanol or 15% ethanol. However, the resistance to organic solvents was greatly reduced at higher temperatures. The enzyme was able to catalyze the malate conversion even in the presence of 1.5% Triton X‐100 or sodium deoxycholate. A number of solvents were found to stimulate the malic activity independent of time. Studies with 50% methanol revealed that the activation was reversible and inversely related to the temperature; moreover, the solvent was demonstrated to exclusively affect the maximal velocity of catalysis, the Km values for both substrates being unchanged. Investigation was made to find out whether there was a correlation between enzyme stability, as well as activation, and hydrophobicity of the organic medium. The residual malic activity after incubation in the water/organic medium correlated inversly with the logarithm of the partition coefficient in octanol/H2O of the mixture used as a hydrophobicity index. On the other hand, the extent of activation depended directly on the logarithm of the molar concentration of the organic solvent required for maximal enzymatic activation. Because of its remarkable resistance to organic solvents and protein denaturants in general, the malic enzyme from Sulfolobus solfataricus can be considered suitable for biotechnological applications.
New dimeric inhibitor of heterologous α‐amylases encoded by a duplicated gene in the short arm of chromosome 3B of wheat (Triticum aestivum L.)
Authors: SANCHEZ‐MONGE Rosa , GOMEZ Luis , GARCÍA‐OLMEDO Francisco , SALCEDO Gabriel
Published: March 2005

A new wheat dimeric α‐amylase inhibitor, designated WDAI‐3, has been characterized. WDAI‐3 is a homodimeric protein active against α‐amylase from human saliva and from the insect Tenebrio molitor, but inactive against that from pig pancreas or against trypsin. Its N‐terminal amino acid sequence is closer to those of the wheat dimeric inhibitors 0.19 and 0.53 (89–91% identical positions in 44 residues) than to that of the monomeric 0.28 inhibitor (69% identical positions). Iha‐BI‐2, the gene encoding the new inhibitor, is located in the short arm of chromosome 3B, where it is part of an intrachromosomal gene duplication that also codes for the 0.53 inhibitor.
Site‐directed mutagenesis of yeast phosphoglycerate kinase
Authors: WALKER Philip A. , LITTLECHILD Jennifer A. , HALL Len , WATSON Herman C.
Published: March 2005

There is evidence, some of it of questionable authenticity, which suggests that phosphoglycerate kinase takes up a more compact form following the binding of substrates. Using this evidence it has been assumed that a conformational rearrangement is required for phosphoryl transfer to occur and that this is brought about by moving the enzyme's two domains towards each other. In order to test this hypothesis we have modified, by sitedirected mutagenesis, an arginine residue thought to be involved in stabilising the transition‐state intermediate. Although some 1.3 nm away from the site of phosphoryl transfer, as seen in the crystallographically determined structure, the substitution of arginine 168 by lysine (R168K) more than halves the specific activity of the enzyme. Substituting the arginine with a methionine (R168M) reduces activity further, but not completely, thus proving that the charge associated with this residue is not essential for catalytic activity. Both mutations raise the Michaelis constants (Km) for ATP and glycerate 3‐phosphate. The largest change is observed with the triose substrate and the methionine mutant, suggesting that the primary function of arginine 168 is to influence the environment of this substrate. The effect on activity of adding sulphate to R168K and R168M mutant enzyme has also been investigated. The sulphate activation effect at low substrate concentrations is reduced for the methionine substitutuion but almost abolished for the lysine substitution. The most reasonable explanation of all these findings in that, in the wild‐type enzyme, the guanidinium group of arginine 168 forms a hydrogen bond with one of the triose substrate's C1 oxygens. This steric arrangement would not be possible in the ‘open form’ of this enzyme as observed in the crystal structure.
Molecular cloning of the nuclear gene for mitochondrial ribosomal protein YmL31 from Saccharomyces cerevisiae
Authors: GROHMANN Lutz , GRAACK Hanns‐Rüdiger , KITAKAWA Madoka
Published: March 2005

The nuclear gene for mitochondrial ribosomal protein YmL31 (MRP‐L31) of Saccharomyces cerevisiae was cloned using synthetic oligonucleotide mixtures which correspond to the N‐terminal amino acid sequence of the mature YmL31. The gene MRP‐L31 codes for a basic protein with a calculated molecular mass of 15.5 kDa and resides on chromosome XI. A comparison of the amino acid sequence deduced from the nucleotide sequence of the MRP‐L31 gene and the N‐terminal sequence of the isolated protein revealed the existence of a leader peptide sequence of 12 amino acid residues. No significant similarity to known ribosomal protein sequences of other organisms was found.
Structural and functional studies on rabbit liver glycogenin
Authors: SMYTHE Carl , VILLAR‐PALASI Carlos , COHEN Philip
Published: March 2005

Glycogenin, the protein primer required for the biogenesis of muscle glycogen, has been isolated from rabbit liver glycogen. The protein comprised 0.0025% of liver glycogen by mass, 200‐fold lower than the glycogenin content of muscle glycogen. Structural analyses, including determination of the amino acid sequence surrounding the glucosylated‐tyrosine residue, showed identity with muscle glycogenin. Catalytically active liver glycogenin was partially purified and, like the skeletal muscle protein, catalysed an intramolecular, Mn2+ ‐ and UDP‐Glcdependent autoglucosylation reaction, forming a primer on which glycogen synthase could act. The results demonstrate that hepatic and muscle glycogenins are almost certainly identical proteins and that liver and skeletal muscle share a common mechanism for the biogenesis of glycogen molecules. The results also indicate that there is about one glycogenin molecule/liver glycogen α particle.
Differences in hydrophobic properties for human α2‐macroglobulin and pregnancy zone protein as studied by affinity phase partitioning
Authors: BIRKENMEIER Gerd , CARLSSON‐BOSTEDT Lena , SHANBHAG Vasant , KRIEGEL Thomas , KOPPERSCHLÄGER Gerhard , SOTTRUP‐JENSEN Lars , STIGBRAND Torgny
Published: March 2005

Human α2‐macroglobulin and pregnancy zone protein are related with regard to primary structure, physicochemical properties, and quarternary structure. Both proteins undergo conformational changes when they form complexes with proteinases or react with primary amines.

The surface properties of the native, chymotrypsin‐treated and methylamine‐treated forms of α2‐macroglobulin and pregnancy zone protein were studied by partitioning in aqueous two‐phase systems composed of 7.5% dextran T70 and 5% poly(ethylene glycol) 8000. All proteins and their derivatives had a high potential for hydrophobic interaction as analyzed in terms of affinity for poly(ethylene glycol) esters of fatty acids included in the phase systems. Treatment of α2‐macroglobulin with methylamine or chymotrypsin increased the surface hydrophobicity significantly compared to that of the native protein. No difference in hydrophobic interaction was found for native and methylamine‐treated pregnancy zone protein, but the chymotrypsin‐treated protein showed a marked increase in binding to the hydrophobic ligand. The changes in surface hydrophobicity parallel changes in receptor binding properties of the derivatized forms of α2‐macroglobulin and could be a signal for binding to cell‐surface receptors, followed by internalization.
Structural changes in oxidised low‐density lipoproteins and of the effect of the anti‐atherosclerotic drug probucol observed by synchrotron X‐ray and neutron solution scattering
Authors: BELLAMY Michael F. , NEALIS Adam S. , AITKEN John W. , BRUCKDORFER K. Richard , PERKINS Stephen J.
Published: March 2005

The atherosclerotic properties of low‐density lipoproteins (LDL) are thought to be strongly enhanced by oxidation. The lipid‐lowering drug probucol reduces the susceptibility of LDL to oxidation. Synchrotron X‐ray and high‐flux neutron solution scattering curves were used to characterise the structural properties of human LDL, before and after modification by oxidation with Cu2+ and the addition of probucol, in order to evaluate these techniques. Analyses based on Guinier plots, simple two‐shell spherical modelling, and the use of cubic splines and indirect transformation show that a 20‐h incubation with Cu2+ ions (but not 6 h) causes some of the LDL to associate to form larger aggregated particles. Gel electrophoresis on Cu2+‐oxidised LDL shows a concomitant degradation of the apolipoprotein B‐100 as well as the formation of high molecular mass forms. These experiments indicate that the apoprotein B‐100 structure has been significantly disrupted by oxidation. The addition of probucol to LDL causes an increase in the polydispersity of LDL, as evidenced by small changes in the Guinier curves and some weakening of the minima in the X‐ray scattering curves. No changes in the quasispherical shape of LDL are observed and gel electrophoresis indicates no changes. It is possible that probucol may exert its effect by increasing the range of sizes of LDL and that the lipid‐lowering effect of probucol in vivo might be caused by the preferential catabolism of the higher molecular mass forms of LDL thus created.
Cholesterol efflux from and high‐density‐lipoproteins binding to cultured bovine vascular endothelial cells are higher than with vascular smooth muscle cells
Authors: SAVION Naphtali , KOTEV‐EMETH Shlomo
Published: March 2005

Cholesterol metabolism was studied and compared in confluent cultures of adult bovine aortic endothelial and bovine aortic smooth muscle cells which were grown under similar conditions. The total cholesterol content/mg protein was only slightly higher in smooth muscle cells than in endothelial cells and upon exposure to [3H]cholesterol the maximal specific activity/mg protein obtained was similar in both cell types. Most (98%) of the incorporated [3H]cholesterol remained in the form of free cholesterol in both cell types, and provided a system for the study of cholesterol efflux. The role of high‐density lipoproteins (HDL) and human serum in cholesterol influx and efflux, in both endothelial and smooth muscle cells, was studied. Net cholesterol transport in the cultures was calculated and net efflux was observed in both cell types. This was higher in endothelial than in smooth muscle cells and HDL was more efficient than human serum in promoting net cholesterol efflux.

During the influx experiments, no conversion of [3H]cholesterol to [3H]cholesteryl ester was observed either in the cell layer or in the incubation medium. On the other hand, during efflux experiments when HDL but not human serum was the acceptor, some (about 6%) conversion of [3H]cholesterol to [3H]cholesteryl ester occurred in the incubation medium.

125I‐HDL3 binding to endothelial and smooth muscle cells was studied and demonstrated saturation at a concentration of about 100 μg protein ml for both cell types. However, endothelial cells bound about six times more 125I‐HDL3 than smooth muscle cells.

These studies indicate that vascular endothelial cells are more protected against cholesterol accumulation than vascular smooth muscle cells. The greater ability of endothelial cells to bind HDL complexes when compared with smooth muscle cells, and thereby to be more susceptible to HDL induced cholesterol efflux, may add a new mechanism through which endothelial cells protect the blood vessel from cholesterol accumulation.
Phosphorylation in vitro of vimentin by protein kinases A and C is restricted to the head domain
Authors: GEISLER Nobert , HATZFELD Mechthild , WEBER Klaus
Published: March 2005

The in vitro phosphorylation of vimentin, the intermediate filament protein of mesenchymal cells, by kinases A and C is serine‐specific and involves only the N‐terminal head domain. In oligomeric protofilament units each kinase recognizes five sites, which have been identified by sequence analysis. Kinase C introduces 1.5 mol phosphate/mol vimentin, while kinase A treatment results in 4 mol phosphate/mol. Kinase‐A‐treated oligomers do not polymerize in standard assays whereas kinase C treatment has no inhibitory effect. Filaments exposed to kinase A remain stable and incorporate only 1.7 mol phosphate/mol vimentin. These phosphates are essentially restricted to two of the five kinase A sites found in protofilament units. Thus the head domain, previously related to in vitro assembly competence and filament stability, changes in accessibility between the oligomeric and polymeric state. We discuss the possibility that in vivo phosphorylation of vimentin filaments by kinase A may not necessarily be accompanied by an extensive depolymerization. It could instead involve a dynamic change of the filament surfaces, which could alter the interaction of the filaments with other cellular structures.