bluuebunny/crossref_metadata_2025 huggingface dataset:
Chromium(III)ATP inactivating (Na++ K+)‐ATPase supports Na+‐Na+ and Rb+‐Rb+ exchanges in everted red blood cells but not Na+,K+ transport
Authors: PAULS Hartmut , SERPERSU Engin Halit , KIRCH Ulrike , SCHONER Wilhelm
Published: March 2005

The chromium(III) complex of ATP, an MgATP complex analogue, inactivates (Na++ K+)‐ATPase by forming a stable chromo‐phosphointermediate. The rate constant k2 of inactivation at 37°C of the β,γ‐bidentate of CrATP is enhanced by Na+ (K0.5= 1.08 mM), imidazole (K0.5= 15 mM) and Mg2+ (K0.5= 0.7 mM). These cations did not affect the dissociation constant of the enzyme‐chromium‐ATP complex.

The inactive chromophosphoenzyme is reactivated slowly by high concentrations of Na+ at 37°C. The half‐maximal effect on the reactivation was reached at 40 mM NaCl, when the maximally observable reactivation was studied. However, 126 mM NaCl was necessary to see the half‐maximal effect on the apparent reactivation velocity constant. K+ ions hindered the reactivation with a Ki of 70 μM.

Formation of the chromophosphoenzyme led to a reduction of the Rb+ binding sites and of the capacity to occlude Rb+.

The β,γ‐bidentate of chromium(III)ATP (Kd= 8 μM) had a higher affinity than the α,β,γ‐tridentate of chromium(III)ATP (Kd= 44 μM) or the cobalt tetramine complex of ATP (Kd= 500 μM). The β,γ‐bidentate of the chromium(III) complex of adenosine 5′‐[β, γ‐methylene]triphosphate also inactivated (Na++ K+)ATPase.

Although CrATP could not support Na+, K+ exchange in everted vesicles prepared from human red blood cells, it supported the Na+‐Na+ and Rb+‐Rb+ exchange.

It is concluded that CrATP opens up Na+ and K+ channels by forming a relatively stable modified enzyme‐CrATP complex. This stable complex is also formed in the presence of the chromium complex of adenosine 5′‐[β,γ‐methylene]triphosphate. Because the β, γ‐bidentate of chromium ATP is recognized better than the α, β, γ‐tridentate, it is concluded that the triphosphate site recognizes MgATP with a straight polyphosphate chain and that the Mg2+ resides between the β‐ and the γ‐phosphorus. The enhancement of inactivation by Mg2+ and Na+ may be caused by conformational changes at the triphosphate site.
Mechanism of renal peritubular extraction of plasma glutathione
Authors: INOUE Masayasu , SHINOZUKA Shigeru , MORINO Yoshimasa
Published: March 2005

To clarify the peritubular mechanism for renal handling of plasma glutathione (GSH), variation of GSH levels in plasma, urine, kidney and liver was examined after intravenous administration of GSH to three groups of animals; control, acivicin‐treated and rats treated with buthionine sulfoximine (BSO). Treatment of animals with BSO, a potent inhibitor of de novo GSH synthesis, markedly reduced hepatorenal GSH levels. Acivicin did not affect these levels. Upon intravenous injection of GSH (0.1 mmol/kg), renal GSH levels did not appreciably change in any of three animal groups. The rate of GSH disappearance from the circulation was rapid in control and BSO‐treated rats, while it was markedly retarded in animals whose renal γ‐glutamyltransferase was extensively inactivated by acivicin. At 30 min after administration a significant amount of injected GSH was localized extracellularly (urine and plasma) in acivicin‐treated animals. By contrast, most of the GSH rapidly disappeared from the extracellular space in control and BSO‐treated animals. Together with the immunocytochemical evidence for the peritubular γ‐glutamyltransferase[Spater, H. W., Poruchynsky, M. S., Quintana, N., Inoue, M. & Novikoff, A. B. (1982) Proc. Natl Acad. Sci. USA 79, 3547–3550] the present results are fully consistent with the contention that the catalytic function of this enzyme is principally responsible for the peritubular mechanism for the renal handling of plasma GSH.
The relationship between light‐induced increases in the H+conductivity of thylakoid membranes and activity of the coupling factor
Authors: HANGARTER Roger , ORT Doland R.
Published: March 2005

In the absence of a transmembrane electric field, about 15 saturating single‐turnover flashes are required for chloroplast thylakoid membranes to accumulate the 80 mmol H+· mol chlorophyll−1that are necessary to form a ΔpH sufficiently large to initiate net ATP synthesis. Lowering the number of turnovers of proton‐producing redox components by decreasing the flash intensity increased the number of flashes required for the onset of ATP formation. Thus, regardless of the intensity, the accumulation of the same number of hydrogen ions was needed for phosphorylation to begin. Since the size of the threshold input was constant over a very wide range of proton accumulation rates, it follows that there were no significant proton leakages during the filling of the pool to its threshold level. However, non‐productive leaks were initiated once phosphorylation began since progressively lower phosphorylation efficiencies were observed at lower and lower flash intensities. It is difficult to explain this observation except in terms of competing, non‐phosphorylating hydrogen ion fluxes only when the threshold accumulation had been reached. We observed an increase in the permeability of thylakoid membrane to hydrogen ions that correlated with indications of coupling factor activity: the onset of ATP synthesis, the release of tightly bound ADP and, in dithiothreitol treated membranes, the initiation of ATPase activity. Our data support the notion that the dependence of coupling factor activation and deactivation on the ΔpH accounts for the substantial changes in the ion conductivity that occur in thylakoid membranes.
Control of ketogenesis in the perfused rat liver by the sympathetic innervation
Authors: BEUERS Ulrich , BECKH Karlheinz , JUNGERMANN Kurt
Published: March 2005

The regulation of ketogenesis by the hepatic nerves was investigated in the rat liver perfused in situ.

Electrical stimulation of the hepatic nerves around the portal vein and the hepatic artery caused a reduction of basal ketogenesis owing to a decrease in acetoacetate release to 30% with essentially no change in 3‐hydroxybutyrate release. At the same time, as observed before [Hartmann et al. (1982) Eur. J. Biochem. 123, 521–526], nerve stimulation increased glucose output, shifted lactate uptake to output and decreased perfusion flow.

Ketogenesis from oleate, which enters the mitochondria via the carnitine system, was also lowered after nerve stimulation owing to a decrease of acetoacetate release to 30% with no alteration in 3‐hydroxybutyrate release.

Ketogenesis from octanoate, which enters the mitochondria independently of the carnitine system, was decreased after nerve stimulation as a result of a drastic decrease of acetoacetate output to 15% and a less pronounced decrease of 3‐hydroxybutyrate release to 65%.

Noradrenaline mimicked the metabolic nerve effects on ketogenesis only at the highly unphysiological concentration of 0.1 μM under basal conditions and in the presence of oleate as well as partly in the presence of octanoate. It was essentially not effective at a concentration of 0.01 μM, which might be reached in the sinusoids owing to overflow from the hepatic vasculature.

Sodium nitroprusside prevented the hemodynamic changes after nerve stimulation; it did not affect the nerve‐dependent reduction of ketogenesis under basal conditions and in the presence of oleate, yet it diminished the nerve effect on octanoate‐dependent ketogenesis.

Phentolamine clearly reduced the metabolic and hemodynamic nerve effects, while propranolol was without effect.

The present data suggest that hepatic ketogenesis was inhibited by stimulation of α‐sympathetic liver nerves directly rather than indirectly via hemodynamic changes or noradrenaline overflow from the vessels and that the site of regulation should be mainly intramitochondrial.
Biochemical comparison of the Neurospora crassa wild type and the temperature‐sensitive and leucine‐auxotroph mutant leu‐5
Authors: KUNUGI Shigeru , UEHARA‐KUNUGI Yukiko , VON DER HAAR Friedrich , SCHISCHKOFF Jörg , FREIST Wolfgang , ENGLISCH Uwe , CRAMER Friedrich
Published: March 2005

The cytoplasmic leucyl‐tRNA synthetases of Neurospora crassa wild type (grown at 37°C) and mutant (grown at 28°C) were purified approximately 1770‐fold and 1440‐fold respectively. Additional enzyme preparations were carried out with mutant cells grown for 24 h at 28°C and transferred then to 37°C for 10–70 h of growth. The mitochondrial leucyl‐tRNA synthetase of the wild type was purified approximately 722‐fold. The mitochondrial mutant enzyme was found only in traces.

The cytoplasmic leucyl‐tRNA synthetase from the mutant (grown at 37°C) in vivo is subject of a proteolytic degradation. This leads to an increased pyrophosphate exchange, without altering aminoacylation. Proteolysis in vitro by trypsin or subtilisin of isolated cytoplasmic wild‐type and mutant leucyl‐tRNA synthetases, however, did not establish any difference in the degradation products and in their catalytic properties.

Comparing the cytoplasmic wild‐type and mutant enzymes (grown at 28°C) via steady‐state kinetics did not show significant differences between these synthetases either. The rate‐determining step appears to be after the transfer of the aminoacyl group to the tRNA, e.g. a conformational change or the release of the product. Besides leucine only isoleucine is activated by the enzymes with a discrimination of ∼ 1:600; however, no Ile‐tRNALeu is released. Similarly these enzymes, when tested with eight ATP analogs, cannot be distinguished. For both enzymes six ATP analogs are neither substrates nor inhibitors. Two analogs are substrates with identical kinetic parameters.

The mitochondrial wild‐type leucyl‐tRNA synthetase is different from the cytoplasmic enzyme, as particularly exhibited by aminoacylating Escherichia coli tRNALeu but not N. crassa cytoplasmic tRNALeu. The presence of traces of the analogous mitochondrial mutant enzyme could be demonstrated.

Therefore, the difference between wild‐type and mutant leu‐5 does not rest in the catalytic properties of the cytoplasmic leucyl‐tRNA synthetases. Differences in other properties of these enzymes are not excluded. In contrast the activity of the mitochondrial leucyl‐tRNA synthetase of the mutant is approximately 1% of that of the wild‐type enzyme.
Glycosylation and secretion of acid phosphatase in Schizosaccharomyces pombe
Authors: SCHWEINGRUBER Anne‐Marie , SCHOENHOLZER Fred , KELLER Lucius , SCHWANINGER Ruth , TRACHSEL Hans , SCHWEINGRUBER M. Ernst
Published: March 2005

We have purified secreted acid phosphatase of Schizosaccharomyces pombe. The enzyme is N‐glycosylated, the associated carbohydrate accounts for 90% of the total molecular mass and the protein moiety has a molecular mass of 54 kDa. The deglycosylated enzyme still exhibits enzymatic activity. Using antibodies recognizing the protein moiety of the enzyme we have identified two intracellular precursors of acid phosphatase: an unglycosylated membrane‐bound 54‐kDa form that accumulates in the presence of tunicamycin and a partially glycosylated 72‐kDa form that accumulates mostly in membranes of cells grown in rich medium. We further showed that the conversion of the 54‐kDa and 72‐kDa forms to partially glycosylated and fully glycosylated acid phosphatase is a regulated process. Growth conditions determine how much of translated 54‐kDa acid phosphatase is glycosylated to the 72‐kDa form and how much remains unglycosylated in membranes. When cells are grown in a rich medium, 5% of the total acid phosphatase protein remains as unglycosylated enzyme and 8% as partially glycosylated 72‐kDa form. In cells grown in the minimal medium, however, all of the 54‐kDa and 72‐kDa forms of acid phosphatase are rapidly processed to fully glycosylated enzyme. The 72‐kDa form and the unglycosylated form of acid phosphatase are not secreted or transported to the plasma membrane.
Reduction of lactoperoxidase by the dithionite anion monomer
Authors: OHLSSON Per‐Ingvar , BLANCK Jürgen , RUCKPAUL Klaus
Published: March 2005

The reduction of lactoperoxidase with sodium dithionite has been studied by means of stopped‐flow spectrophotometry in an anaerobic system. Under pseudo‐first‐order conditions the rate constant was found to be linearly dependent on the square root of the dithionite concentration, which confirms the monomeric radical, SO⋅−2 as the reducing species. The second‐order rate constant is moderately influenced by increased ionic strength but drastically increased at lower pH. The pH dependence supports the previously suggested existence of a carboxyl group, essential to the different enzymatic functions of lactoperoxidase.

The second‐order rate constant for the reduction of lactoperoxidase at pH 7.0 (k1= 1.3 × 105 M−1 s−1) was about three times higher than the rate constant for the reduction of cyanide‐bound lactoperoxidase and two times the rate constant for the reduction of the fluoride‐lactoperoxidase complex.
Activation in vitro of respiratory nitrate reductase of Escherichia coli K12 grown in the presence of tungstate
Authors: SARACINO Laurence , VIOLET Michele , BOXER David H. , GIORDANO Gerard
Published: March 2005

The chlorate‐resistant (chlR) mutants are pleiotropically defective in molybdoenzyme activity. The inactive derivative of the molybdoenzyme, respiratory nitrate reductase, present in the cell‐free extract of a chlB mutant, can be activated by the addition of protein FA, the probable active product of the chlB locus. Protein FA addition, however, cannot bring about the activation if 10 mM sodium tungstate is included in the culture medium for the chlB strain. The inclusion of a heat‐treated preparation of a wild‐type or chlB strain prepared after growth in the absence of tungstate, restores the protein‐FA‐dependent activation of nitrate reductase. All attempts to activate nitrate reductase in extracts prepared from tungstate‐grown wild‐type Escherichia coli strains failed. It appears that during growth with tungstate, the possession of the active chlB gene product leads to the synthesis of a nitrate reductase derivative which is distinct from that present in the tungstate‐grown chlB mutant. Heat‐treated preparations from chlA and chlE mutants which do not possess molybdenum cofactor activity fail to restore the activation. Fractionation by gel filtration of the heat‐treated preparation from a wild‐type strain produced two active peaks in the eluate of approximate Mr 12000 and ≤ 1500. The active material in the heat‐treated extract was resistant to exposure to proteinases, but after such treatment the active component, previously of approximate Mr 12000, eluted from the gel filtration column with the material of Mr≤ 1500. The active material is therefore of low molecular mass and can exist either in a protein‐bound form or in an apparently free state. Molybdenum cofactor activity, assayed by the complementation of the apoprotein of NADPH:nitrate oxidoreductase in an extract of the nit‐1 mutant of Neurospora crassa, gave a profile following gel filtration similar to that of the ability to restore respiratory nitrate reductase activity to the tungstate‐grown chlB mutant soluble fraction. This was the case even after proteinase treatment of the heat‐stable fraction. Analysis of the chlC (narC) mutant, defective in the structural gene for nitrate reductase, revealed that heat treatment is not necessary for the expression of the active component. Furthermore both the active component and molybdenum cofactor activity are present in corresponding bound and free fractions in the non‐heat‐treated soluble subcellular fraction. However, a heat‐treated extract from a chlG mutant, while possessing molybdenum cofactor as assayed by N. crassa nit‐1 complementation, was unable to function in the activation of nitrate reductase in the tungstate‐grown chlB mutant extract. We conclude that the active component in the heat‐stable fraction is either the molybdenum cofactor or a closely related compound. This is consistent with the lack of molybdenum cofactor activity in extracts from tungtstate‐grown cells.
Tissue‐specific and light‐dependent changes of chromatin organization in barley (Hordeum vulgare)
Authors: STEINMÜLLER Klaus , BATSCHAUER Alfred , APEL Klaus
Published: March 2005

The DNase I sensitivity of the nuclear genes encoding the NADPH‐protochlorophyllide oxidoreductase, the light‐harvesting chlorophyll a/b protein (LHCP), the hordeins and a 15‐kDa protein of unknown function was assayed in chromatin of etiolated and green leaves and endosperm tissue of barley (Hordeum vulgare L.).

A tissue‐specific differentiation of chromatin structure was found for the LHCP, hordein and 15‐kDa protein genes. The genes for the LHCP and the 15‐kDa protein, which are expressed in leaf tissue, display DNase I sensitivity in leaves but not in endosperm. Hordein genes which are expressed solely in endosperm, were insensitive to low levels of digestion with DNase I in leaves but sensitive in endosperm. The effect of light on chromatin structure was determined by comparing leaves of etiolated plants and plants which had been grown under a day/night cycle. Only in the case of the 15‐kDa protein is there a remarkable change from a DNAse‐I‐sensitive configuration in etiolated leaves to a more resistant one in leaves from illuminated plants. The gene for the NADPH‐protochlorophyllide oxidoreductase was found to be equally sensitive to DNase I in leaves and endosperm.
The Mr‐50000 polypeptide of mammalian pyruvate dehydrogenase complex participates in the acetylation reactions
Authors: DE MARCUCCI Olga G. L. , HODGSON Jeffery A. , LINDSAY John G.
Published: March 2005

The mammalian pyruvate dehydrogenase complex, Mr 8.5 × 106, contains an additional tightly bound 50000‐Mr polypeptide, component X, which copurifies with the intact assembly.

Small amounts of the individual E2 and X polypeptides were obtained by elution of the protein bands from SDS/polyacrylamide gels. One‐dimensional peptide mapping studies with 125I‐labelled lipoyl acetyltransferase (E2) and component X subunits indicate that these two proteins are structurally distinct entities. Similar analysis of purified subunits, initially radiolabelled in the intact complex in the presence of [2‐14C]pyruvate and N‐ethyl‐[2,3‐14C]maleimide confirm that distinct 14C‐labelled peptides are generated from these two species. These protein‐chemical data supplement recent immunological findings, which demonstrate that component X is not a proteolytic fragment of the larger lipoyl acetyltransferase (Mr 70000) subunit.

Incubation of the native PDC in the presence of [2‐14C]pyruvate leads to rapid uptake of radiolabel, presumably as acetyl groups, into both E2 and protein X. Specific incorporation of acetyl groups declines to a similar extent on both polypeptides after inhibiting pyruvate dehydrogenase (E1) activity by phosphorylation or omitting thiamine diphosphate (TPP) from the assay mixture. Addition of CoASH promotes the parallel deacetylation of both lipoyl acetyltransferase and protein X in a reaction which displays sensitivity to N‐ethylmaleimide.
Anomeric specificity of D‐glucose metabolism in rat adipocytes
Authors: MALAISSE‐LAGAE Francine , MALAISSE Willy J.
Published: March 2005

The anomeric specificity of D‐glucose metabolism was investigated in rat adipocytes exposed for 60 min at 8°C to pure α‐ or β‐D‐glucose or to equilibrated D‐glucose. The rate of D‐[5‐3H]glucose utilization was higher with α‐ than β‐D‐glucose. However, as judged from the oxidation of D‐[1‐14C]glucose and D‐[6‐14C]glucose anomers, the fraction of D‐glucose catabolism occurring via the pentose cycle was higher with β‐ than α‐D‐glucose. In the presence of equilibrated D‐glucose, the utilization of α‐D‐[5‐3H]glucose and the oxidation of both α‐D‐[1‐14C]glucose and α‐D‐[6‐14C]glucose were higher, relative to the anomer concentration, than the corresponding values for β‐D‐glucose. It is concluded that the anomeric specificity of D‐glucose metabolism is operative in adipocytes, even when they are exposed to equilibrated D‐glucose.
Hormonal regulation of the tricarboxylic acid cycle in the isolated perfused rat liver
Authors: PATEL Tarun B.
Published: March 2005

The effect of Ca2+‐mobilizing hormones, vasopressin, angiotensin II and the α‐adrenergic agonist phenylephrine, on the metabolic flux through the tricarboxylic acid cycle was investigated in isolated perfused rat livers. All three Ca2+‐mobilizing agonists stimulated 14CO2 production and gluconeogenesis in livers of 24‐h‐fasted rats perfused with [2‐14C]pyruvate. Prazosin blocked the phenylephrine‐elicited stimulation of 14CO2 and glucose production from [2‐14C]pyruvate whereas the α2‐adrenergic agonist, BHT‐933, did not affect the rates of 14CO2 and glucose production from [2‐14C]pyruvate indicating that the phenylephrine‐mediated response involved α1‐adrenergic receptors. Phenylephrine, vasopressin and angiotensin II stimulated 14CO2 production from [2‐14C]acetate in livers derived from fed rats but not in livers of 24‐h‐fasted rats. In livers of 24‐h‐fasted rats, perfused with [2‐14C]acetate, exogenously added pyruvate was required for an increase in the rate of 14CO2 production during phenylephrine infusion. This last observation suggests increased pyruvate carboxylation as one of the mechanisms involved in stimulation of tricarboxylic acid cycle activity by the Ca2+‐mobilizing agonists, vasopressin, angiotensin II and phenylphrine.
Tarantula hemocyanin mRNA
Authors: VOIT Renate , SCHNEIDER Hans‐Jürgen
Published: March 2005

Following induction of hemopoiesis, poly(A)–rich RNA was prepared from the heart of the tarantula, Eurypelma californicum, and translated in rabbit reticulocyte lysates. In vitro translation products were immunoprecipitated with antiserum against whole dissociated Eurypelma hemocyanin. Analysis of the immunoprecipitate by sodium dodecyl sulfate/polyacrylamide gel electrophoresis revealed a set of polypeptides comigrating with authentic Eurypelma hemocyanin. The mRNA was transcribed into cDNA, clones were constructed using the pUC9 vector and probed with a synthetic 17–mer oligonucleotide probe complementary to the amino acid sequence of the ‘copper A’ binding site of chelicerate hemocyanins. One clone, pHC4, contained a 1.62–kb cDNA insert, which was subcloned into phage M13. Sequence analysis by the dideoxynucleotide chain–termination method yielded a nucleotide sequence coding for 526 amino acids of Eurypelma hemocyanin subunit e.
Natural selection versus primitive gene structure as determinant of codon usage
Authors: WONG J. Tze‐Fei , CEDERGREN Robert
Published: March 2005

Different codons are not utilized equally in known gene sequences. One of the important biases of codon usage is observed in the from of an enrichment of RNY codons, especially within RNN codon families. Such biases could represent the residue of a primitive repeating‐RNY gene structure, or the outcome of natural selection, or both. Analyses based on the rates of silent substitutions, the requencies of base doublets, and synonymous codon rations for Escherichia coli, yeast, Drosophila and Xenopus proteins have been performed. The results rule out any significant support for a primitive repeating‐RNY or repeating‐RNY gene structure, and establish the important role of natural selection in determining the choice of codons. With strong intervention by natural selection, the relationship between primitive gene structure and codon usage necessarily becomes minimal.
Cytosolic adenylates and adenosine release in perfused working heart
Authors: BÜNGER Rolf , SOBOLL Sibylle
Published: March 2005

Free cytosolic adenylates were examined in relation to adenosine plus inosine released from perfused working guinea‐pig hearts. Whole‐tissue adenylate data from freeze‐clamped hearts were quantitatively compared with corresponding values obtained by subcellular fractionation of homogenized myocardium in non‐aqueous media. Adenosine and inosine in venous cardiac effuents were measured by high‐performance liquid chromatography. Hearts, perfused at their natural flows, were subjected to various workloads, substrates and catecholamines to alter myocardial energy metabolism and respiration over a wide physiological range. Non‐aqueous cytosolic ATP and creatine phosphate (CrP) accounted for more than 80% of the respective total myocardium content. The cytosolic CrP/Pi ratio was in near‐quantitative agreement with the overall tissue CrP/Pi ratio when the latter parameter was corrected for extracellular Pi. This was conclusive evidence that ATP, CrP and Pi were pre‐dominantly located in the cytosol of the well‐oxygenated cardiomyocyte. Measured myocardial oxygen uptake (MVO2) was reciprocally related to the phosphorylation state of CrP ([CrP]/[Cr] × [Pi]) and hence that of ATP ([ATP]/[ADP] × [Pi]) assuming the creatine kinase at near‐equilibrium at a near‐constant pH of 7.2. On the other hand, calculated mean free cytosolic ADP concentrations increased essentially linearily up to threefold with increasing MVO2 in the presence of virtually unchanged or only slightly decreased ATP levels; this was found both according to the whole tissue and the special subcellular fractionation data. Employing the myokinase mass‐action ratio and substituting total cardiac ADP by the mean free cytosolic ADP concentrations, the mean free cytosolic AMP concentrations proved to be in the nanomolar range, i.e. up to three orders of magnitude lower than the overall tissue AMP content. We propose, therefore, that in the normoxic heart, AMP is located predominantly in the mitochondrial compartment. Nevertheless, both free cytosolic AMP concentration and release of adenosine plus inosine were apparently square or even higher‐power functions of the rate of cardiac respiration. On the other hand, the mean purine nucleoside release seemed linearily correlated (r= 0.920) with the calculated free cytosolic AMP concentration.

Our observations seem to suggest that the concentrations of free ADP and AMP in the cytosol are major determinants of the production of inosine and coronary vasodilator adenosine. Such kinetic control of AMP degradation can conceivably render the free adenylate/adenosine system highly adaptive to mechanical perfor‐mance, total energy output, and/or the energy state of the myocardium. The energy‐linked changes in the free ATP/ADP system also seemed to produce an obligatory association between stimulated respiration and decreased cytosolic ATP potential concomitant with an increase in the concentration of free ADP.
Multispecific DNA methyltransferases from Bacillus subtilis phages
Authors: GÜNTHERT Ursula , LAUSTER Roland , REINERS Luzia
Published: March 2005

Temperate Bacillus subtilis phages SPR, φ3T, ϱ11 and SPβ code for DNA methyltransferases, each having multiple sequence specificities. The SPR wild‐type and various mutant methyltransferases were overproduced 1000‐fold in Escherichia coli and were purified by three consecutive chromatographic steps.

The stable form of these multispecific enzymes in solution are monomers with a relative molecular mass (Mr) of about 50000. The methyl‐transfer kinetics of the SPR wild‐type and mutant enzymes were determined with DNA substrates carrying either none or one of the three recognition sequences (GGCC, CCGG, CCATGG).

Evaluation of the catalytic properties for DNA and S‐adenosylmethionine binding suggested that the NH2‐terminal part of the protein is important for both non‐sequence‐specific DNA binding and S‐adenosylmethionine binding as well as transfer of methyl groups. On the other hand, mutations in the COOH‐terminal part lead to weaker site‐specific interactions of the enzyme.

Antibodies raised against the purified SPR enzyme specifically immunoprecipitated the φ3T, ϱ11 and SPβ methyltransferases, but failed to precipitate the chromosomally coded enzymes from B. subtilis (BsuRI) and B. sphaericus (BspRI). Immunoaffinity chromatography is an efficient purification step for the related phage methyltransferases.
Sulfite oxidase from chicken liver
Authors: RITZMANN Martin , BOSSHARD Hans Rudolf
Published: March 2005

Oxidation of sulfite to sulfate by sulfite oxidase is inhibited when the enzyme is treated with reagents known to modify imidazole and carboxyl groups. Modification inhibits the oxidation of sulfite by the physiological electron acceptor cytochrome c, but not by the artificial acceptor ferricyanide. This indicates interference with reaction steps that follow the oxidation of sulfite by the enzyme's molybdenum cofactor.

Reaction with diethylpyrocarbonate modifies ten histidines per enzyme monomer. Loss of activity is concomittant to the modification of only a single histidine residue. Inactivation takes place at the same rate in free sulfite oxidase and in the sulfite‐oxidase – cytochrome‐c complex.

Blocking of carboxyl groups with water‐soluble carbodiimides inactivates the enzyme. But none of the enzyme's carboxyl groups seems to be essential in the sense that its modification fully abolishes activity.

The pattern of inactivation by chemical modification of sulfite oxidase is quite similar to that observed previously for cytochrome c peroxidase from yeast [Bosshard, H. R., Bänziger, J., Hasler, T. and Poulos, T. L. (1984) J. Biol. Chem. 259, 5683–5690; Bechtold, R. and Bosshard, H. R. (1985) J. Biol. Chem. 260, 5191–5200]. The two enzymes have very different structures yet share cytochrome c as a common substrate of which they recognize the same electron‐transfer domain around the exposed heme edge.
Control of a futile urea cycle by arginine feedback inhibition of ornithine carbamoyltransferase in Agrobacterium tumefaciens and Rhizobia
Authors: VISSERS Stephan , LEGRAIN Christiane , WIAME Jean‐Marie
Published: March 2005

In Agrobacterium tumefaciens and Rhizobia arginine can be used as the sole nitrogenous nutrient via degradation by an inducible arginase. These microorganisms were found to exhibit arginine inhibition of ornithine carbamoyltransferase activity. This inhibition is competitive with respect to ornithine (Km for ornithine = 0.8 mM; Ki for arginine = 0.05 mM). This type of urea cycle regulation has not been observed among other microorganisms which degrade arginine via an arginase. The competitive pattern of this inhibition leads to its being inoperative in ornithine‐grown cells, where the intracellular concentration of ornithine is high. In arginine‐grown cells, however, the intracellular arginine and ornithine concentrations are compatible with inhibition and ornithine recycling appears to be effectively blocked in vivo.
Carbohydrate‐dependent induction of fatty acid synthase in primary cultures of rat hepatocytes
Authors: GIFFHORN‐KATZ Susanne , KATZ Norbert R.
Published: March 2005

The glucose and insulin‐dependent long‐term regulation of fatty acid synthase was studied in primary cultures of hepatocytes from adult female rats.

Under basic culture conditions, i.e. 5.5 mM glucose and 0.5 nM insulin, the enzyme activity was continuously decreased over 6 days. In the presence of 100 nM insulin this decrease was reduced but it was not prevented. Enhancement of glucose to 20 mM was followed by an increase of the enzyme activity; after 5 days of treatment the activity was three times higher than under basic culture conditions. The simultaneous presence of 20 mM glucose and of 100 nM insulin resulted in a much more pronounced increase of the activity; after 5 days of treatment the activity was eight times higher than under basic culture conditions.

The enhancement was prevented by inhibition of glycolysis. This may indicate that the increase of fatty acid synthase was mediated by a metabolite of glucose rather than by glucose itself.

The coordinate regulation of fatty acid synthase and of other lipogenic enzymes was specific as demonstrated by comparison with the activity of lactate dehydrogenase and with synthesis and degradation of cytosolic proteins.

The enhancement of the enzyme protein, demonstrated by rocket immunoelectrophoresis, was due to an increase in the rate of enzyme synthesis by 600% as well as to a prolongation of the apparent half‐life of the enzyme by 50% (45 h).
Epidermin: sequencing of a heterodet tetracyclic 21‐peptide amide antibiotic
Authors: ALLGAIER Hermann , JUNG Günther , WERNER Rolf G. , SCHNEIDER Ursula , ZÄHNER Hans
Published: March 2005

Epidermin is a large peptide antibiotic, which is synthesized in the ribosome via a precursor protein, followed by enzymatic modifications. It was isolated from the culture filtrate of Staphylococcus epidermidis Tü 3298 by adsorption on Amberlite XAD‐8. The basic heneicosapeptide amide was chromatographed on Sephadex LH‐20 and purified to homogeneity via multiplicative counter‐current distributions in one acidic and one neutral system. Tryptic digestion gave the soluble N‐terminal fragment epidermin‐(1–13)‐peptide (P1) and the insoluble C‐terminal fragment 2‐oxobutyryl‐epidermin‐(15–21)‐peptide amide (P2), each possessing two sulfide ring systems. The heterodetic rings consisted of meso‐lanthionine and (2S,3S,6R)‐3‐methyllanthionine (P1), meso‐lanthionine and C‐terminally the new amino acid S‐(2‐aminovinyl)‐D‐cysteine (P2). The complex sequence was elucidated via a combination of desulfurization with Raney nickel, enzymatic and acidolytic degradations, gas‐phase sequencing, fast‐atom bombardment and field‐desorption mass spectrometry and NMR spectroscopy.
1H and 13C NMR assignment of benzothiophenquinones from the sulfur‐oxidizing archaebacterium Sulfolobus solfataricus
Authors: LANZOTTI Virginia , TRINCONE Antonio , GAMBACORTA Agata , DE ROSA Mario , BREITMAIER Eberhard
Published: March 2005

From Sulfolobus solfataricus, a sulfur‐oxidizing thermophilic member of archaebacteria, three unusual benzothiophenquinones were isolated. Detailed NMR studies on these quinones, including multipulse monodimensional and two‐dimensional techniques, were performed to obtain carbon and proton assignments, onebond, geminal and vicinal coupling constants and T1 relaxation times. This report extends the known quinone composition of thermophilic archaebacteria and further supports the concept that these biomolecules can serve as a useful chemotaxonomic tool.
Studies on the reaction mechanism of general acyl‐CoA dehydrogenase
Authors: POHL Brigitte , RAICHLE Thomas , GHISLA Sandro
Published: March 2005

The kinetic properties of general acyl‐CoA dehydrogenase from pig kidney have been investigated using normal butyryl‐CoA as well as a α‐deutero, β‐deutero‐and perdeutero‐butyryl‐CoA. In turnover catalysis, isotope effects of 2, 3.6, and 9 were found respectively. In the reductive half reaction the isotope effects were 2.5, 14, and 28 for the same substrates, and 21 for (2R,3R)‐(2,3‐D2)butyryl‐CoA. No intermediates are apparent during the reduction of oxidized enzyme to the presumed complex of reduced enzyme and crotonyl‐CoA. The results are interpreted as indicating a high degree of concertedness during the rupture of the α and β C‐H bonds. They are compatible with a mechanism in which simultaneously the α‐hydrogen is abstracted as a proton, while the β‐hydrogen is transferred to the oxidized flavin as a hydride.
The effect of citrate/cis‐aconitate on oxidative metabolism during transformation of Trypanosoma brucei
Authors: OVERATH Peter , CZICHOS Joachim , HAAS Claudia
Published: March 2005

Monomorphic bloodstream forms of Trypanosoma brucei, grown in the mammal, are deficient in aconitase and 2‐oxoglutarate dehydrogenase and they do not respire in the presence of the substrates citrate, cis‐aconitate, succinate, proline or 2‐oxoglutarate. When grown in vitro low levels of aconitase, succinate oxidase and proline oxidase are detected.

Addition of citrate/cis‐aconitate at 37°C to bloodstream forms leads to the formation of aconitase and proline oxidase. Most cells undergo an ‘abortive’ transformation to non‐dividing procyclic‐like cells while some cells adapt to the presence of the citric acid cycle intermediates and continue to multiply as bloodstream forms.

At 27°C and in the presence of citrate/cis‐aconitate bloodstream forms transform synchronously to dividing procyclic cells. Within 72 h the rate of respiration with proline, succinate and 2‐oxoglutarate becomes similar to that in established procyclic cells while the rate of glucose oxidation decreases.

The possible role of citric acid cycle intermediates in determining whether a trypanosome will retain the properties of a bloodstream trypomastigote or differentiate to a procyclic trypomastigote is discussed.
Effects of guanine nucleotides on the properties of 5‐hydroxytryptamine secretion from electropermeabilised human platelets
Authors: KNIGHT Derek E. , SCRUTTON Michael C.
Published: March 2005

1. Guanosine 5′‐[γ‐thio]triphosphate and guanosine 5′‐[β,γ‐imido]triphosphate enhance Ca2+‐dependent 5‐hydroxytryptamine secretion from electropermeabilised human platelets. GTP has little such effect except when the platelets are permeabilised, and incubated with this nucleotide, at 2°C and pH 7.4.

2. The lag phase observed in the time course of 5‐hydroxytryptamine secretion induced by addition of guanosine 5′‐[γ‐thio]triphosphate is markedly longer than that characterising secretion induced by Ca2+ alone, by thrombin ± GTP or by guanosine 5′‐[γ‐thio]triphosphate in the presence of thrombin.

3. GTP causes competitive inhibition of the enhancement of the Ca2+‐dependent secretory response induced by guanosine 5′‐[γ‐thio]triphosphate when both nucleotides are added simultaneously. The extent of inhibition is decreased if guanosine 5′‐[γ‐thio]triphosphate is added prior to GTP.

4. GTP markedly enhances the effect of thrombin on Ca2+‐dependent 5‐hydroxytryptamine secretion by increasing the maximal extent of the response and decreasing the thrombin concentration required to give half‐maximal response. A similar effect is observed on addition of guanosine 5′‐[γ‐thio]triphosphate in the presence of thrombin at short incubation times. On more prolonged incubation the effects of thrombin and guanosine 5′‐[γ‐thio]triphosphate are additive.

5. Guanosine 5′‐[β‐thio]diphosphate completely inhibits the response induced by guanosine 5′‐[γ‐thio]triphosphate or guanosine 5′‐[β,γ‐imido]triphosphate but has little effect on the response induced by Ca2+ when added alone or in the presence of thrombin. Partial inhibition is observed for the response induced by thrombin + GTP.

6. Cyclic‐AMP effectively inhibits the response induced by thrombin + GTP but has little effect on that induced by guanosine 5′‐[γ‐thio]triphosphate or guanosine 5′‐[β,γ]imidotriphosphate.

7. The results provide further support for the proposal [Haslam, R. J. & Davidson, M. M. L. (1984) FEBS Lett. 170, 90–95], that receptor–phospholipase‐C coupling in platelets is mediated in part by a guanine‐nucleotide‐binding (Np) protein but that a coupling mechanism may also exist which is independent of such a protein. The properties of guanine‐nucleotide‐dependent coupling resemble those previously described for receptor–adenylate‐cyclase coupling.
Some aspects of the phosphorylation of α‐crystallin A
Authors: VOORTER  , MULDERS John W. M. , BLOEMENDAL Hans , DE JONG Wilfried W.
Published: March 2005

The cAMP‐dependent phosphorylation of α‐crystallin was investigated. The major products of in vitro phosphorylation of total bovine lens homogenate are the αA1 and αB1 polypeptides, but in addition a minor labeled spot is present which might correspond with a double phosphorylated αB chain. It is demonstrated that the A1 and B1 subunits of α‐crystallin from bovine eye lenses are solely the result of phosphorylation of the primary gene products αA2 and αB2, respectively, as judged from the stoichiometry of the phosphate content of these polypeptides. Both the in vitro and in vivo phosphorylation sites of the A chain of bovine α‐crystallin were determined and found to be the same. After in vitro incubation the majority of the 32P label was found in the tryptic peptides T17a and T16‐T17a, the latter being the result of incomplete tryptic cleavage between T16 and T17a. The in vivo phosphorylation site is also located in T17a, as could be concluded from the retention times on reversed‐phase HPLC of T16–17a and T17a from αA1 as compared to those from αA2, and from the differences in their mobilities on high‐voltage paper electrophoresis at pH 6.5. Furthermore, both T17a and T16–17a of αA1 contain approximately 1 mol phosphate/mol peptide. Thermolytic digestion of T16–17a of both αA2 and αA1, followed by separation on RP‐HPLC, demonstrated that Ser‐122 is the phosphorylation site of the A chain of bovine lens α‐crystallin. The replacement of this phosphorylation site or the lack of basic amino acids at the N‐terminal side of Ser‐122 in some vertebrate species apparently results in the absence of phosphorylation of α‐crystallin A both in vitro and in vivo.
Effects of serum vitamin‐D‐binding protein on actin in the presence of plasma gelsolin
Authors: COUÉ Martine , CONSTANS Jacques , OLOMUCKI Anna
Published: March 2005

The interaction of serum vitamin‐D‐binding protein (DBP) and plasma gelsolin with actin was studied using fluorescent 7‐chloro‐4‐nitrobenzo‐2‐oxa‐1,3‐diazole‐actin or N‐pyrenylcarboxyamidomethyl‐actin. DBP and gelsolin formed very tight complexes with one or two monomeric actin subunits respectively. Kd values of about 10 nm have been found for both complexes. When DBP and gelsolin were added simultaneously to G‐actin no ternary complex was observed and the DBP‐actin complex was formed preferentially. In the presence of CaCl2 no transfer of actin occurred from the 1:2 gelsolin:actin molar complex to free DBP while in the presence of EGTA one actin monomer could be transferred. DBP did not affect the severing activity of gelsolin. The effects of a mixture of gelsolin and DBP on F‐actin suggest that only individual interactions of these two plasma proteins with actin occurred in solution.
Purification and properties of tauropine dehydrogenase from the shell adductor muscle of the ormer, Haliotis lamellosa
Authors: GÄDE Gerd
Published: March 2005

Tauropine dehydrogenase (tauropine: NAD oxidoreductase) was purified from the shell adductor muscle of the ormer, Haliotis lamellosa. The enzyme was found to utilize stoichiometrically NADH as co‐enzyme and pyruvate and taurine as substrates producing tauropine [rhodoic acid; N‐(D‐1‐carboxyethyl)‐taurine]. The enzyme was purified to a specific activity of 463 units/mg protein using a combination of ammonium sulphate fractionation, ion‐exchange and affinity chromatography. The relative molecular mass was 38000 ± 1000 when assessed by gel filtration on Ultrogel AcA 54 and 42000 ± 150 by electrophoresis on 5–10% polyacrylamide gels in the presence of 1% sodium dodecyl sulphate; the data suggest a monomeric structure. Tauropine and pyruvate were found to be the preferred substrates. Among the amino acids tested for activity with the enzyme, only alanine is used as an alternative substrate, but with a rate less than 6% of the enzyme activity with taurine. Of the oxo acids tested, 2‐oxobutyrate and 2‐oxovalerate were also found to be substrates. Apparent Km values for the substrates NADH, pyruvate and taurine are 0.022 ± 0.003 mM, 0.64 ± 0.07 mM and 64.7 ± 5.4 mM, respectively, at pH 7.0 and for the products, NAD+ and tauropine, are 0.29 ± 0.01 mM and 9.04 ± 1.27 mM, respectively, at pH 8.3. Apparent Km values for both pyruvate and taurine decrease with increasing co‐substrate (taurine or pyruvate) concentration. NAD+ and tauropine were found to be product inhibitors of the forward reaction. NAD+ was a competitive inhibitor of NADH, whereas tauropine gave a mixed type of inhibition with respect to pyruvate and taurine. Succinate was found to inhibit non‐competitively with respect to taurine and pyruvate with an apparent Ki value in the physiological range of this anaerobic end product. The inhibition by L‐lactate, not an end product in the ormer, was competitive with respect to pyruvate. The physiological role or tauropine dehydrogenase during anaerobiosis is discussed.
Rat liver nuclear protein kinases NI and NII
Authors: DELPECH Marc , LEVY‐FAVATIER Florence , MOISAND Françoise , KRUH Jacques
Published: March 2005

Rat liver nuclear protein kinases NI and NII have been purified to homogeneity by an improved method. This method includes a casein‐phosvitin‐Sepharose column step, which separates the enzymes from the other chromosomal non‐histone proteins, and a gel filtration at high ionic strength in the presence of a high concentration of protease inhibitors to separate the two enzymes from each other. NI has an apparent molecular mass of approximately 50 kDa and is composed of a single subunit. NII has an apparent molecular mass of 133 kDa and is composed of two subunits of identical molecular mass. The V and the Km of the two enzymes were determined for several substrates. Both enzymes phosphorylate chromosomal non‐histone proteins with partly different specificities as shown by two‐dimensional electrophoreses. When incubated in the absence of protease inhibitors, the enzymes were degraded into discrete polypeptides.

Autophosphorylation of a polypeptide derived from NII was observed after incubation of the enzyme with ATP. This phosphorylation stimulated the enzyme activity.

Several chromosomal proteins coeluted with NII from the casein‐phosvitin‐Sepharose column. They remained associated with the enzyme in sucrose gradients, during gel filtration performed at physiological ionic strength, and are dissociated at high ionic strength. These proteins were highly phosphorylated when the protein‐NII complex was incubated with ATP.
1H‐NMR study of mobility and conformational constraints within the proline‐rich N‐terminal of the LC1 alkali light chain of skeletal myosin
Authors: BHANDARI Dhan G. , LEVINE Barry A. , TRAYER Ian P. , YEADON Mary E.
Published: March 2005

Analysis by 1H‐NMR spectroscopic techniques of the conformation of the N‐terminal segment of the LC1 alkali light chain of rabbit skeletal muscle has shown that this portion of the molecule adopts a well‐defined elongated configuration. This rod‐like feature is a consequence of the Ala/Pro‐rich composition and the functional aspects of such conformational preference in this and similar segments in other proteins are discussed.
Differential detergent‐solubilization of integral thylakoid membrane complexes in spinach chloroplasts
Authors: MORRISSEY Peter J. , McCAULEY Steven W. , MELIS Anastasios
Published: March 2005

Progressive solubilization of spinach chloroplast thylakoids by Triton X‐100 was employed to investigate the domain organization of the electron transport complexes in the thylakoid membrane. Triton/chlorophyll ratios of 1:1 were sufficient to disrupt fully the continuity of the thylakoid membrane network, but not sufficient to solubilize either photosystem I (PSI), photosystem II (PSII) or the cytochrome b6‐f (Cyt b6‐f) complex. Progressive with the Triton concentration increase (Triton/Chl >1:1), a differential solubilization of the three electron transport complexes was observed. (a) Solubilization of the Cyt b6‐f complex from the thylakoid membrane preceded that of PSI and apparently occurred early in the solubilization of stroma‐exposed segments of the chloroplast lamellae. (b) The initial removal of chlorophyll (up to 40% of the total) occurred upon solubilization of PSI from the stroma‐exposed lamella regions in which PSI is localized. (c) The tightly appressed membrane of the grana partition regions was markedly resistant to solubilization by Triton X‐100. Thus, solubilization of PSII from this membrane region was initiated only after all Cyt b6‐f and PSI complexes were removed from the chloroplast lamellae. The results support the notion of extreme lateral heterogeneity in the organization of the electron transport complexes in higher plant chloroplasts and suggest a Cyt b6‐f localization in the membrane of the narrow fret regions which serve as a continum between the grana and stroma lamellae.