bluuebunny/crossref_metadata_2025 huggingface dataset:
The Function fo the Subunits of the Fumarate Reductase Complex of Vibrio succinogenes
Authors: UNDEN Gottfried , KRÖGER Achim
Published: March 2005

The membrance‐bound fumarate reductase complex of Vibrio succinogenes catalyzes the reduction fo fumarate by 2,3‐dimethyl‐1,4‐naphthohydrquinone (dimethylnaphthohydroquinone) and consists of three different keptides (Mr 79000, Mr 31000 and Mr 25000), the smallest of which is cytochrome b [Unden, G., Hackenberg, H. and Kröger, A.(1980) Biochem. Biophys.Acta 591, 275–288]. The complex was cleaved with guanidinium chloride, the resulting subunits characterized and their functions within the complex investigated by reconstitutional experiments.

The Mr‐79000 subunits catalyzed the reduction fo fumarate by benzylviologen radicals as well as the oxidation of succinate by methylene blue, but not fumarate reduction by dimethylnaphthohydroquinone.

The spectral and the redox properties of the isolated cytochrome b(Mr 25000) were equivalent to those of the high‐protential cytochrome b of the bacteria. The isolated cytochrome b had a midpoint potential of ‐15 mV and was reducible by dimethylnaphthohydroquinone in the absence of the other subunits.

The Mr‐31000 subunit did not catalyze any of the ractions mentioned above. For the reduction of cytochrome b by succinate in the presence of the Mr‐79000 subunits, an amount of the Mr‐31000 subunits was required which was equimolar to cytochrome b.

The activity of fumarate reduction by dimethylnaphthohydroquinone could be restord by coprecipitation of the three subunits.

It is concluded that the fumarate reductase complex has two different reactive sites, which are essential for its function in the phosphorylative electron transport of the bacterium. The site reacting with the substrates fumarate and succinate is situated on the Mr‐79000 subunit, and that reacting with dimethylnaphthohydroquinone is cytochrome b. The Mr‐31000 subunit mediates the electron transport between cytochrome b and the Mr‐31000 subunits mediates the electron transport between cytochrome b and the Mr‐79000 subunit.
cAMP Receptor Proteins and protein Kinases in Human Lymphocytes: Fundamental Alterations in Chronic Lymphocytic Leukemia Cells
Authors: WEBER Wolfgang , SCHWOCH Grehild , WIELCKENS Klaus , GARTEMANN Axel , HILZ Helmuth
Published: March 2005

To study the significance of cAMP receptor proteins and protein kinase activities in tumor dys‐differentiation peripheral blood lymphocytes from normal donors and from patients with chronic lymphocytic leukemia were isolated by Percoll gradient centrifugation, and analyzed, Both normal and tumor lymphocyte populations were devoid of cell proliferation.

Normal human lymphocytes contained about equal activities of histone kinase and ‘casein kinase’, Of the total histone kinase, cAMP‐dependent activity in normal and tumor lymphocytes comprised about 50% as shown by the use of the heat‐stable inhibitor. The tumor lymphocytes, however, exhibited drastically reduced values of all three protein kinase activities, the most pronounced decrease (to 7% of the normal control cells) being observed with the ‘casein kinase’.

Total high‐affinity camp‐binding sites (= regulatory subunits R of camp‐dependent protein kinases type I and II) in leukemic lymphocytes were also strongly reduced (to <20%) Immunotitration with specific antibodies raised against rabbit muscle RI regulatory subunits in both normal and leukemic lymphocytes. Immuno‐reactive RII components comprised about 40% of total binding sites in lymphocytes o chronic lymphocytic leukemia, but only 20% in normal cells, the residual 20% being represented by RI protein of low immunoreactivity. Combination of gel elctrophoretic analysis of the camp‐binding proteins labeled with [32P]n3camp and binding to the R‐specific antibodies allowed the identification fo individual type I and type II R proteins: Besides the regulatory subunit RI of 49 kDa present in both cell types, additional isoproteins wwere found, the predominat RII form being a 52kDa subunit in normal lymphocytes and a 50‐kDa subunit in leukemic lymphocytes.

The findings in lymphocytes of chronic lymphocytic leukemia cannot be explained by the fact that they are B‐type lymphocytes.Control experiments reveled that normal B cell do not differ significantly from total blood lymphocytes with respect to the parameters mentioned above.

Biochemical correlates of the dys‐differentiated status of these non‐proliferating (G0) cell from patients with chronic lymphocytic leukemia, then, are represented by reduced levels of protein kinases, of basal cAMP as well as by changes of the R‐protein pattern. The data indicate that both protein kinases type I and type II may relate to the state of differentiation, independently from cell proliferation.
Stereochemical Investigation of the Phosphoketolase Reaction
Authors: MERKLER Ingeborg , RÉTEY János
Published: March 2005

d‐(1R)‐(1‐2H1)[1‐3H] Fructose 6‐phosphate was prepared by the action of glucosephosphate isomerase on d‐(1‐2H)glucose 6‐phosphate in tritiated water. Using the same enzyme but interchanging the labeling pattern of the substrate d‐(1S)‐(1‐2H)[1‐3H] fructose 6‐phosphate was also obtained.

The doubly labeled samples of d‐fructose 6‐phophate were converted into chiral acetylphosphate on phosphoketolase from Bifidium globosum (ATCC 25864), and these were reacted in situ with phosphotransacetylase and malate synthase in the presence of coenzyme A and glyoxylate.

The steric distributin of the tritium in the samples of malate, as shown by the action of fumarase, revealed that d‐(1R)‐and d‐(1S)‐(1‐2H1)[1‐3H] fructose 6‐phosphate was converted into (r) and (S)‐(2H1)[3H] acetylphosphate, repectively.

The results are discussed in terms of the mechanism of action of phosphoketolase.
The Fatty Acid Composition of Subcellular Membranes of Rat Liver, Heart, and Brain : Diet‐Induced Modifications
Authors: TAHIN Quivo S. , BLUM Max , CARAFOLI Ernesto
Published: March 2005

The influence of diets having different fatty acids composition on the fatty acid content of (the phospholipids) of rat liver mitochondria and microsomes, heart mitochondria, brain mitochondria and microsonies has been analyzed. It has been found that each organelle has its own peculiar composition in fatty acids. This composition may be profoundly influenced by the diet, but to different degrees in different organelles. Those of brain are most resistant.

The changes observed are rather rapid, being generally already maximal after three weeks of treatment. The parallel between fatty acid composition of diets, and the changes observed in the organelles, is not strict, and is probably influenced by the metabolic competition among oleic acid, linoleic acid, linolenic acid. Unusual fatty acids like crucic acid. trans‐oleic acid. and trans‐linoleic acid can also become incorporated into the membranes of cell organelles.
Larger Precursors of Mitochondria1 Translation Products in Neurospora crassa
Authors: Van't SANT Peter , MAK J. Frdncisca C , KROON Albert M.
Published: March 2005

Specific labeling in vivo of the formylated N termini of mitochondrial translation products revealed that some mitochondrially synthesized proteins were not labeled this way. As a consequence, it was worthwhile considering that larger precursor proteins of mitochondrial translation products exist. Although we used a rapid isolation procedure, only after 2‐h of labeling in the presence of cycloheximide, could three additional mitochondrial translation products (molecular mass 45, 36 and 25 kilodaltons) be detected. Preincubation with cycloheximide indicated that these proteins might be larger precursors which were no longer processed due to the prolonged presence of cycloheximide.

To prevent processing of the precursors during isolation, cells of the slime mutant were directly lysed in boiling sodium dodecyl sulphate solution. In this way, the same three additional mitochondrial translation products were detected after a pulse‐labeling of 1 min. These proteins behave in a precursor‐like fashion. Labeling at 9 °C resulted in a partial accumulation of the three additional proteins.

Finally protein blots treated with antibodies and 251‐labeled protein A, support the idea that the 45‐kDa protein is a precursor of subunit 1 of cytochrome c oxidase; 50–80% of this precursor could be detected in the post‐mitochondria1 supernatant, indicating that this polypeptide is not tightly bound to the membrane.
Mössbauer Studies on the Active Fe …[2Fe‐2S] Site of Putidamonooxin, Its Electron Transport and Dioxygen Activation Mechanism
Authors: BILL Eckhard , BERNHARDT Frithjof‐Hans , TRAUTWEIN Alfred Xaver
Published: March 2005

Putidamonooxin, the oxygenase of a 4‐methoxybenzoate monooxygenase enzyme system, catalyzes the oxidative O‐demethylation of the substrate 4‐methoxybenzoate in conjunction with the NADH: putidamonooxin oxidoreductase. Putidamonooxin is a conjugated iron‐sulfur protein which needs iron ions as cofactors for its enzymatic activity.

Putiamonooxin was isolated from Pseudornonus putida, which was grown on a 57Fe‐enriched culture medium. Thus putidamonooxin was enriched in vivo with 57Fe up to about 80%.

During our Mössbauer study of putidamonooxin a number of parameters have been varied: (a) the oxidation state of putidamonooxin (oxidized, reduced and aerobically reoxidized); (b) the substrate bound to putidamonooxin (Cmethoxybenzoate, benzoate, 4‐tert‐butylbenzoate); (c) the temperature between 2.7 K and 245 K; (d) the applied magnetic field between 0 and 0.1 T and (e) the amount of iron cofactor.

From our Mössbauer results it is obvious that the iron‐sulfur centers of putidamonooxin are [2 Fe‐2 S] clusters similar to those of the plant‐type ferredoxins. Further, we have evidence for the existence of iron ions (one per [2 Fe‐2S] cluster), which serve as cofactors for the dioxygen activation, functioning as the dioxygen binding site and mediating the electron flow from the [2 Fe‐2 S] cluster to dioxygen.
Analysis of Proteinase A Function in Yeast
Authors: MECHLER Bernd , WOLF Dieter H.
Published: March 2005

Yeast mutants lacking proteinase A were isolated. One of these mutants (Hb I) is characterized in detail. The mutation called pral segregates 2:2 in meiotic tetrads indicating a single gene mutation. No anti‐(proteinase A) cross‐reacting material can be detected. Diploids heterozygous for pral show gene dosage. Thus, it appears that PRAI might be the structural gene for proteinase A.

Results obtained with this mutant show that proteinase A is not a vital component of the vegetative cell cycle. The mutant exhibits normal mitotic growth under rich and poor growth conditions and shows normal mating. Enzymes subject to carbon catabolite inactivation and inactivation of NADP‐dependent glutamate dehydrogenase, processes which were proposed to be of proteolytic nature, are not affected by the absence of proteinase A. However, protein degradation under sporulation conditions is about 30;(reduced in proteinasc A mutant cells. The differentiation process of spordation is also disturbed leading to a 40 reduced sporulation frequency in mutant cells.
Modulation of Phosphofructokinase Behavior by Chemical Modifications during the Immobilization Process
Authors: CAMBOU Bernard , LAURENT Michel , HERVAGAULT Jean‐François , THOMAS Daniel
Published: March 2005

Phosphofructokinase was immobilized within a protein membrane or on soluble protein polymers using glutaraldehyde as cross‐linking reagent. The native enzyme was also modified chemically, using the cross‐linking reagent alone. A comparative kinetic investigation of these preparations was carried out.

The catalytic activity of the chemically modified enzyme and its affinity towards fructose 6‐phosphate decreased significantly; the modified enzyme lost its cooperative properties and the allosteric regulation by AMP was affected. When the chemical treatment was performed in the presence of effectors (AMP or ATP) the allo‐ steric transition induced by AMP was restored, suggesting that the cross‐linking reagent modified the AMP regulatory sites, albeit no higher‐substrate‐affinity enzyme conformation was frozen. Molecular data showed that glutaraldehyde produced intramolecular then intermolecular bonds as its concentration increased.

When the enzyme was immobilized into protein membranes or on soluble polymers, the enzyme behavior was quite similar: decrease of affinity towards fructose 6‐phosphate but no changes in cooperative properties and modifications of allosteric transition induced by AMP. When AMP was present during the immobilisation process, the enzyme immobilized in this way was no longer sensitive to effectors, either AMP or ATP. It showed Michaelian behavior and higher substrate affinity quite similar to that of the native enzyme. The data suggested that a higher‐ substrate‐affinity enzymatic form was most probably stabilized by immobilization.
Equilibrium and Kinetic Measurements of the Binding of Pyridoxal 5′‐Phosphate to Hybrid Tryptophan Synthase from Escherichia coli
Authors: BALK Hubert , FRANK Albert , BARTHOLMES Peter , JAENICKE Rainer
Published: March 2005

Hybrid tryptophan synthase was prepared that contains a β, subunit with one functional active site while the internal aldimine between cofactor and enzyme of the other protomer was reduced with sodium borohydride (α,ββ*). The modified enzyme shows a specific activity for the l‐serine+indole to l‐tryptophan reaction of about 50% compared to the native enzyme [Hathaway, G. M., Kida, S. and Crawford, I. P., Biochemistry 8, 989—997 (1969)]. The binding of pyridoxal 5′‐phosphate (pyridoxal‐P) to the α2apo ββ* complex and to the apo ββ* subunit was studied by equilibrium methods and rapid mixing experiments.

The equilibrium binding curves for the hybrid species are hyperbolic (i. e. non‐cooperative), yielding apparent microscopic dissociation constants of 1.6±O.2 × 10−6 M for the α2 holoββ* subunit.

The rate of formation of the internal aldimine in the unmodified active site was followed by fast reduction with sodium borohydride, L‐tryptophan synthesis was used to monitor the rate of formation of active enzyme.

Cofactor binding to the α2apo ββ* complex is characterized by three sequential steps of decreasing rate: formation of a non‐covalent initial complex, which reacts to an enzymatically inactive internal aldimine, followed by a conformational change leading to the active α2holo ββ* bienzyme complex. A similar series of consecutive steps with decreasing reaction rates is observed for the binding of pyridoxal‐P to the apoββ* subunit. The formation of active enzyme, however, is biphasic. The faster process, which parallels the formation of the internal aldimine and which accounts for 85% of the total amplitude, obviously involves an already active high‐affinity state of the hybrid molecule whereas the second phase parallels the activation process as observed for cofactor binding to the native dimer.

These findings are discussed with respect to the recently proposed mechanism for pyridoxal‐P binding to the unmodified apoβ2 subunit [Bartholmes, P. Balk, H. and Kirschner, K, Biochemistry 19, 4527–4533 (1980]
Reconstitution of Lactic Dehydrogenase after Acid Dissociation
Authors: ZETTLMEISSL Gerd , RUDOLPH Rainer , JAENICKE Rainer
Published: March 2005

Reconstitution of lactic dehydrogenase from pig muscle after dissociation at acidic pH produces native tetramers and inactive aggregates, both of which have been previously described with respect to their structural properties and the kinetics of their formation. The ratio of the two fractions is determined by the relative rates of reassociation and aggregation [G. ZettlmeißI, R. Rudolph and R. Jaenicke (1979) Biochemistry 18, 5567–5571]. The present study is concerned with the influence of residual structural elements within the acid‐denatured monomers on the process of reconstitution.

Long incubation at low pH leads to a marked decrease of the rate and yield of reactivation, caused by conformational changes within the partially unfolded monomers. These slow rearrangements are reflected by slight changes of the far‐ultraviolet circular dichroism of the acid‐dissociated monomers, indicating a decrease in helix content from 30 observed immediately after the transfer to low pH, to 25% after completion of the acid transition. The unchanged near‐ultraviolet circular dichroism and fluorescence properties prove local structural alterations to be of lesser importance compared to the changes of the residual backbone folding. Varying the experimental conditions during denaturation proves the slow decrease in the rate and yield of reactivation, as well as the changes in the far‐ultraviolet circular dichroism to be enhanced with incubation time and temperature (0–30°C). The first‐order rate constants of the decrease in the reactivation yield after incubation in 1 M glycine/H3PO4 pH 2.3, 0.1 M sodium phosphate pH 2.3, and 1 M H3PO4 were found to be in the same time range as the respective changes in the far‐ultraviolet circular dichroism.

Apart from the similarity of the rate constants, the activation energy for both the acid induced changes of the yield of reactivation and the far‐ultraviolet circular dichroism is found to be identical (80 kJ/mol).

The effects of low pH on the reconstitution of lactic dehydrogenase can be eliminated by complete disruption of the structure of the polypeptide chain, e.g. by adding 6 M guanidine · HCl. This proves unambiguously that the above‐mentioned structural rearrangements at acid pH are responsible for the observed decrease of the rate and yield of reactivation.
The Aryl Acylamidases and Their Relationship to Cholinesterases in Human Serum, Erythrocyte and Liver
Authors: GEORGE Shaji T. , BALASUBRAMANIAN Aiylam S.
Published: March 2005

Human serum aryl acylamidase associated with serum cholinesterase was purified to homogeneity. Evidence for the identity of the two enzymes was based on coelution profiles, co‐purification in the different steps including affinity chromatography with constant ratios of specific activity and percentage recoveries, co‐migration on gel electrophoresis, parallel inhibition by typical cholinesterase inhibitors and co‐precipitation by antibody raised against the purified enzyme. Human liver aryl acylamidase was partially purified. Based on the elution profiles, purification data, inhibitory characteristics and gel electrophoresis it was concluded that aryl acrylamidase of liver was not associated with liver cholinesterase. More conclusive evidence for the non‐association of the liver aryl acylamidase and cholinesterase came from their clear‐cut separation on procainamide‐Sepharose affinity chroma‐ tography. Both the serum and liver aryl acylamidase were compared with the purified erythrocyte aryl acylamidase associated with acetylcholinesterase. While the erythrocyte and serum aryl acylamidases showed some similarities in their sensitivities to amines like serotonin or tryptamine and choline derivatives, the liver enzyme was unaffected by any of these compounds. A notable observation was the activation by tyramine of the serum aryl acylamidase but not the erythrocyte and liver aryl acylamidases. The liver aryl acylamidase also differed from the other two in its relative insensitivity to inhibition by eserine, neostygmine and other cholinesterase inhibitors. Immunodiffusion and immunoprecipitation studies showed that the aryl acylamidases from the liver and erythrocytes were immunologi‐cally non‐identical with the serum enzyme.
Interaction of Fluorescein Isothiocyanate with Nucleotide‐Binding Sites of the Ca‐ATPase from Sarcoplasmic Reticulum
Authors: PICK Uri
Published: March 2005

We have previously shown that fluorescein 5′ isothiocyanate is a potent inhibitor of the Ca‐ATPase from sarcoplasmic reticulum [Pick, U. and Karlish, S. J. D. (1980) Biochim. Biophys. Acta, 626, 255–261] and completely blocks Ca uptake with ATP but not with acetyl phosphate [Pick, U. and Bassilian, S. (1981) FEBS Lett. 123, 127–130]. Here we show that fluorescein isothiocyanate inhibits in parallel all the ATP‐dependent partial reactions: the Ca‐ATPase, ATP‐dependent Ca uptake and phosphorylation by ATP whereas the phosphorylation reaction by inorganic phosphate is resistant to fluorescein isothiocyanate and Ca uptake with acetyl phosphate is only partly inhibited by the modification.

The time course of inactivation by fluorescein isothiocyanate is biphasic and the rate of inactivation is markedly increased at alkaline pH. The observations that a lysine‐specific reagent (2,4,6‐trinitrobenzene sulfonic acid) com‐ petes with binding of fluorescein isothiocyanate and the pH dependence of inactivation strongly suggests binding to a lysine E‐amino group.

Mg ions accelerate the rate of inactivation at pH 7.5–8.0 and Ca ions slightly inhibit the inactivation and the binding of fluorescein isothiocyanate.

The adenine nucleotides ATP and AdoPP [NH]P protected against inactivation by fluorescein isothiocyanate. A kinetic analysis of the initial rates of inactivation indicates competition between ATP and fluorescein isothiocyanate (the apparent dissociation constant KATP= 38 μM) and suggests a common binding site. Ca ions increase the apparent affinity of the non‐hydrolyzable analog AdoPP [NH]P by an order of magnitude but have no appreciable effect on the apparent affinity of ATP. Mg ions decrease the apparent affinity for adenine nucleotides both in the presence and absence of Ca.

It is suggested that (a) fluorescein isothiocyanate binds to and modifies the ATP‐binding site of the enzyme. (b) The enzyme has only one ATP‐binding site but the site exists in two different configurations which sequentially appear at the E2 and the El conformations of the enzymes; and correspond to the ‘regulatory’ ATP‐binding site and to the phosphorylation site and (c) the site is blocked by fluorescein isothiocyanate in both configurations.
Proton Nuclear‐Magnetic‐Resonance Spectroscopy of Myosin Subfragment 1 Isoenzymes
Authors: PRINCE H. Paul , TRAYER Hylary R. , HENRY Gillian D. , TRAYER Ian P. , DALGARNO David C. , LEVINE Barry A. , CARY Peter D. , TURNER Christopher
Published: March 2005

High‐resolution proton NMR spectrocopy has been used to study the solution structures of the subfragment 1 (S1)isoenzymes (containing either the A1 or A2 light chains) from rabbit skeletal muscle myosin and to investigate their interaction with actin. Superimposed upon broad components, the narrow signals of the S1 spectra are unexpectedly sharp, indicating that domains of varying sidechain mobility occur in the conformation adopted solution. These observations are in agreement with previous studies of the mixed isoenzymes [Highsmith et al. (1979) Biochemistry, 18, 4238 ‐42431]. Peptide amide exchange studies show also that the S1 structure accommodates fluctuations of sufficient amplitude to allow most of the peptide groups to come into contact with the solvent on the time scale‐of the 1H‐NMR experiment. The overall impression is that S1 is a molecule possessing backbone motility well as domains of different sidechain mobility.

Close comparison of the Sl(A1) and Sl(A2) spectra indicate that the N‐terminal41 residues of the A1 light chain, rich in lysine, proline and alanine, display a high degree of segmental mobility. The difference spectrum [S1(A1)‐S1(A2)] obtained closely resembles the spectral simulation of the 41‐residue segment. Upon addition of actin, many of the narrow S1 resonances decrease in intensity or progressively disappear altogether, indicative of intermediate‐ slow exchange conditions consistent with the recognised high affinity between the two proteins. These changes are interpreted as an overall modulation in the observed and hence more mobile regions of S1 as has been suggested earlier 1H‐NMR studies referred to above. In particular, the differences noted between S1(A1) and S1(A2) have now largely disappeared in their complexes with actin indicating a marked reduction in the segmental mobility of the N‐terminal region of the light chain in S1(A1). Together with other affinity chromatography results [Winstanley and Trayer (1979) Biochem. Soc. Trans. 7, 703–704], this is good evidence for a direct interaction between this area of Sl(A1) and actin.
Conformational Analysis of Peptide Substrates and Inhibitors of the Zn2+ G and Serine R61 d‐Alanyl‐d‐alanine Peptidases
Authors: DE COEN Jean‐Louis , LAMOTTE‐BRASSEUR Josette , GHUYSEN Jean‐Marie , FRÉRE Jean‐Marie , PERKINS Harold R.
Published: March 2005

The tripeptide Nα,Nɛ‐diacetyl‐l‐lysyl‐d‐alanyl‐d‐alanine (Ac2‐ l‐LLys1‐dAIa2‐dAIa3), which is the standard substrate of the Zn2+ G and serine R61 d‐alanyl‐d‐alanine peptidases, and several ldd tripeptide analogues where the size and/or the electrical charge of the side chains at position 1, 2 or 3 have been modified (alterations affecting more than one position at the same time were not investigated) have been submitted to conformational analyses based on both short‐range and long‐range interactions. Among the many backbone conformers of minimal energy of the øii space that have been characterized, four types of conformers are the most probable ones. Depending on the peptides, these conformers may have varying relative probability P values so that the leader conformer is not always the same, but, in all cases, the sum of their P values is 90% or more. With the Gly1, Gly2 or Gly3 analogues (which encompass a larger conformational space), the above ∑P values are still as high as 35–50%. All the above tripeptides bind to the serine d‐alanyl‐d‐alanine peptidase and with the exception of the Gly3 and Gly2 analogues, to the Zn2+d‐alanyl‐d‐alanine peptidase with virtually the same efficacy, at least within a range of variation of the Km values for the substrates or the Ki values for the inhibitors, which is less than one order of magnitude. Structural variations at position 1, 2 or 3 in the peptides that are compatible with efficient binding are not necessarily compatible with substrate activity, thus converting the modified peptides into competitive inhibitors. In particular, substrate activity requires a long side chain at position 1 in the peptides. Conformational analyses of Ac2‐lLys‐dAla‐dAla show that the main backbone has a tendency to adopt a ring‐like shape from which the lLYS side chain protrudes as an extended structure. This latter structure forms with the C‐terminal d‐alanyl‐d‐alanine an angle varying between 120° and 180° (depending on the conformers) so that its N‐terminal acetyl group is about 1–1.5 nm apart from the scissile amide bond. High turnover numbers (at enzyme saturation) also require a dAla at position 2 with both d‐alanyl‐d‐alanine peptidases and at position 3 in the case of the serine d‐alanyl‐d‐alanine peptidase. Finally, all the conformers of the lAla2 and lAla3 analogues of Ac2‐lLys‐dAla‐dAla fall outside the backbone conformational space that comprises the φiφi angles exhibited by the four types of conformers of the ldd tripeptides. The lAla2 and lAla3 tripeptide analogues do not bind to the serine d‐alanyl‐d‐alanine peptidase (at least at a 10 mM concentration) but they behave as noncompetitive inhibitors of the Zn2+d‐alanyl‐d‐alanine peptidase.
Glycoproteins Containing Peanu t‐Agglutinin Receptors from Human‐Peripheral‐ Blood T‐Lymphocyte Plasma Membranes
Authors: FARRAR Graham H. , HOLZ Gisela , UHLENBRUCK Gerhard
Published: March 2005

A glycoprotein fraction possessing peanut agglutinin receptors has been isolated from detergent extracts of neuraminidase‐treated human peripheral blood T‐lymphocyte plasma membranes with affinity matrices comprising the peanut agglutinin co‐valently immobilised on Sepharose 4B.

This fraction could be specifically eluted from affinity columns using buffer solutions supplemented with 0.2 M d‐galactose and was shown, by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate (0.1 %), to contain four major glycoprotein components with apparent molecular weights of 200000, 190000, 110000 and 95000.

It is suggested, from the observed reactivity of these glycoproteins with various lectins in double‐diffusion experiments, that they possess both O‐glycosidically and N‐glycosidically linked carbohydrates.
Purification and Properties of 3‐Methyladenine‐DNA Glycosylase from L‐Cells
Authors: MALE Rune , NES Ingolf F. , KLEPPE Kjell
Published: March 2005

3‐Methyladenine‐DNA glycosylase from L‐cells has been purified approximately 800‐fold. The enzyme is present primarily in the nucleus of the cells. The enzymatic reaction was sensitive to changes in the assay conditions and optimum activity was found at pH 6.5 and at 100 mM KCl. Mg2+ did not effect the enzymatic reaction, which also worked in the presence of EDTA. The activity on denatured methylated DNA was 20–40% of that of the native double‐stranded form. Sephadex gel filtration of the most purified fraction revealed enzyme species with molecular weights of 68000, 47000 and 27000, which differ from those reported for corresponding enzymes from other organisms. Addition of the product, 3‐methyladenine, to the reaction mixture resulted in inhibition of the glycosylase activity of up to 60%. The remaining activity could not be abolished by increasing the concentration of 3‐methyladenine.
Nucleotide Sequence and Secondary Structure of Citrus Exocortis and Chrysanthemum Stunt Viroid
Authors: GROSS Hans J. , KRUPP Guido , DOMDEY Horst , RABA Manfred , JANK Peter , LOSSOW Christine , ALBERTY Heidemarie , SÄNGER Heinz L. , RAMM Karla
Published: March 2005

The complete nucleotide sequence of citrus exocortis viroid (CEV, propagated in Gymura) and chrysanthemum stunt viroid (CSV, propagated in Cineraria) has been established, using labelling in vitro and direct RNA sequencing methods and a new screening procedure for the rapid selection of suitable RNA fragments from limited digests.

The covalently closed circular single‐stranded viroid RNAs consist of 371 (CEV) and 354 (CSV) nucleotides, respectively. As previously shown for potato spindle tuber viroid (PSTV, 359 nucleotides), CEV and CSV also contain a long polypurine sequence. Maximal base‐pairing of the established CEV and CSV sequences results in an extended rod‐like secondary structure similar to that previously established for PSTV and as predicted from detailed physicochemical studies of all these viroids. Although the three viroid species sequenced to date differ in size and nucleotide sequence, there is 60–73% homology between them. As PSTV, CEV and CSV also contain conserved complementary sequences which are separated from each other in the native secondary structure. We postulate that the resulting ‘secondary’ hairpins, being formed and observed in vitro during the complex process of thermal denaturation of viroid RNA, must have a vital, although yet unknown, function in vivo. The possible origin and function of viroids are discussed on the basis of the characteristic structural features and of a considerable homology with Ula RNA found for a region highly conserved in the three viroids.
Glutathione Reductase from Human Erythrocytes
Authors: KRAUTH‐SIEGEL R. Luise , SALEH Mansoor , UNTUCHT‐GRAU Renate , SCHIRMER R. Heiner , BLATTERSPIEL Rosi , SCHILTZ Emile
Published: March 2005

Sequence analysis of the NADPH domain (residues 158 – 293) and of the interface domain (365 – 478) was based on 12 CNBr fragments, which were isolated using ion‐exchange chromatography and paper methods. Fragments with more than 15 residues were digested further with trypsin and chymotrypsin. The isolated peptides were sequenced by automated solid‐phase Edman degradation. All sequenced peptides were ordered and overlapped by computerized comparisons with a complete sequence guessed from the electron density map of the protein. In the case of short CNBr fragments, this alignment was confirmed by the sequence analysis of protein fragments resulting from incomplete CNBr cleavage.

In the NADPH domain, residue 197, which is involved in an induced‐fit mechanism, was identified as a tyrosine. The structure of the NADPH domain is probably homologous with the NAD domain of lipoamide dehydrogenase and with the FAD domain of several proteins, but not with NADPH domains of known chainfold in other proteins.

The paper completes the sequence analysis of glutathione reductase so that the enzyme is now known in atomic detail. The numbering scheme of the chemically determined sequence will be used henceforth in crystallo‐ graphic studies also. As inferred from the sequence data each of the two identical chains contains 478 amino acid residues, the composition being Cys10, Asp21, Asn17, Thr31, Ser31, Glu29, Gln11, Pro24, Gly43, Ala42, Val44, Met15, He29, Leu34, Tyr13, Phe14, Lys34, His16, Argl7, and Trp3. From these data an Mr of 2 × 51600 was calculated for the FAD‐free apoenzyme and an Mr of 2 × 52400 for the holoenzyme.
Evidence for the Existence of Actomyosin ATPase in the Rat Pancreas
Authors: VANDERMEERS André , VANDERMEERS‐PIRET Marie‐Claire , HEPBURN Anne , RATHE Jean , CHRISTOPHE Jean
Published: March 2005

In a crude extract of rat pancreas, myosin was associated with a protein having the same electrophoretic mobility as actin. This myosin was purified after dissociation of the actomyosin complex with KI‐ATP. On sodium dodecyl‐sulfate/acrylamide gel electrophoresis, the isolated pancreatic myosin showed a major component of approxi‐ mately 200 kDa, and two smaller components with apparent molecular weight of 22 and 15 kDa, respectively.

This purified myosin exhibited high ATPase activity in the presence of K++ EDTA or Ca2+ and very little activity in the presence of Mg2+. (K++ EDTA)‐ATPase activity showed one pH optimum at 8.0, while Ca2+‐ATPase activity showed two pH optima at 6.0 and 9.0, respectively. (K++ EDTA)‐stimulated enzyme activity was specific for ATP whereas Ca2+‐stimulated activity showed low specificity for nucleoside triphosphates.
The Growth Inhibition in Friend Erythroleukemia Cells Induces Cycling of Preexisting Nonhistone Proteins
Authors: YANCHEVA Nina , DJONDJUROV Lalju
Published: March 2005

The present investigation performed on Friend erythroleukemia cells provides evidence that the cellular reprogramming associated with transition of the cells from the growing to the resting state is accompanied by an intranuclear cycling of preexisting proteins migrating from the cytoplasm. The model system developed for this study affords an opportunity to follow these events in a conveniently short period. The flow microfluorometric analysis revealed that, with respect to DNA content, the quiescent nuclei exhibit distribution in GI, S and G2 phases, as do the nuclei of the control, exponentially growing cells. It was found further that thc induction of quiescence arouses two waves of migration of preexisting cytoplasmic proteins toward the nucleus : an early one, during the transition to quiescence clearly showing density dependence, and a late one, in the established resting state. While the early‐cycled proteins are undetectable in mass and once they have reached the nucleus have a short life time and, thus, could be considered as regulatory molecules, the late‐cycled proteins accumulate within the nucleus and are associated most probably with structural reorganization of the resting nuclei; moreover, the late‐cycled nonhistone proteins were found localized, at least partly, in the nuclear protein matrix structure. Control experiments confirmed that the early and late‐cycled proteins have been synthesized in the cytoplasm in the proliferative state but are cycled intranuclearly only if the resting state has been induced. When finally the resting cells were stimulated to proliferate, the accumulated presynthesized and newly synthesized nonhistone proteins undergo a rapid degradation which suggests that the new cellular programme may require a complete elimination of the ‘resting’ nonhistone proteins.
Tetramethyl‐p‐phenylenediamine Oxidase of Pseudomonas aeruginosa
Authors: YANG Tsanyen
Published: March 2005

An oxidase complex has been solubilized and partially purified from the membrane particle of Pseudomonas aeruginosa grown under limited oxygen condition. The oxidase consists of two major cytochrome components, cytochrome c554 and cytochrome o (b561), with a molar ratio of about 9: 1 in terms of c‐heme to protoheme content. Ninety percent of the cytochrome c+o complex, corresponding to all of the cytochrome c554, is reducible by reduced N,N,N′,N′‐tetramethyl‐p‐phenylenediamine. This partially purified oxidase exhibited a maximal specific activity about 5 μmol O2 uptake × min−1× mg protein−1, with a Km, (of reduced N,N,N′,N′‐tetramethyl‐p‐phenyl‐ enediamine) =7.2 × 10−4 M at 30 °C. The oxidase is sensitive to KCN, NaN3 and NaNO2. Oxidation‐reduction potentiometric titration shows that cytochrome c554 has a midpoint potential of 289 mV and cytochrome o of +25 mV at pH;7.2 in the partially purified oxidase preparation. The purity of the preparation has been estimated to be about 85–90 %by gel electrophoresis.
Photooxidase System of Rhodospirillum rubrum 111. The Role of Rhodoquinone and Ubiquinone in the Activity of Preparations of Chromatophores and Photoreaction Centers
Authors: GIMENEZ‐GALLEGO Guillermo , RAMÍREZ‐PONCE M. Pilar , LAUZURICA Pilar , RAMÍREZ Juan M.
Published: March 2005

The role of rhodoquinone and ubiquinone in the oxygen photoreducing (photooxidase) activity of Rhodospirillum rubrum was investigated. The sole addition of purified rhodoquinone restored photooxidase activity in isolated chromatophores which had been extracted with organic solvents and which were apparently free of secondary acceptor ubiquinone. Rhodoquinone also enhanced photooxidase activity in photoreaction center preparations from which secondary ubiquinone seemed to have been removed. Those results suggest that rhodoquinone accepts electrons direαly from primary ubiquinone during chromatophore photooxidase activity. In contrast, rhodoquinone does not participate in the basal activity of photoreaction center preparations, which seems to result from the autooxidation of both primary and secondary ubiquinone.
Catalase is Induced by Ecdysterone and Ethanol in Drosophila Cells
Authors: BEST‐BELPOMME Martin , ROPP Michkle
Published: March 2005

Clones sensitive or resistant to ecdysterone and cultured in vitro were isolated from established cell line of Drosophila melanoguster. Ecdysterone (20‐hydroxyecdysone), a hormonal steroid of critical importance in insect physiology, induces catalase activity in the sensitive clones. This catalase induction does not occur in clones known to be resistant to the hormone. The important role of catalase as a scavenger for H2O2 in the aerobic cells (Drosophila cells in culture are consuming oxygen) led to the demonstration of catalatic properties of this induced enzyme. The peroxidatic property of catalase, useful in alcohol metabolism, was the reason for showing that ethanol can also induce catalase in Drosophila cells.
Incorporation of Deoxyribonucleosides into DNA of Coryneform Bacteria and the Relevance of Deoxyribonucleoside Kinases
Authors: AULING Georg , PRELLE Hans , DIEKMANN Hans
Published: March 2005

In order to obtain basic knowledge of the salvage pathways for DNA synthesis, the ability of Brevibacterium ammoniagenes ATCC 6872 and Micrococcus luteus ATCC 15932 for incorporation of nucleobases and nucleo–sides was investigated. Only adenine and uracil are incorporated by B. ammoniagenes, whereas M. luteus additionally can utilize deoxyadenosine and, less efficiently, thymidine.

In M. luteus, the demonstration of deoxyadenosine kinase and thymidine kinase explains the incorporation data. Uptake of thymidine is of short duration because of rapid breakdown of exogenously supplied thymidine to thymine. At a 540–fold excess pyrimidine deoxyribonucleosides inhibit 14C incorporation from thymidine nearly totally and purine deoxyribonucleosides cut by half the uptake rate, probably by interfering with transport of thymidine. However, as no cessation of thymidine incorporation occurs at these concentrations of purine deoxyribonucleosides, incorporation is finally enhanced. During the initial period of this reduced uptake considerable protection of thymidine from breakdown to thymine is provided by deoxyguanosine, but not by deoxyadenosine. At a 108–fold excess there is actually no inhibition of thymidine uptake by deoxyguanosine and only an insignificant impairment by deoxyadenosine resulting in an ultimate enhancement of 14C incorporation up to 20%of the exogenously supplied thymidine.

As there is no salvage pathway for thymidine in B. ammoniagenes due to the absence of thymidine kinase, labelling with adenine and hydrolyzing of the ‘contaminated’ RNA fraction with 1 M KOH is recommended for measurements of overall DNA synthesis in this strain.
The Composition and Properties of the Escherichia coli 5–S RNA–Protein Complex
Authors: METSPALU Ene , USTAV Mart , MAIMETS Toivo , VILLEMS Richard
Published: March 2005

At a high concentration of MgCl2 (30 mM) and a low concentration of proteins from the 50–S subunit (0.2 mg/ml), only three proteins, L15, L18 and L25, bind to 5–S RNA in significant amounts. On the other hand, in a buffer containing only 1 mM Mg C12, but otherwise at the same ionic strength (0.2 M), or at a protein concentration about 1.5 mg/ml, a large, stable complex can form between immobilized 5–S RNA and 50–S ribosomal proteins. This complex contains proteins L2, L3, L5, L15, L16, L17, L18, L21, L22, L25, L33 and L34, and it possesses properties relevant to the function of the 50–S subunit: it has a binding site for deacylated tRNA, with a dissociation constant of 4.5 × 10−7 M. The complex formed with 5–S RNA immobilized on an affinity column interacts also with 30–S subunits. The 5–S RNA–protein complex is interpreted as a sub–ribosomal domain which includes a considerable fraction of the peptidyl transferase center of the Escherichiu coli ribosome.
The Substrate Binding Site of Aldehyde Reductase from Pig Liver
Authors: BRANLANT Guy
Published: March 2005

Aldehyde reductase I from pig liver is strongly inhibited by cyclized NADP–2‐oxodiacid adducts. This result, in conjunction with those showing a strong inhibitory effect of certain diacid derivatives, such as (±)‐ dimethylsuccinic acid, towards aldehyde reductase I, suggests the presence of two anion sites in or near the substrate binding site. As previously shown by our group [Eur. J. Biochem. 116, 505–512 (1981)], at least one of the two anion sites is an arginine residue. The fact that (2R,3R)‐dimethylsuccinic acid is bound more tightly to aldehyde reductase I than the meso and (2S,3S)‐dimethylsuccinic acids is indicative of a particular spatial orientation of the two anion sites relative to those of the coenzyme and substrate binding sites of the reductase. This result also supports the idea that one of the two diastereomers of the cyclized adducts, corresponding to the A‐side addition product, should exert a stronger inhibitory effect on aldehyde reductase than the other. On the basis of the NMR study made recently by L. J. Arnold et al. [Biochemislry, 18, 2787–2793 (1979)], who determined the conformation and configuration of cyclized pyridine nucleotide adducts, a tentative design of the hydrophobic pocket of the substrate binding site of aldehyde reductase I, containing the two anion sites, is presented.
The Role of Biotin and Sodium in the Decarboxylation of Oxaloacetate by the Membrane‐Bound Oxaloacetate Decarboxylase from Klebsiella aerogenes
Authors: DIMROTH Peter
Published: March 2005

The biotin‐containing oxaloacetate decarboxylase from Klebsiella aerogenes catalyzed the Na+‐dependent decarboxylation of oxaloacetate to pyruvate and bicarbonate (or CO2) but not the reversal of this reaction, not even in the presence of an oxaloacetate trapping system.

The enzyme catalyzed an avidin‐sensitive isotopic exchange between [1‐14C]pyruvate and oxaloacetate, which indicated the intermediate formation of a carboxybiotin enzyme. Sodium ions were not required for this partial reaction, but promoted the second partial reaction, the decarboxylation of the carboxybiotin enzyme, thus accounting for the Na+ requirement of the overall reaction. Therefore, the 14CO2‐enzyme which was formed upon incubation of the decarboxylase with [4‐14C]oxaloacetate, could only be isolated if Na+ ions were excluded. Preincubation of the decarboxylase with avidin also prevented its labelling with 14CO2.

The isolated 14CO2‐labelled oxaloacetate decarboxylase revealed the following properties. It was slowly decarboxylated at neutral pH and rapidly upon acidification. The 14CO2 residues of the 14CO2‐enzyme could be transferred to pyruvate yielding [4‐14C]oxaloacetate. In the presence of Na+ this 14CO2 transfer was repressed by the simultaneous decarboxylation of the 14CO2‐enzyme. However, Na+ alone was insufficient as a cofactor for the decarboxylation of the isolated 14CO2‐enzyme, since this required pyruvate in addition to Na+. It is therefore concluded that the decarboxylation of oxaloacetate proceeds over a CO2‐enzyme‐pyruvate complex and that free CO2‐enzyme is an abortive reaction intermediate.

The activation energy of the enzymic decarboxylation of oxaloacetate changed with temperature and was about 113 kJ below 11 °C, 60 kJ between 11 °C and 31 °C and 36 kJ between 31–45 °C.
The Generation of an Electrochemical Gradient of Sodium Ions upon Decarboxylation of Oxaloacetate by the Membrane–Bound and Na+–Activated Oxaloacetate Decarboxylase from Klebsiella aerogenes
Authors: DIMROTH Peter
Published: March 2005

Inverted membrane vesicles from Klebsiella arragenes containing the Na+‐activated oxaloacctate decarboxylase accumulated 22Na+ in response to the decarboxylation of oxaloacetate. Complexation of the biotin prosthetic group of the decarboxylase with avidin completely prevented the oxaloacetate‐dependent Na+ uptake, whereas avidin saturated with biotin was without effect.

The rapid accumulation of 22Na+ inside the vesicles following oxaloacetate addition was succeeded by a slower efflux of 22Na+ after exhaustion of oxaloacetate. Other substrates, e.g. d‐lactate, were unable to replace oxaloacetate in accumulating 22Na+ to a comparable extent, with the exception of (S)‐malate, which acted through its conversion to oxaloacetate. The amount of Na+ which was transported into the vesicles by a limited amount of oxaloacetate critically depended on the rate of oxaloacetate decarboxylation. This rate was retarded by using a low temperature (0 °C), by inhibiting the enzyme with oxalatc or by using oxaloacetate‐generating systems, where the activity of the enzyme catalyzing oxaloacetate formation was rate‐determining. With these retarded systems of oxaloacetate decarboxylation the efficiency of the Na+ transport was significantly increased. Under optimized conditions Na+could be concentrated inside the inverted vesicles eight‐times higher than in the incubation medium.

The Na+‐carrying ionophores nigericin and trinactin effectively prevented the oxaloacetate‐dependent Na+ accumulation in a K+‐free incubation medium. In the presence of valinomycin and K+, however, the amount of Na+ which was taken up by the vesicles following oxaloacetate addition increased by a factor of two, which indicated limitation of Na+ uptake by electrical parameters. The generation of a membrane potential was determined from the distribution of the lipophilic anion [14C]SCN‐between interior and exterior vesicular spaces. The oxaloacetate‐decarboxylase‐driven Na+ transport into inverted vesicles at 0°C generated a membrane potential of 65 mV, an Na+ gradient equivalent to 49 mV and a total driving force of 114 mV. The proton carrier carbonylcyanide‐p‐trifluoromethoxy phenylhydrazone had essentially no effect on the oxaloacetate‐de‐carboxylase‐driven Na+ uptake, indicating that thc gcneration of a proton gradicnt is not involved. It is concluded from these results that oxaloacetate decarboxylase acts as a primary active Na+ pump which generates an electrochemical gradient of Na+ ions.