bluuebunny/crossref_metadata_2025_split huggingface dataset:
Purification, characterization and revised amino acid sequence of a second thioredoxin from Corynebacterium nephridii
Authors: McFARLAN Sara C. , HOGENKAMP Harry P. C. , ECCLESTON Eric D. , HOWARD James B. , FUCHS James A. 
Published: March 2005

A second thioredoxin, distinct from the one reported by Meng and Hogenkamp in 1981 (J. Biol. Chem. 256, 9174–9182), has been purified to homogeneity from an Escherichia coli strain containing a plasmid encoding a Corynebacterium nephridii thioredoxin. Thioredoxin genes from C. nephridii were cloned into the plasmid pUC13 and transformants were identified by complementation of a thioredoxin negative (trxA−) E. coli strain. The abilities of the transformants to support the growth of several phages suggested that more than one thioredoxin had been expressed [Lim et al. (1987) J. Biol. Chem. 262, 12114–12119]. In this paper we present the purification and characterization of one of these thioredoxins.The new thioredoxin from C. nephridii, designated thioredoxin C‐2, is a heat‐stable protein containing three cysteine residues/molecule. It serves as a substrate for C. nephridii thioredoxin reductase and E. coli and Lactobacillus leichmannii ribonucleotide reductases. Thioredoxin C‐2 catalyzes the reduction of insulin disulfides by dithiothreitol or by NADPH and thioredoxin reductase and is a hydrogen donor for the methionine sulfoxide reductase of E. coli. Spinach malate dehydrogenase (NADP+) and phosphoribulokinase are activated by this thioredoxin while glyceraldehyde‐3‐phosphate dehydrogenase (NADP+) is not. Like the thioredoxin first isolated from C. nephridii, this new thioredoxin is not a reducing substrate for the C. nephridii ribonucleotide reductase. The complete primary sequence of this second thioredoxin has been determined. The amino acid sequence shows a high degree of similarity with other thioredoxins. Surprisingly, in contrast to the other sequences, this new thioredoxin contains the tetrapeptide ‐Cys‐Ala‐Pro‐Cys‐ at the active site. With the exception of the T4 thioredoxin, this is the first example of a thioredoxin that does not have the sequence ‐Cys‐Gly‐Pro‐Cys‐. Our results suggest that, like plant cells, bacterial cells may utilize more than one thioredoxin.
CF0, the proton channel of chloroplast ATP synthase
Authors: LILL Holger , JUNGE Wolfgang 
Published: March 2005

The discharge of the flash‐induced transmembrane voltage through the exposed proton channel, CF0, of the chloroplast ATP synthase, CF0CF1 was investigated. EDTA treatment of thylakoid membranes exposed approximately 50% of total CF0 by removal of the CF1 counterparts. This greatly accelerated the decay of the transmembrane voltage, as was apparent from electrochromic‐absorption changes of intrinsic pigments and by pH‐indicating‐absorption changes of added dyes. Two decay processes were discernible, one rapid with a typical half‐decay time of 2 ms, and a slower one with a half‐decay time variable between 20 – 100 ms. Both were sensitive to CF0 inhibitors, but only the rapid decay process was also inhibited by added CF1. CF1 was effective in surprisingly small amounts, which were significantly lower than those previously removed by EDTA treatment. This finding corroborated our previous conclusion that the rapid decay of the transmembrane voltage was attributable to only a few high‐conductance channels among many CF0 molecules, typically in the order of one channel CF1‐depleted EDTA vesicle.Inhibition of photophosphorylation in control thylakoids was measured as function of the concentration of CF0 inhibitors. It was compared with the inhibition of proton conduction through exposed CF0 in EDTA vesicles. Photophosphorylation and proton conduction by the high‐conductance form of CF0 were inhibited by the same low inhibitor concentrations. This suggested that the high‐conducting form of CF0 with a time‐averaged single‐channel conductance of 1 pS [Lill, H., Althoff, G. & Junge, W. (1987) J. Membrane Biol. 98, 69–78] represented the proton channel in the integral enzyme, which acted as a low‐impedance access from the thylakoid lumen to the coupling site in CF0CF1.The slow decay process was attributed to a majority of low‐conductance CF0 channels, i. e. about 50 molecules/vesicle. The conductance of these channels was more than 100‐fold lower and they did not compete with the very few highly conducting channels for rebinding of added CF1. The low proton conduction of the majority of exposed CF0 molecules, possibly due to a structural rearrangement, may be protecting the thylakoid membrane against rapid energy dissipation caused by accidental loss of CF1. It may also explain the low single‐channel conductance of bacterial F0 reported in the literature.
Comparative characterization of membrane‐associated and cytosolic Tyr‐protein kinases in human erythrocytes
Authors: CLARI Giulio , MORET Vittorio 
Published: March 2005

In recent years, two protein‐tyrosine kinase activities, phosphorylating tyrosine residues on the transmembrane band‐3 protein, have been isolated from human erythrocyte membranes and partially characterized by different laboratories, i. e. one extracted by non‐ionic detergent (Triton X‐100 or Nonidet P‐40), the other solubilized by 0.25 M NaCl from the detergent‐insoluble residue.The present paper shows that these two membrane‐associated Tyr‐protein kinases purified, in the presence of bovine serum albumin, by phosphocellulose chromatography followed by heparin‐Sepharose chromatography, have the same apparent molecular mass (36 kDa) determined by Ultrogel Ac44 filtration. Moreover, both Tyrprotein kinases exhibit several identical properties, including Km values for band 3, the random acidic copolymer poly(Glu, Tyr)4:1 and angiotensin II, pH dependence, response to Mn2+ and Mg2+, response to NaCl and 2,3‐bisphosphoglycerate.All these properties are identical or very similar to those exhibited by the Tyr‐protein kinase previously isolated by us from human erythrocyte cytosol.These results suggest that the two membrane‐associated and the cytosolic Tyr‐protein kinase activities are mediated by the same enzyme, distributed between the cytosol and the membrane structures.
The primary structure of the Chloroflexus aurantiacus reaction‐center polypeptides
Authors: SHIOZAWA Judith A. , LOTTSPEICH Friedrich , OESTERHELT Dieter , FEICK Reiner 
Published: March 2005

The complete nucleotide sequence of two Chloroflexus aurantiacus reaction‐center genes has been obtained.The amino acid sequence deduced from the first gene showed 40% similarity to the L subunit of the Rhodobacter sphaeroides reaction center. This L subunit was 310 amino acids long and had an approximate molecular mass of 35 kDa. The second gene began 17 bases downstream from the first gene. The amino acid sequence deduced from it (307 amino acids; 34950 Da) was 42% similar to the M subunit of the Rhodobacter sphaeroides reaction center. 20% of the deduced primary structure were confirmed through automated Edman degradation of cyanogen bromide peptide fragments or N‐chlorosuccinimide peptide fragments isolated from the purified reaction‐center complex or from the individual subunits. The peptides were isolated by preparative gel electrophoresis combined with molecular sieve chromatography in the presence of a mixture of formic acid, acetonitrile, 2‐propanol and water. This method appeared to be applicable to the isolation of other hydrophobic proteins and their peptides.
Structural studies on the fucosamine‐containing O‐specific polysaccharide of Proteus vulgaris O19
Authors: VINOGRADOV Evgeny V. , KACA Wieslaw , KNIREL Yuriy A. , RÓZLSKI Antoni , KOCHETKOV Nikolay K. 
Published: March 2005

The polysaccharide chain of Proteus vulgaris O19 lipoppolysaccharide contains D‐galactose, N‐acetyl‐D‐glucosamine, N‐acetyl‐D‐galactosamine and N‐acetyl‐L‐fucosamine in the ratio 1:1:1:1. The structure of the polysaccharide was established by full acid hydrolysis and methylation analysis, as well as by non‐destructive methods. i.e. the computer‐assisted evaluation of the 13C‐NMR spectrum and computer‐assisted evaluation of the specific optical rotation by Klyne's rule. The polysaccharide is regular and built up of tetrasaccharide repeating units of the following structure: →3)‐α‐L‐FucNAcp‐(1→3)‐β‐D‐GlcNAcp‐(1→3)‐α‐D‐Galp‐(1→4)‐α‐D‐GalNAcp‐(1→The O19‐antiserum cross‐reacts with lipopolysaccharide from P. vulgaris O42, the structure of which is still unknown. No cross‐reactions were observed with O‐polysaccharides Pseudomonas aeruginosa O7 and Salmonella arizonae O59 in spite of some structural similarities.
Zonal distribution of fatty acid synthase in liver parenchyma of male and female rats
Authors: KATZ Norbert , THIELE Jutta , GIFFHORN‐KATZ Susanne 
Published: March 2005

1. A sensitive radiochemical assay was established to determine the activity of fatty acid synthase in microdissected liver tissue of less than 1 μ dry mass.2. In female rats, the enzyme activity in perivenous tissue was twice that in periportal liver tissue while it was homogeneously distributed in livers of male animals. The overall activity was higher in female than in male animals.3. The absolute activity, as well as the perivenous/periportal ratio, was reduced during starvation and in diabetes. They were greatly increased after refeeding to values above those observed in animals during normal feeding.4. Overectomy or administration of testosterone to female rats resulted in a significant reduction of the zonal heterogeneity.5. Castration of administration of estradiol to male animals was followed by an increase in the enzyme activity exclusively in the perivenous tissue, resulting in a zonal heterogeneity as observed in female rats.
Purification and characterization of 6‐pyruvoyl tetrahydropterin synthase from salmon liver
Authors: HASLER Thomas , CURTIUS Hans‐Christoph 
Published: March 2005

Salmon liver was chosen for the isolation of 6‐pyruvoyl tetrahydropterin synthase, one of the enzymes involved in tetrahydrobiopterin biosynthesis. A 9500‐fold purification was obtained and the purified enzyme showed two single bands of 16 and 17 kDa on SDS/PAGE. The native enzyme (68 kDa) consists of four subunits and needs free thiol groups for enzymatic activity as was shown by reacting the enzyme with the fluorescent thiol reagent N‐(7‐dimethylamino‐4‐methylcoumarinyl)‐maleimide. The enzyme is heat‐stable up to 80°C, has an isoelectric point of 6.0–6.3, and a pH optimum at 7.5. The enzyme is Mg2+‐dependent and has a Michaelis constant for its substrate dihydroneopterin triphosphate of 2.2 μ. The turnover number of the purified salmon liver enzyme is about 50 times as high as that of the enzyme purified from human liver. It does not bind to the lectin concanavalin A, indicating that it is free of mannose and glucose residues.Polyclonal antibodies raised against the purified enzyme in Balb/c mice were able to immunoprecipitate enzyme activity. The same polyclonal serum was not able to immunoprecipitate enzyme activity of human liver 6‐pyruvoyl tetrahydropterin synthase, nor was any cross‐reaction in ELISA tests seen.
Trypanothione reductase from Trypanosoma cruzi
Authors: JOCKERS‐SCHERÜBL Maria C. , SCHIRMER R. Heiner , KRAUTH‐SIEGEL R. Luise 
Published: March 2005

Tryanothione reductase of Trypanosoma cruzi is a key enzyme in the antioxidant metabolism of the parasite. Here we report on the enzymic and pharmacological properties of trypanothione reductase using glutathionylspermidine disulfide as a substrate.

Both pH optimum (7.5) and the ionic strength optimum (at 30 mM) are unusually narrow for this enzyme. 40 mM Hepes, 1 mM EDTA, pH 7.5 was chosen as a standard assay buffer because in this system the kcat/Km ratio had the highest values for both natural substrates, glutathionylspermidine disulfide (2.65 × 106 M–1 s–1) and trypanothione disulfide (4.63 × 106 M–1 s–1).
Using the standardized assay, trypanothione reductase and the phylogenetically related host enzyme, human glutathione reductase, were studied as targets of inhibitors. Both enzymes, in their NADPH‐reduced forms, were irreversibly modified by the cytostatic agent, 1,3‐bis(2‐chloreethyl)‐1‐nitrosourea (BCNU). Nifurtimox, the drug used in the treatment of Chagas' disease, is a stronger inhibitor of glutathione reductase (Ki= 40μM) than of trypanothione reductase (IC50= 200 μM).
Of the newly synthesized trypanocidal compounds [Henderson, G. B., Ulrich, P., Fairlamb, A. H., Rosenberg, I., Pereira, M., Sela, M. & Cerami, A. (1988) Proc. Natl Acad. Sci., 85, 5374–5378] a nitrofuran derivative, 2‐(5‐nitro‐2‐furanylmethylidene)‐N,N′ ‐ [1,4‐piperazinediylbis (1,3‐propanediyl)]bishydrazinecarboximidamide tetrahydrobromide, was found to be a better inhibitor for trypanothione reductase (Ki= 0.5 μM) than for glutathione reductase (IC50= 10μM). A naphthoquinone derivative, 2,3‐bis[3‐(2‐amidinohydrazono)‐butyl]‐1,4‐naphthoquinone dihydrochloride, turned out to be both an inhibitor (IC50= 1 μM) and an NADPH‐oxidation‐inducing substrate (Km= 14 μM). This effect was not observed with human glutathione reductase. Such compounds which lead to oxidative stress by more than one mechanism in the parasite are promising starting points for drug design based on the three‐dimensional structures of glutathione and trypanothione reductases.
Mutant proteins of human interleukin 2
Authors: WEIGEL Ulrich , MEYER Marita , SEBALD Walter 
Published: March 2005

Muteins, i.e. proteins altered by mutation of their genes, of interleukin 2 (I12) were generated by oligonucleotide‐directed mutagenesis in vitro. All acidic and basic residues conserved between man and mouse were exchanged as well as four lipophilic residues contained within four hydrophobic segments of the protein. The muteins were produced in Escherichia coli and submitted to a renaturation and purification protocol, before bioactivity and receptor binding of each of them was determined.All muteins besides two (K44/T125 and Q110/T125) could be renatured and purified. One mutein (K94/T125) exhibited a more than tenfold‐improved renaturation yield.One amino exchange (Asp‐20 to Asn) resulted in an about 20‐fold reduction in proliferative activity and high‐affinity receptor binding. The binding to the low‐affinity I12‐binding protein (Tac antigen) was unimpaired.A second exchange (Arg‐38 to Gln) had no effect on proliferative activity. The binding to both the high‐ and the low‐affinity receptor, however, was reduced about 20‐fold.Preliminary trials on the stability of these muteins by guanidinium hydrochloride denaturation studies detected no differences between wild‐type interleukin 2 and muteins.It is suggested that Asp‐20 forms part of the binding site for the large receptor subunit whereas Arg‐38 is involved in the contact site to the small subunit.
A human melanoma‐derived cell line (IGR39) with a very high number of vasoactive‐intestinal‐peptide (VIP) receptors
Authors: MARTIN Jean‐Michel , LUIS José , MARVALDI Jacques , PICHON Jacques , PIC Pierre 
Published: March 2005

Vasoactive intestinal peptide (VIP) stimulated in a dose‐dependent manner the accumulation of cAMP in human melanoma‐derived cell line IGR39. The maximal effect (about 100 times the basal level) was observed with 10 nM VIP. Half‐maximum cAMP production was obtained at 0.78 nM VIP. VIP‐related peptides were also potent in stimulating the cAMP production in IGR39 cells. The order of potency was VIP ≫ peptide histidine–methioninamide > human growth‐hormone‐releasing factor(1–44) > secretin > glucagon. Using the same conditions, IGR37 cells, a metastasic counterpart of IGR39 cells, displayed a weak stimulation of cAMP production.After exposure of IGR39 cells to 10 nM VIP, the cAMP response to a new stimulation by VIP was strongly reduced. This desensitization of IGR39 cells to VIP was rapid (t1/2 < 2 min) and homologous.Preincubation of IGR39 cells in the presence of native VIP induced disappearance of the VIP‐binding sites at the cell surface. This phenomenon was dependent on time and VIP concentration. Maximum effect (loss of 80% of binding capacity) was obtained after exposure of the cells at 37°C with a VIP concentration of 1 μM. The t1/2 of maximum disappearance was less than 2 min and the concentration of VIP giving half‐maximum decrease in binding of mono[125I]iodinated VIP (125I‐VIP) was 8 nM. This phenomenon was also reversible since 85% of the VIP‐binding capacity could be restored in less than 1 h by incubating IGR39 cells in a VIP‐free medium.The IGR39 cell line should be a useful model for further study of the structure and function of the human VIP receptor.
Specific dephosphorylation by phosphatases 1 and 2A of a nuclear protein structurally and immunologically related to nucleolin
Authors: SCHNEIDER Helge R. , MIESKES Gottfried , ISSINGER Olaf‐Georg 
Published: March 2005

A new nuclear substrate (N‐60) for phosphatase 1 and 2Ac has been described. In contrast to nucleolin (C23), to which it is structurally and immunologically related, N‐60 becomes dephosphorylated to 51% and 41% by phosphatases 1 and 2Ac, respectively, within 10 min.Incubation up to 20 min led to a complete dephosphorylation of N‐60. The two other phosphatases tested (2B and 2C) did not dephosphorylate protein N‐60 to the same extent as phosphatases 1 and 2Ac.In the case of nucleolin only 18% phosphate was released by all four phosphatases tested. The activity of both phosphatases, 1 and 2A, could be blocked by tumour promoter okadaic acid (100 nM) when N‐60 was used as a substrate. These results support the notion that the observed okadaic‐acid‐induced hyperphosphorylation of N‐60 in intact human fibroblasts may be caused by specific inhibition of phosphatases involved in the process of rDNA transcription.
The glycogen‐binding subunit of protein phosphatase‐1g from rabbit skeletal muscle
Authors: HUBBARD Michael J. , COHEN Philip 
Published: March 2005

The glycogen‐bound form of protein phosphatase‐1 (PP‐1g) was previously purified as a heterodimer composed of a 37‐kDa catalytic (C) subunit and a proteolytically sensitive 103‐kDa glycogen‐binding (G) subunit [Stråhlfors, P., Hiraga, A. & Cohen, P. (1985) Eur. J. Biochem. 149, 295–303]. In this paper we demonstrate by a variety of criteria that the intact G subunit is a 161‐kDa protein, and that the 103‐kDa species (now termed G′) is itself a product of proteolysis.A second phosphorylation site for cAMP‐dependent protein kinase (termed site 2) was identified on the G subunit. The site 2 serine was phosphorylated at a comparable rate to site 1, and near stoichiometric phosphorylation could be achieved in the presence and absence of glycogen. Site 2 was dephosphorylated by PP‐1 at a slow rate, whereas site 1 was resistant to autodephosphorylation.PP‐1G, as well as the proteolytic activity responsible for degradation of the G subunit, remained tightly associated with glycogen‐protein particles during washing with a variety of solvents. The PP‐1G holoenzyme was released from glycogen‐protein particles by dilution, with a dissociation half point corresponding to about 10 nM PP‐1G. Binding experiments with purified PP‐1G. Binding was not significantly affected by increasing ionic strength to 0.5 M or variation of pH from 6 to 8. The results are consistent with a high‐affinity glycogen‐binding domain on the G subunit, and indicate that at physiological concentrations of phosphatase and glycogen, PP‐1G should be almost entirely bound to glycogen.
Dephosphorylation of the human T lymphocyte CD3 antigen
Authors: ALEXANDER Denis , GORIS Jozef , MARAIS Richard , ROTHBARD Jonathan , MERLEVEDE Wilfried , CRUMPTON Michael J. 
Published: March 2005

Previous studies demonstrated that activation of T lymphocytes by phorbol ester or mitogenic lectin leads to phosphorylation of Ser 126 of the CD3 antigen γ chain, whereas treatment with ionomycin results in phosphorylation of both Ser 123 and 126 [Davies, A. A. et al. (1987) J. Biol. Chem. 262, 10918 – 10921]. In the present study, the dephosphorylation of Ser 123 and Ser 126 of the γ chain was investigated. Phorbol‐ester‐induced phosphorylation of the γ‐chain Ser 126 in vivo was reversed following removal of phorbol ester. Dephosphorylation of both Ser 123 and 126 was also observed in vitro using the microsome fraction of T lymphocytes. In order to identify the phosphatases acting at these two sites, the immunoprecipitated γ chain was used as substrate either following treatment with protein kinase C in vitro, in which case phosphorylation occurs mainly at Ser 123, or following in vivo phosphorylation of Ser 126. Purified oligomeric forms of the polycation‐stimulated phosphatases were more effective in dephosphorylating both phosphorylated forms of the γ chain compared with equivalent amounts of ATP, Mg2+ ‐dependent phosphatases or calcineurin. By using phosphopeptide analogues of the CD3 γ chain containing Ser 123 or Ser 126 as substrates (A3 and A6), it was shown that polycation‐stimulated phosphatases selectively dephosphorylated Ser 123 compared to Ser 126.In order to determine which phosphatases dephosphorylate the γ chain in microsomes, A3 and A6 were used as substrates for characterising phosphatases in microsomes from human T leukaemia Jurkat 6 cells. Three phosphopeptide phosphatases (250—400 kDa) co‐eluted through five purification steps with three forms of polycation‐stimulated phosphorylase phosphatase. The partially purified A3/A6 phosphopeptide phosphatases were insensitive to Ca2+, calmodulin and inhibitor‐1, and dephosphorylated A3 preferentially compared with A6. A latent form of microsomal ATP, Mg2+ ‐dependent phosphorylase phosphatase was stimulated 10‐fold by trypsinisation, but did not dephosphorylate phosphopeptides A3 and A6. The results show that high‐Mr forms of polycation‐stimulated phosphatases are the only enzymes in human T leukaemia cell microsomes which dephosphorylate γ chain phosphopeptides.The data point to an important role for polycation‐stimulated phosphatases in regulating the phosphorylation state, and so function(s), of the CD3 antigen.
Positive and negative regulatory elements control the expression of the UGA4 gene coding for the inducible 4‐aminobutyric‐acid‐specific permease in Saccharomyces cerevisiae
Authors: VISSERS Stephan , ANDRE Bruno , MUYLDERMANS Francine , GRENSON Marcelle 
Published: March 2005

In Saccharomyces cerevisiae, the pathway of 4‐aminobutyric acid catabolism, for use as a nitrogen source, involves a specific permease (encoded by the UGA4 gene) and two enzymes (encoded by the UGA1 and UGA2 genes, respectively). The synthesis of these proteins is induced by 4‐aminobutyrate. It also requires the product of the UGA3 gene. Here, we describe four additional regulatory mutations which provide evidence for the existence of both positive and negative regulatory elements which control the final expression of the UGA4 gene. Some of them simultaneously control the expression of the UGA1 and UGA2 genes. Three classes of mutant with a constitutive 4‐aminobutyrate‐specific permease have been isolated. (a) Recessive mutations in the UGA43 gene suggest that the product of the UGA43 gene behaves like a trans‐acting negative regulator of UGA4 gene expression. (b) The semi‐dominant mutation (uga11), closely linked to the UGA4 gene, might affect the receptor of the UGA43 gene product. In these two classes of mutant, only the permease is constitutive. (3) The uga81 mutation, closely linked to the UGA3 gene. makes the whole UGA regulon constitutive.On the other hand, recessive mutations at the UGA3 gene locus lead to non‐inducibility of the UGA regulon. Hence the UGA35 gene product behaves like a second trans‐acting positive regulator in addition to UGA3.