bluuebunny/crossref_metadata_2025 huggingface dataset:
Cell‐free‐synthesized interleukin‐6 (BSF‐2/IFN‐β2) exhibits hepatocyte‐stimulating activity
Authors: GEIGER Thomas , ANDUS Tilo , BAUER Joachim , NORTHOFF Hinnak , GANTER Ursula , HIRANO Toshio , KISHIMOTO Tadamitsu , HEINRICH Peter C.
Published: March 2005

Secretory products of cultured human blood monocytes contain a hepatocyte‐stimulating factor which is able to induce the acute‐phase proteins α2‐macroglobulin and fibrinogen in rat liver cells. Total RNA was isolated from unstimulated and lipopolysaccharide‐stimulated human monocytes and translated in a reticulocyte lysate. The capability of the cell‐free synthesized proteins to induce the acute‐phase proteins α2‐macroglobulin and fibrinogen was assayed in rat hepatocyte primary cultures and in the rat hepatoma cell line Fao. The products translated from the mRNA of lipopolysaccharide‐stimulated human monocytes induced mRNAs for α2‐macro‐globulin and fibrinogen and therefore contain hepatocyte‐stimulating factor. The translation products of unstimulated monocytes had no effect.

A cDNA containing the coding sequence for interleukin‐6 (B‐cell stimulatory factor 2, interferon‐β2/26‐kDa protein, interleukin HP1) derived from human T‐cells cloned into the transcription vector pGEM4 was transcribed in vitro. Translation of the isolated RNA in a reticulocyte lysate led to the synthesis of a protein of about 25 kDa. This cell‐free synthesized interleukin‐6 exhibited hepatocyte‐stimulating activity measured by the induction of β‐fibrinogen mRNA in Fao cells. Using an antibody against interleukin‐6, two proteins of 22 kDa and 23 kDa were immunoprecipitated from the culture medium of lipopolysaccharide‐stimulated human monocytes. These two proteins were not synthesized by unstimulated monocytes.

When total RNA from unstimulated human monocytes and lipopolysaccharide‐stimulated human monocytes and lymphocytes was subjected to Northern analysis and hybridized with the interleukin‐6 cDNA, a strong hybridization signal corresponding to an RNA of about 1300 bases was detected only in the RNA from lipopolysaccharide‐stimulated human monocytes, indicating that human monocytes express the interleukin‐6 gene after stimulation. The data presented in this paper strongly suggest that hepatocyte‐stimulating factor from human monocytes and interleukin‐6 from T‐cells are identical.
Stimulation of uracil nucleotide synthesis in mouse liver, intestine and kidney by ammonium chloride infusion
Authors: ZAHAREVITZ Daniel W. , NAPIER Elizabeth A. , ANDERSON Lawrence W. , STRONG John M. , CYSYK Richard L.
Published: March 2005

De novo pyrimidine synthesis was studied in mouse liver, intestine, and kidney by intraperitoneal infusion of 15NH4C1 and analysis of 15N incorporation into uracil nucleotide pools. When the dose of a 1‐h infusion of 15NH4C1 was increased from 50 μmol to 250 μmol the fraction of the total uracil nucleotide pool formed by de novo synthesis increased 4.0‐fold in liver to 8.4% and 2.3‐fold in intestine to 13.7%. The increase in intestine was independent of the increase in liver as evidenced by the lack of correlation between the increase observed in the intestine and liver of the same animal and the different distributions of label in the uracil ring nitrogens. A 2.4‐fold increase in newly formed uracil nucleotides was observed in kidney when the infusion dose was raised from 150 μmol to 250 μmol. The increase in kidney was correlated with the increase in liver in the same animal and the distribution of label in the uracil ring nitrogens was similar to the distribution in liver. These results suggest that the increase in newly formed uracil nucleotides in intestine is due to increased de novo synthesis of pyrimidines in the intestine, while the increase in the kidney is due to increased salvage synthesis of uracil nucleotides from uridine synthesized in the liver and output to the circulation.
Import of proteins into mitochondria: a multi‐step process
Authors: PFANNER Nikolaus , HARTL Franz‐Ulrich , NEUPERT Walter
Published: March 2005

Translocation of precursor proteins from the cytosol into mitochondria is a multi‐step process. The generation of translocation intermediates, i.e. the reversible accumulation of precursors at distinct stages of their import pathway into mitochondria (‘translocation arrest’), has allowed the experimental characterization of distinct functional steps of protein import. These steps include: ATP‐dependent unfolding of precursors; specific recognition of precursors by distinct receptors on the mitochondrial surface; interaction of precursors with a general insertion protein (‘GIP’) in the outer mitochondrial membrane; membrane‐potential‐dependent translocation into the inner membrane at contact sites between both membranes; proteolytic processing of precursors; and intramitochondrial sorting of precursors via the matrix space (‘conservative sorting’). The functional characteristics unveiled by studying mitochondrial protein import appear to be of general interest for investigations on intracellular protein sorting.
Immunological and biochemical studies on the relationship between two actin‐binding proteins, phosphofructokinase and gelsolin
Authors: BUSS Folma , HINSSEN Horst , JOCKUSCH Brigitte M.
Published: March 2005

Phosphofructokinase and gelsolin‐like proteins coexist in many muscle and non‐muscle tissues. They are both actin‐binding proteins, and some of their biochemical parameters are remarkably similar. In a previous report [Füchtbauer, A., Jockusch, B. M., Leberer, E. & Pette, D. (1986) Proc. Natl Acad. Sci. USA 83, 9502–9506] it was shown that phosphofructokinase preparations contained actin‐filament‐severing activities characteristic for gelsolin, Therefore, we investigated a possible relationship between these proteins with respect to their actin‐binding properties. Immunoblotting experiments with specific and non‐cross‐reacting antibodies to both proteins revealed two distinct polypeptides with slightly different molecular mass in SDS‐PAGE of crude extracts from rabbit skeletal muscle, indicating that phosphofructokinase and gelsolin are not identical. An actin‐filament‐severing activity as well as the component detected by anti‐gelsolin were found to copurify with phosphofructokinase during its preparation. However, the presumptive gelsolin was completely eliminated after a heat‐denaturation step leaving the phosphofructokinase activity unaffected. Purified phosphofructokinase had no effects on the polymer state of preformed actin filaments. Unlike gelsolin, phosphofructokinase did not promote nucleation of actin polymerization but delayed the nucleation step. We therefore conclude that phosphofructokinase and gelsolin are functionally and structurally distinct proteins.
Assembly of asparagine‐linked oligosaccharides in baby hamster kidney cells treated with castanospermine, an inhibitor of processing glucosidases
Authors: FODDY Louise , HUGHES R. Colin
Published: March 2005

We have shown previously that the processing of asparagine‐linked oligosaccharides in baby hamster kidney (BHK) cells is blocked only partially by the glucosidase inhibitors, 1‐deoxynojirimycin and N‐methyl‐l‐deoxynojirimycin [Hughes, R. C., Foddy, L. & Bause, E. (1987) Biochem. J. 247, 537–5441. Similar results are now reported for castanospermine, another inhibitor of processing glucosidases, and a detailed study of oligosaccharide processing in the inhibited cells is reported. In steady‐state conditions the major endo‐H‐released oligosaccharides contained glucose residues but non‐glycosylated oligosaccharides, including Man9GlcNAc to Man5GlcNAc, were also present. To determine the processing sequences occurring in the presence of castanospermine, BHK cells were pulse‐labelled for various times with [3H]mannose and the oligosaccharide intermediates, isolated by gel filtration and paper chromatography, characterized by acetolysis and sensitivity to jack bean α‐mannosidase. The data show that Glc3Man9GlcNAc2 is transferred to protein and undergoes processing to produce Glc3Man8GlcNAc2 and Glc3Man7GlcNAc2 as major species as well as a smaller amount of Man9GlcNAc2. Glucosidase‐processed intermediates, GlclMan8GlcNAc2 and GlclMan7GlcNAc2, were also obtained as well as a Man7GlcNAc2 species derived from Glc1Man7GlcNAc2 and different from the Man7GlcNAc2 isomer formed in the usual processing pathway. No evidence for the direct transfer of non‐glucosylated oligosaccharides to proteins was obtained and we conclude that the continued assembly of complex‐type glycans in castanospermine‐inhibited BHK cells results from residual activity of processing glucosidases.
Identification by amino acid sequencing of three major regulatory phosphorylation sites on rat acetyl‐CoA carboxylase
Authors: MUNDAY Michael R. , CAMPBELL David G. , CARLING David , HARDIE D. Grahame
Published: March 2005

We have examined the sites phosphorylated on acetyl‐CoA carboxylase by three protein kinases which have been shown to inactivate the enzyme, i.e. cyclic‐AMP‐dependent protein kinase, acetyl‐CoA carboxylase kinase‐2 (ACK2, purified from rat mammary gland) and the AMP‐activated protein kinase (formerly called acetyl‐CoA carboxylase kinase‐3, purified from rat liver). Each protein kinase phosphorylates two out of three sites (termed 1–3) which have been established by amino acid sequencing. The two sites phosphorylated by each kinase can be recovered on separate peptides, TC1 and TC2, derived by combined digestion of the native enzyme by trypsin and chymotrypsin: TC1 = Ser‐2Ser(P)‐Met‐3Ser(P)‐Gly‐Leu; TC2 = Arg‐Met‐1Ser(P)‐Phe‐Cyclic‐AMP‐dependent protein kinase phosphorylates sites 1 and 2 exclusively, whereas the AMP‐activated protein kinase phosphorylates sites 1 and 3, plus at least one other minor site. ACK2 phosphorylates site 1 and, more slowly, an unidentified site(s) within TC1.

We have also established the structures of the single major phosphopeptides (T1 and C1 respectively) which are recovered by HPLC after acetyl‐CoA carboxylase phosphorylated by cyclic‐AMP‐dependent protein kinase is digested with trypsin or chymotrypsin alone. T1 is related to TC1, and has the structure: Ser‐Ser(P)‐Met‐Ser‐Gly‐Leu‐His‐Leu‐Val‐Lys. C1 is identical with TC2.

We have carried out studies on the correlation of the activity of acetyl‐CoA carboxylase with the occupancy of sites 1, 2 and 3 during phosphorylation by each of the three protein kinases. The results suggest that phosphorylation of site 3 is primarily responsible for the large decrease in Vmax produced by the AMP‐activated protein kinase, while phosphorylation of site 1 may be primarily responsible for the increase in A0.5 for citrate and more modest depression of Vmax produced by cyclic‐AMP‐dependent protein kinase and ACK2.

Our results emphasize that amino acid sequence information is essential in the unequivocal interpretation of data from phosphopeptide mapping experiments and allow a more complete interpretation of previous data on phosphorylation of acetyl‐CoA carboxylase in intact cells. They also open the way to experiments which could establish the physiological roles of these protein kinases in the control of fatty acid synthesis.
Analysis of sites phosphorylated on acetyl‐CoA carboxylase in response to insulin in isolated adipocytes
Authors: HAYSTEAD Timothy A. J. , CAMPBELL David G. , HARDIE D. Grahame
Published: March 2005

We have examined the sites phosphorylated on acetyl‐CoA carboxylase in response to insulin in isolated adipocytes. Two tryptic peptides derived from the enzyme become more radioactive after treatment of 32P‐labelled cells with insulin. One of these (T4a) accounts for a large part of the total increase in phosphate observed after insulin treatment, and comigrates with the pep tide containing the sites phosphorylated in vitro by casein kinase‐2. The other may correspond to the T site peptide originally described by Brownsey and Denton in 1982: labelling of this peptide is stimulated at least threefold by insulin treatment, but it is a minor phosphopeptide and, even after insulin treatment, accounts for only about 2.5% of the enzyme‐bound phosphate (equivalent to < 0.1 mol phosphate/mol 240‐kDa subunit). Two other major tryptic phosphopeptides (T1 and T4b) labelled in adipocytes do not change significantly in response to insulin, and comigrate with peptides containing sites phosphorylated in vitro by cyclic‐AMP‐dependent protein kinase and calmodulin‐dependent multiprotein kinase respectively.

We have sequenced peptides T4a and T4b from acetyl‐CoA carboxylase derived from control and insulin‐treated adipocytes, and also after phosphorylation in vitro with casein kinase‐2 and the calmodulin‐dependent multiprotein kinase. The results show that T4a and T4b are forms of the same peptide containing phosphate groups on different serine residues: Phe‐Ile‐Ile‐Gly‐Ser4‐Val‐Ser5‐Gln‐Asp‐Asn‐Ser6‐Glu‐Asp‐Glu‐Ile‐Ser‐Asn‐Leu‐. Site 5 was phosphorylated by the calmodulin‐dependent protein kinase and site 6 by casein kinase‐2. Migration in the T4a position was exclusively associated with phosphorylation in site 6, irrespective of the presence of phosphate in sites 4 and 5. Sites 5 and 6 were partially phosphorylated in control adipocytes, and there were also small amounts of phosphate in site 4. On stimulation with insulin, phosphorylation appeared to occur primarily at site 6, thus accounting for the increase in 32P‐labelling of T4a. We were unable to isolate sufficient quantities of the other insulin‐sensitive peptide to determine its sequence.

Our results are consistent with the idea that insulin activates either casein kinase‐2, or a protein kinase which has the same specificity as casein kinase‐2. The function of this modification is not clear, since phosphorylation by casein kinase‐2 has no direct effect on acetyl‐CoA carboxylase activity.
Destabilization of tubulin mRNA during heat shock in Tetrahymena pyriformis
Authors: CÓIAS Rui , GALEGO Lisete , BARAHONA Isabel , RODRIGUES‐POUSADA Claudina
Published: March 2005

The regulation of tubulin gene expression was studied in Tetrahymena pyriformis cells during heat shock (shift from 28°C to 34°C). Fluorograms of two‐dimensional gels of radiolabelled proteins synthesized during thermal stress revealed that tubulin synthesis is highly repressed when compared with that of exponentially growing cells. The variation in the levels of α and β‐tubulin mRNAs was analyzed by Northern‐blot hybridization using homologous genomic probes (αTT and βTT1). The results obtained show that heat shock induces a drastic and coordinate reduction in the amount of α and β‐tubulin mRNAs isolated from polysomes. This decrease is not due to a shift from the polysomes to the post‐polysomal fraction because it was also observed when total cytoplasmic mRNAs were analyzed. Run‐on transcription experiments were performed in order to examine whether repression of transcription in heat‐shocked cells could explain that reduction. The results obtained show that the apparent rates of tubulin gene transcription are not significantly modified, but on the contrary increase slightly in cells heat‐shocked for 15 min and 30 min. The effects of inhibitors of protein synthesis, cycloheximide and pactamycin, on the destabilization of tubulin mRNAs were tested in heat‐shocked Tetrahymena cells. Our results revealed that in the presence of these inhibitors, tubulin mRNAs become more stable thus suggesting that an induced factor may be involved in the degradation of α and β‐tubulin mRNAs during heat shock.
Peripheral inactivation of neurotensin
Authors: BARELLI Hélène , VINCENT Jean‐Pierre , CHECLER Frédéric
Published: March 2005

A peptidase that inactivated neurotensin by cleaving the peptide at the Pro10‐Tyr11 bond, generating the biologically inactive fragments neurotensin(1–10) and neurotensin(11–13) was purified from whole rat ileum homogenate. The purified enzyme behaved as a 70–75‐kDa monomer as determined by SDS‐PAGE analysis in reducing or non‐reducing conditions and gel permeation on Ultrogel AcA34. The peptidase was insensitive to thiol‐blocking agents and acidic and serine protease inhibitors but could be strongly inhibited by 1,10‐phenanthroline, EDTA, dithiothreitol and heavy metal ions such as zinc, copper and cobalt. Zinc was the only divalent cation able potently to reactivate the apoenzyme. This enzyme could be distinguished from endopeptidases EC 3.4.24.15 and EC 3.4.24.11, angiotensin‐converting enzyme, proline endopeptidase, aminopeptidase and pyroglutamyl–peptide hydrolase since it was not affected by micromolar concentrations of their specific inhibitors. The peptidase displayed a high affinity for neurotensin (1.6 μM). Studies concerning the specificity of the enzyme towards the sequence of neurotensin established the following. (a) Neurotensin(9–13) was the shortest partial sequence that fully inhibited tritiated neurotensin degradation; shortening the C‐terminal part of the neurotensin molecule led to inactive fragments. (b) Amidation of the C‐terminal end of the peptide did not prevent the recognition by the peptidase. (c) There existed a strong stereospecificity of the peptidase for the residues in positions 8, 9 and 11 of the neurotensin molecule. (d) Pro‐Xaa dipeptides (where Xaa represented aromatic or hydrophobic residues) were the most potent inhibitors of tritiated neurotensin degradation while all the Xaa‐Pro dipeptides tested were totally ineffective. (e) The neurotensin‐related peptides: neuromedin N, xenopsin and [Lys8‐Asn9]neurotensin(8–13), as well as angiotensins I and I1 and dynorphins(1–8) and (1–13) were as potent as neurotensin in inhibiting [3H]neurotensin hydrolysis.
Effect of intracellular pH and potassium ions on a primary transport system for glutamate/aspartate in Streptococcus mutans
Authors: NOJI Sumihare , SATO Yutaka , SUZUKI Ryuta , TANIGUCHI Shigehiko
Published: March 2005

We have studied the mechanism of l‐glutamate/l‐aspartate transport in a fermentative oral bacterium of Streptococcus mutans (strain Ingbritt). The transport rate stays virtually constant throughout the pH range 5.5–8.5 and followed Michaelis‐Menten type kinetics. At high pH values from 7 to 8.5, transport was essentially insensitive to N,N'‐dicyclohexyl‐carbodiimide (DCCD), an inhibitor of ATPase, and to carbonyl cyanide‐p‐trifluoromethoxyphenyl‐hydrazone (FCCP), an ionophore dissipating proton motive force indicating that S. mutans transports glutamate by a primary transport system at the expense of ATP or an alternative energized metabolite. At lower external pH (7–5.), DCCD (100 μM) or FCCP (10 μM) significantly inhibited l‐glutamate transport while the intracellular ATP level was hardly affected, indicating that the activity of the primary transport sytem was decreased at lower intracellular pH. The glutamate transport was stimulated in the presence of potassium ion at an external pH of 6. The stimulation can be explained partly by the regulation of intracellular pH with concomitant potassium ion movement.
Tertiary structure of Escherichia coli tRNAThr3 in solution and interaction of this tRNA with the cognate threonyl‐tRNA synthetase
Authors: THEOBALD Anne , SPRINGER Mathias , GRUNBERG‐MANAGO Marianne , EBEL Jean‐Pierre , GIEGE Richard
Published: March 2005

The solution structure of Escherichia coli tRNAThr3 (anticodon GGU) and the residues of this tRNA in contact with the α2 dimeric threonyl‐tRNA synthetase were studied by chemical and enzymatic footprinting experiments. Alkylation of phosphodiester bonds by ethylnitrosourea and of N‐7 positions in guanosines and N‐3 positions in cytidines by dimethyl sulphate as well as carbethoxylation of N‐7 positions in adenosines by diethyl pyrocarbonate were conducted on different conformers of tRNAThr3. The enzymatic structural probes were nuclease S1 and the cobra venom ribonuclease. Results will be compared to those of three other tRNAs, tRNAAsp, tRNAPhe and tRNATrp, already mapped with these probes.

The reactivity of phosphates towards ethylnitrosourea of the unfolded tRNA was compared to that of the native molecule. The alkylation pattern of tRNAThr3 shows some similarities to that of yeast tRNAPhe and mammalian tRNATrp, especially in the D‐arm (positions 19 and 24) and with tRNATrp, at position 50, the junction between the variable region and the T‐stem. In the T‐loop, tRNAThr3, similarly to the three other tRNAs, shows protections against alkylation at phosphates 59 and 60. However, tRNAThr3 is unique as far as very strong protections are also found for phosphates 55 to 58 in the T‐loop. Compared with yeast tRNAAsp, the main differences in reactivity concern phosphates 19, 24 and 50.

Mapping of bases with dimethyl sulphate and diethyl pyrocarbonate reveal conformational similarities with yeast tRNAPhe. A striking conformational feature of tRNAThr3 is found in the 3′‐side of its anticodon stem, where G40, surrounded by two G residues, is alkylated under native conditions, in contrast to other G residues in stem regions of tRNAs which are unreactive when sandwiched between two purines. This data is indicative of a perturbed helical conformation in the anticodon stem at the level of the 30–40 base pairs.

Footprinting experiments, with chemical and enzymatic probes, on the tRNA complexed with its cognate threonyl‐tRNA synthetase indicate significant protections in the anticodon stem and loop region, in the extra‐loop, and in the amino acid accepting region. The involvement of the anticodon of tRNAThr3 in the recognition process with threonyl‐tRNA synthetase was demonstrated by nuclease S1 mapping and by the protection of G34 and G35 against alkylation by dimethyl sulphate. These data are discussed in the light of the tRNA/synthetase recognition problem and of the structural and functional properties of the tRNA‐like structure present in the operator region of the thrSmRNA.
Purification and characterization of natural and recombinant human plasminogen activator inhibitor‐1 (PAI‐1)
Authors: ALESSI Marie Christine , DECLERCK Paul J. , DE MOL Maria , NELLES Luc , COLLEN Désiré
Published: March 2005

Human plasminogen activator inhibitor‐1 (PAI‐1) was purified from the conditioned medium of endotoxin‐stimulated umbilical vein endothelial cell cultures by combinations of zinc‐chelate‐Sepharose chromatography, gel filtration on Sephacryl S‐300 and immunoadsorption on an insolubilized murine monoclonal antibody (MA‐7D4). The final product was obtained with a recovery of approximately 20% from conditioned medium containing about 3 μ;g/ml PAI‐1. The yield of PAI‐1 was 15 – 100 μ;g/umbilical cord, depending on the culture and harvest conditions. SDS gel electrophoresis revealed a main band with Mr= 46000 both under reducing and non‐reducing conditions. On gel filtration on Sephacryl S‐300, however, the material was separated in two fractions, one eluting at the void volume, which contains active PAI‐1, and one with Mr= 46000 containing inactive material that could be reactivated with 12 M urea. SDS gel electrophoresis of the isolated high‐Mr fraction revealed several bands including a main 46000‐Mr component, which reacted with anti‐(PAI‐1) antibodies on immunoblotting and neutralized tissue‐type plasminogen activator (t‐PA). The active high‐Mr fraction and the reactivated low‐Mr fraction of PAI‐1 inhibited t‐PA very rapidly with an apparent second‐order rate constant of (1.5–4) × 107 M−1 S−1.

The cDNA of endothelial cell PAI‐1 was cloned and expressed in Chinese hamster ovary cells. The translation product, purified from conditioned medium of transfected cells, also revealed a high‐Mr and a low‐Mr fraction on gel filtration, which were indistinguishable from the natural proteins by physicochemical, immunochemical and functional analysis. On reduced SDS gel electrophoresis, the high‐Mr fraction was separated into the Mr‐46000 low‐Mr PAI‐1 and two other components with Mr 65000 and one barely entering the gel.

When reactivated low‐Mr PAI‐1 was added to plasma, PAI activity and PAI‐1 antigen eluted with an apparent Mr 300000 on gel filtration, indicating that active PAI‐1 complexes with one or more binding proteins in plasma.
Molecular mechanics calculation of geometries of NAD+ derivatives, modified in the nicotinamide group, in a ternary complex with horse liver alcohol dehydrogenase
Authors: de KOK Peter M. T. , BEIJER Nicoline A. , BUCK Henk M. , SLUYTERMAN Lamoraal A. AE. , MEIJER Emmo M.
Published: March 2005

The geometry of seven NAD+ analogues bound to horse liver alcohol dehydrogenase (LADH) modified only in their nicotinamide group, have been studied using AMBER molecular mechanics energy‐minimization procedures. Starting geometries were taken from X‐ray crystallographic data for NAD+/Me2SO/LADH reported by Eklund and co‐workers. In this study the NAD+ analogues were encaged by the constituent amino acids of the enzyme within a range of 0.6 nm from the initial NAD+/Me2SO/Zn2+ complex. The calculational method used is able to rationalize individual substituent effects and to evaluate the essential interactions between NAD+ analogue, enzyme, Me2SO and Zn2+ without the necessity of additional X‐ray data. The results presented here demonstrate that the reactivity of NAD+ derivatives as reported in literature can be qualitatively related to the position of the pyridine moiety in the active site.
Phosphorylation of a 51‐kDa envelope membrane polypeptide involved in protein translocation into chloroplasts
Authors: HINZ Giselbert , FLÜGGE Ulf Ingo
Published: March 2005

In this report we demonstrate that a 51‐kDa outer‐envelope membrane protein (P51) is involved in protein translocation into chloroplasts. Furthermore it is shown that phosphorylation of P51 is functionally related to the process of binding and/or importing precursor proteins into chloroplasts. Several lines of evidence have been obtained supporting this suggestion. First, protein import into chloroplasts was inhibited by the membrane‐impermeable agent pyridoxal 5′‐phosphate, which has been shown to react with a component of the protein‐import apparatus. Phosphorylation of envelope membrane polypeptides using [γ‐32P]ATP in the presence of pyridoxal 5′‐phosphate resulted in an increased incorporation of 32P radiolabel into a 51‐kDa membrane polypeptide (P51). A close correlation between the inhibition of protein import and the increase in the phosphorylation state of P51, both as a function of PLP concentration, was observed. Second, binding of purified precursor proteins to chloroplasts resulted in a specific increase in the phosphorylation state of P51. This effect was not exerted by the mature form of the precursor protein lacking the presequence. Third, internally generated ATP was able to compete specifically with externally added [γ‐32P]ATP for the phosphorylation of P51. Fourth, digestion of the outer‐envelope membrane with low amounts of thermolysin resulted in a loss of protein import activity, which was associated with the removal of the phosphorylation site of P51.

Phosphorylation of P51 proceeds with an apparent Km (ATP) of about 5 μM, which is much lower than the ATP concentration required for the protein translocation itself. We suggest that two different ATP‐dependent processes are involved in protein translocation into chloroplasts. P51 represent presumably a regulatory component of the protein‐import apparatus or the protein receptor itself.
Partial purification and characterization of cytosolic Tyr‐protein kinase(s) from human erythrocytes
Authors: CLARI Giulio , BRUNATI Anna Maria , MORET Vittorio
Published: March 2005

Tyrosine‐protein kinase, phosphorylating tyrosine residues of transmembrane band 3 protein, has been partially purified from human erythrocyte cytosol by DEAE‐Sepharose chromatography followed by heparin‐Sepharose chromatography.

Such a Tyr‐protein kinase (36 kDa), as distinct from the Ser/Thre‐protein kinases (casein kinase S and TS), appears to display a broader site specificity than does the previously described human erythrocyte P‐Tyr‐protein phosphatase, dephosphorylating band 3 protein. That is, it is able to phosphorylate not only the highly acidic copolymer poly(Glu–Tyr)4:1 but also angiotensin II, lacking an acidic amino acid sequence around the target Tyr residue. Moreover, the phosphorylation of these two substrates exhibits a different pH dependence and a different response to NaCl and 2,3‐bisphosphoglycerate.

These results suggest that in intact erythrocytes the cytosolic Tyr‐protein kinase might phosphorylate band 3 not only on Tyr‐8, surrounded by several acidic side‐chains (as demonstrated preferentially to occur in isolated ghosts), but also on other Tyr residues surrounded by other amino acid sequences.
The regulation of the cytoplasmic free Ca2+ concentration in aortic smooth muscle cells (A7r5 line) after stimulation by vasopressin and bombesin
Authors: VIGNE Paul , BREITTMAYER Jean‐Philippe , LAZDUNSKI Michel , FRELIN Christian
Published: March 2005

The characteristics of intracellular Ca2+ transient induced by vasopressin adn bombesin in aortic smooth muscle cells were studied using flow cytometric analysis of indo‐1 loaded cells. The two hormones induced a rapid and tranisient rise in [Ca2+]i. This Ca2+ transient was independent of the presence of extracellular Ca2+. Addition of bombesin to cells that have already been stimulated by vasopressin (or conversely the addition of vasopressin to bombesin‐stimulated cells) resuls in a second Ca2+ transient that has smaller amplitude. This transient is the same when the external Ca2+ concentration is lowered from 1.8 mM to 50 nM, suggesting the agonistsensitive pool reloaded using the Ca2+ that has been previously released into the cytoplasm. Intracellular Ca2+ pools that have been depleted by a prolonged incubation of the cessl in a low Ca2+ medium cna be refilled by shifting cells to a high Ca2+ medium. The reloading was analyzed in detail and found to be a slow process. It is hardly affected by Ni2+ or by (‐)D888, a potent inhibitor of the voltage‐dependent Ca2+ channels. It is accelerated when Ca2+ uptake by the Na+/Ca2+ exchange system is stimulated. The results suggest that Ca2+ homeostasis in aortic smooth muscle cells is achieved using mechanisms that are distince from thsoe operating in various acini and in striated muscles.
Protein phosphorylation in Mycoplasma gallisepticum
Authors: PLATT Mark W. , ROTTEM Shlomo , MILNER Yoram , BARILE Michael F. , PETERKOFSKY Alan , RE1ZER Jonathan
Published: March 2005

Incubation of the soluble fraction derived from Mycoplasma gallisepticum cells with [γ‐32P]ATP results in the phosphorylation of several endogenous proteins. One protein with an apparent molecular mass of 55 kDa was the acceptor of more than 95% of the radioactive phosphate. This protein was also found to be radiolabeled in intact cells grown in the presence of [32P]orthophosphate. Acid hydrolysis of the phosphorylated 55‐kDa protein followed by two‐dimensional electrophoresis revealed that the 32P‐labelcd material co‐migrated with phosphoserine. The in vitro phosphorylation of the 55‐kDa protein has an optimum pH of 5.5‐6.0 and is not affected by various metabolites of glycolysis, by cAMP or by calmodulin with or without Ca2+. The phosphorylation is dependent upon divalent cations, a dependency that is best fulfilled by the simultaneous addition of Ca2+ and Zn2+ that act in a specific and cooperative manner. Of a variety of possible exogenous protein acceptors tested, the endogenous protein kinase was capable to phosphorylate only phosvitin. The phosphorylation of the 55‐kDa protein is reversible through the activity of a phosphoprotein phosphatasc present in the soluble fraction of M. gallisepticum. The phosphoprotein phosphatase has an optimum pH of 7.5‐8.0, is inhibited by NaF and stimulated to a large extent by inorganic phosphate and arsenate and to a lesser extent by pyrophosphate ATP and ADP. The possible association of the reversible protein phosphorylation to cell shape and gliding motility of M. gallisepticum are discussed.
Bovine endothelial cell plasminogen activator inhibitor
Authors: KATAGIRI Kazuya , OKADA Kenji , HATTORI Hiroyuki , YANO Milsuo
Published: March 2005

A plasminogen activator inhibitor (PAI) was purified from bovine endothelial cell conditioned medium by a simple procedure in the absence of protein denaturant The yield was 2.2 mg from 1.6 1 conditioned medium in a typical experiment. The purified inhibitor showed a single band on sodium dodecyl sulfate/polyacrylamide gel electrophoresis and reverse fibrin autography with an apparent molecular mass of 45 kDa. The amino‐terminal 40‐amino‐acid sequence was determined and found to be 70% similar to the reported corresponding sequence of human PAI‐1. The amino acid composition also revealed a close relationship between bovine PAI and human PAI‐1.

The purified PAI was substantially inactive (570 U/mg) but it could be activated by treatment with protein denaturants such as 1% SDS (1.8 × 105 U/mg) and 4 M guanidine‐HCl (l.5 × 105 U/mg). A more effective activation of this latent PAI was achieved by heat treatment at 100°C for 2.S min, generating the specific activity of 1.0 × 106 U/mg. The heat‐activated PAI lost its activity during incubation at 56°C for 30 min. but repeated heat at 100°C for 2.5 min could regenerate about 70% of the initial activity. Treatment at 37°C, 56°C and 80°C. however, failed to activate the latent PAI at all. These findings suggest that the buried reactive site of the latent PAI is exposed as a result of a heat‐induced, specific conformational change, but tends to be masked again during renaturalion under mild conditions, i.e. the PAI protein takes on preferentially a latent form.
Intracellular serine‐protease zymogen, factor C, from horseshoe crab hemocytes
Authors: NAKAMURA Takanori , TOKUNAGA Fuminori , MORITA Takashi , IWANAGA Sadaaki , KUSUMOTO Shoichi , SHIBA Tetsuo , KOBAYASHI Tetsuyuki , INOUE Kcizo
Published: March 2005

An intracellular clotting factor, factor C, found in the horseshoe crab hemocytes is a lipopolysaccharide‐sensitive serine‐protease zymogen, which participates in the initiation of the hemolymph clotting system [T. Nakamura et al. (1986) Eur. J. Biochem. 154, 511‐521]. The subsequent study of this zymogen, using various synthetic lipid A analogues, revealed that the zymogen factor C is rapidly activated by acylated (β1‐6)‐D‐glucosa‐mine disaccharide bisphosphate (synthetic Escherichia coil‐type lipid A), and the corresponding 4′‐monophosphate analogues. However, the corresponding non‐phosphorylated lipid A did not activate factor C, indicating that a phosphate ester group linked with the (β1‐6)‐D‐glucosamine disaccharide backbone is required for the zymogen activation. During these studies we also found that the zymogen factor C is significantly activated by acidic phospholipids, such as phosphatidylinositol, phosphatidylglycerol and cardiolipin, but not at all by neutral phospholipids. The rate of this activation, however, was affected markedly by ionic strength in the reaction mixture, although such an effect was not observed in the lipid‐A‐mediated activation of factor C. A varietv of negatively charged surfaces, such as sulfatide, dextran sulfate and ellagic acid, which are known as typical initiators for activation of the mammalian intrinsic clotting system, did not show any effect on the zymogen factor C activation. These results suggest that lipid A is the most effective trigger to initiate the activation of the horseshoe crab hemolymph clotting system.
Enzymes as biosensors
Authors: SOULIE Jean‐Michel , RIVIERE Mireille , RICARD Jacques
Published: March 2005

Oxidized chloroplastic fructose‐bisphosphatase is almost totally inactive at pH 7.5, that is under pH conditions that prevail in the chloroplast stroma. When preincubated for different time periods with fructose 2, 6‐bisphosphate and assayed in the absence of this ligand, it displays an activity which is directly related to the duration of the preincubation phase. This implies that fructose 2, 6‐bisphosphate induces enzymes conformers that appear in sequence and may be competent for catalytic activity. Upon desorption of fructose 2, 6‐bisphosphate the enzyme may retain its active conformation for a time period whose duration depends on magnesium concentration. It thus appears that reduction of the enzyme is not an obligatory prerequisite for its acitivity.

Fructose 2, 6‐bisphosphate behaves as a competitive inhibitor of the reduced, active enzyme, with respect to the real substrate. When assayed with the oxidized enzyme, however, its behaves as an acitvator. Moreover, the apparent steady‐state rate that may be measured experimentally depends on both fructose 2, 6‐bisphosphate concentration and the direction of a concentraion change. The reaction velocity experimentally measured is thus a meta‐steady‐state rate and depends on the initial conditions of the system. The fructose‐bisphosphatase system thus displays, with respect to fructose 2, 6‐bisphosphate. a hysteresis loop and may then sense whether the concentration of that ligan is increased ordecreased.

A model has been proposed which allows one to explain these results. This model is based on the view that the substrateand fructose 2, 6‐bisphosphate compete for the same site of the enzyme and that this latter ligand stabilizes a conformation competent for enzyme activity. After the ligand has been chased away, the enzyme retains the active conformation for a while and slowly relapses to the initial inactive conformation. The time‐scale of this slow relaxation overlaps that of the steady state of product appearance and this generates meta‐steady‐state kinetic, which is dependent on the initial state and therefore on the history of the system.
Calcium‐dependetn inactivation of the Ca2+‐ATPase from sarcoplasmic reticulum by chemically reactive adenosine triphosphate
Authors: BILL Eberhard , GUTOWSKI Zeynep , BÄUMERT Hans G.
Published: March 2005

ATP γP‐imidazolidate, synthesized from ATP and carbonyldiimidazole, inhibits the Ca2+‐ATPase of sarcoplasmic reticulum in a biphasic;manne.r A fast first phase is concentration‐dependent while a slower second phase is independent of the inhibitor concentration.

The inhibnition is calcium‐ and magnesium‐dependent. No inhibition occurs in the absence of either cation.

Inhibition of the Ca2+‐ATPase can be prevented by the protection with ATP.

The loss of ATPase activity is pH‐dependent. Maximal inhibition coincides with maximal ATPase activity It indicates a participation of the reacting amino acid side chain in the catalytic cycle.

The incorporation of radioactive inhibitor is reversedin a time‐dependent fashion while the Ca2+ATPase remains inhibited.

We conclude that ATP γP‐imidazolixate initially reacts covalently with an amino acid side chain, probably Asp‐351, but is subsequently expelled by a reaction with a second amino acid.

This two‐step reaction induces an intramolecular cross‐link with can be shown by the creation of a new protein band on SDS‐PAGE which originates in the Ca2+‐ATPase.
Sequence of the Saccharomyces cerevisiae CATI gene and amino acid sequence of catalase A derived from it
Authors: CHOEN Gerald , RAPATZ Winfried , RUIS Helmut
Published: March 2005

The nucleotide sequence of a 2785‐base‐pair stretch of DNa containing the Saccharomyces cerevisiae catalase A (CATI) gene has been determined. This gene contains an uninterrupted open reading frame encoding a protein of 515 amino acids (relative molecular mass 58490). Catalase A, the peroxisomal catalase of S. cerevisiae was compared to the peroxisomal catalases from bovine liver and from Candida tropicalis and to the non‐peroxisomal, presumably cytoplasmic, catalase T of S. cerevisiae. Whereas the peroxisomal catalases are almost colinear, three major insertions have to be introduced in the catalase T sequence to obtain an optimal fit with the other proteins. Catalase A is most closely related tothe C. tropicalis enzyme. It is also more similar to the bovine liver catalase than to the second S. cerevisiae catalase. The differences between the two S. cerevisiae enzymes are most striking within four blocks of amino acids consisting of a total of 37 residues with high homology between the three peroxisomal, but low conservation betwen the S. cerevisiae catalases. The results obtained indicate that the peroxisomal catalases compared have very similar three‐dimensional structures and might have similar targetting signals.
A 1H‐NMR study on the blue copper protein amicyanin from Thiobacillus versutus
Authors: LOMMEN Arjen , CANTERS Gerard W. , van BEEUMEN Jozef
Published: March 2005

A number of resonances in the 1H‐NMR spectra of reduced and oxidised amicyanin from Thiobacillus versutus have been identified by one‐ and two‐dimensional NMR techniques. The second‐order electron self‐exchange rate constant (8.5 × 104 M−1; s−1; pH = 7.4; T= 308.5 K) was determined by measuring the line broadening of six singlets in slightly oxidised solutions of the protein. A large increase in electron exchange rate is observed in the presence of ferrocyanide. The copper atom in the reactive centre of the protein appears to be coordinated by nitrogens from two histidines and sulfurs from a methionine and a cysteine. One of the ligand histidines becomes protonated at low pH [pK**= 6.74 (± 0.02)], the asterisk indicating value uncorrected for the deuterium isotope effect] in reduced amicyanin. This is the First example of a non‐photosynthetic blue copper protein in which a ligand histidine becomes protonated at low pH. A small pH‐independent conformational rearrangement occurs upon oxidation.
Detergentless microemulsions as media for enzymatic reactions
Authors: KHMELNITSKY Yuri L. , HILHORST Riet , VERGER Cees
Published: March 2005

Catalytic acitivity and stability of cholesterol oxidase dissolved in ternary systems composed on n‐hexane, isopropanol, and water were studied. The dependence of catalytic acitivity on the composition of the system revealed two maxima, in contrast to the behaviour of previously studied enzymes where a single maximum has been observed. The stability profile of cholesterol oxidase showed a single sharp maximum coinciding with the microemulsion region of the phase diagram. Both catalytic activity and the first‐order inactivation rate constant of cholesterol oxidase dissolved in n‐hexane/isopropanol/water ternary systems were found to decrease with decreasing temperature. This decrease was more rapid for the inactivation rate constant than for catalytic activity, the activation energies being 200 and 60 kJ mol−1, respectively.

Preparative conversion of cholesterol to cholestenone catalyzed by cholesteroloxidase in n‐hexane/isopropanol/water ternary systems was carried out with 100% yield. Decreased temperature and the presence of catalase were required to achieve high degrees of cholesterol conversion. A simple procedure suitable for rapid separation of the reaction product and recovery of the enzyme was developed.
The role of aspartic acid‐49 in the active site of phospholipase A2
Authors: VAN DEN BERGH Card J. , SLOTBOOM Arend J. , VERHEIJ Hubertus M. , DE HAAS Gerard H.
Published: March 2005

In order to probe the role of Asp‐49 in the active site of procine pancreatic phospholipase A2 two mutant proteins were constructed containing either Glu or Lys at position 49. Their enzymatic activities and their affinities for substrate and for Ca2+ions were examined in comparision with the native enzyme. Enzymatic charaterization indicated that the presence of Asp‐49 is essential for effective hydrolysis of phospholipids. Conversion of Asp‐49 to either Glu or Lys strongly reduces the binding of Ca2+ions in particular for the lysine mutant but the affinity for substrate analogues is hardly affected. Extensive purification of [Lys49] phospholipase A2 form the venom of Agkistrodon piscivorus piscivorus yielded a protein which was 4000 times less active than the basic [Asp49] phospholipase A2 from this venom. Inhibition studies with p‐bromophenacyl bromide showed that this residual activity was due to a small amount of contaminating enzyme and that the Lys‐49 homologue itself is inactive. The results obtained both with the procine pancreatic phospholipase A2 mutant with the native venom enzymes show that Asp‐49 is essential for the catalytic action of phospholipase A2.
Binding of ellipticine base and ellipticinium cation to calf‐thymus DNA
Authors: DODIN Guy , SCHWALLER Marc‐Antoine , AUBARD Jean , PAOLETTI Claude
Published: March 2005

The acid‐basic properties of ellipticine have been reestimated. The apparent pK of protonation at 3 mUM drug concentration is 7.4 ± 0.1. The ellipticine free base (at pH 9, I = 25 mM) intercalates into calf‐thymus DNA with an affinity constant of 3.3 ± 0.2 × 105 M−1 and a number of binding sites per phosphate of 0.23. The ellipticinium cation (pH 5, I = 25 mM) binds also to DNA with a constant of 8.3 ± 0.2 × 105 M−1 and at a number of binding sites (n = 0.19). It is postulated that the binding of the drug to DNA at pH 9 is driven by hydrophobic and/or dipolar effects. Even at pH 5, where ellipticine exists as a cation, it is thought that the hydrophobic interaction is the main contribution to binding. The neutral and cationic forms share common binding within DNA sites but yield to structurally different complexes. The free base has 0.04 additional specific binding sites per phosphate. As determined from temperature‐jump experiments, the second‐order rate constant of the binding of the free base (pH 9) is 3.4 × 107 M−1 s−1 and the residence time of the base within the DNA is 8 ms. The rate constant for the binding of the ellipticinium cation is 9.8 × 107 M‐−1 s−1 when it is assumed that drug attachment occurs via a pathway in which the formation of an intermediate ionic complex is not involved (competitive pathway).
Purification and characterization of a rotenone‐insensitive NADH: Q6 oxidoreductase from mitochondria of Saccharomyces cerevisiae
Authors: de VRIES Simon , GRIVELL Leslie A.
Published: March 2005

A mitochondrial NADH: Q6 oxidoreductase has been isolated from cells of Saccharomyces cerevisiae by a simple method involving extraction of the enzyme from the mitochondrial membrane with Triton X‐100, followed by chromatography on DEAE‐cellulose and blue Sepharose CL‐6B. By this procedure a 2000‐fold purification is achieved with respect to whole cells or a 150‐fold purification with respect to the mitochondrion. The purified NADH dehydrogenase consists of a single subunit with molecular mass of 53 kDa as indicated by SDS/poly‐acrylamide gel electrophoresis. The enzyme contains FAD, non‐covalently linked, as the sole prosthetic group with Em,7.67.6 = ‐370 mV and no iron‐sulphur clusters. The enzyme is specific for NADH with apparent Km= 31 μM and was found to be inhibited by flavone (I50= 95 mUM), but not by rotenone or piericidin. The purified enzyme can use ubiquinone‐2, ‐6 or ‐10, menaquinone, dichloroindophenol or ferricyanide as electron acceptors, but at different rates. The greatest turnover of NADH was obtained with ubiquinone‐2 as acceptor (2500 s−1). With the natural ubiquinone‐6 this value was 500s‐1. The NADH:Q2 oxidoreductase activity shows a maximum at pH 6.2, the NADH:Q6 oxidoreductase activity is constant between pH 4.5‐9.0. The amount of enzyme in the cell is subject to glucose repression: it increases slightly when cells, grown on glucose or lactate, enter the stationary phase. The experiments performed so far suggest that the enzyme purified in this study is the external NADH:Q6 oxidoreductase, bound to the mitochondrial inner membrane and that it is involved in the oxidation of cytosolic NADH. The relation of this enzyme with respect to various other NADH dehydrogenases from yeast and plant mitochondria is discussed.