bluuebunny/crossref_metadata_2025 huggingface dataset:
Purification and characterization of the ferredoxin‐glutamate synthase from the unicellular cyanobacterium Synechococcus sp. PCC 6301
Authors: MARQUÉS Silvia , FLORENCIO Francisco J. , CANDAU Pedro
Published: March 2005

Ferredoxin‐glutamate synthase from the unicellular cyanobacterium Synechococcus sp. PCC 6301 has been purified using, as main steps, ethanol fractionation in the presence of high ionic strength, ion‐exchange chromatography and ferredoxin‐Sepharose affinity chromatography. The overall process yielded an homogeneous enzyme with a specific activity of 30 U/mg protein, after a purification of 2800‐fold with a recovery of 43%. The molecular mass of the native protein was 156 kDa, as calculated from its Stokes radius (rs, 4.32 nm) and sedimentation coeficient (s20,w, 8.46 S). The size was also estimated by SDS/PAGE as 160 kDa, indicating that the native protein was a monomer. The enzyme exhibited absorption maxima at 279, 370 and 438 nm and a A279/A438 absorbance ratio of 11. One molecule of FMN, but not FAD, was found/molecule native protein. The addition of dithionite resulted in the loss of the absorption peak at 438 nm, which was restored by the addition of 2‐oxoglutarate, thus indicating that the prosthetic group is functional in catalysis. Classical hyperbolic kinetics with substrate inhibition was seen for 2‐oxoglutarate. The Km values determined for glutamine and ferredoxin were 0.7 mM and 7 μM, respectively, and the apparent Km for 2‐oxoglutarate was estimated to be 1.7 mM. Azaserine and 6‐diazo‐5‐oxo‐l‐norleucine were potent inhibitors of the activity, while pyridoxal 5‐phosphate, known to react with Lys residues, partially inactivated the enzyme. This ferredoxin‐dependent glutamate synthase is, as far as we know, the first purified from prokaryotic organisms and resembles its counterpart from chloroplasts, suggesting that cyanobacterial glutamate synthase may have been the ancestor of ferredoxin‐glutamate synthase in plants.
Dynamics of the exposure of epitopes on OmpF, an outer membrane protein of Escherichia coli
Authors: FOUREL Didier , MIZUSHIMA Shoji , PAGÈS Jean‐Marie
Published: March 2005

The OmpF protein is the major outer membrane trimeric porin of Escherichia coli B. The exposure of several cell‐surface‐exposed epitopes, that are recognized by various monoclonal antibodies directed against the protein, is investigated. Kinetic analyses show that two epitopes (E18 and E19) appear early during the in‐vivo assembly on the folded monomer, just after the removal of the signal peptide, and are conserved in the native trimer. The trimerization that immediately follows or occurs in conjunction with the folding of monomers exposes another antigenic site (E21) at the surface of metastable forms. The binding of nascent lipopolysaccharide promotes the conversion of the heat‐modifiable intermediate to a stable trimer and ensures the exposure of E20, E1, E3, E4 and E7. Late epitopes, E1, E3, E4 and E7 are only detected in the outer membrane fraction. These results suggest that different steps induce the sequential exposure of native antigenic sites. The detection of these epitopes depends on conformational changes occurring during the OmpF insertion into the outer membrane.
The glutamate dehydrogenase gene of Clotridium symbiosum
Authors: TELLER Jan K. , SMITH Rachel J. , McPHERSON Michael J. , ENGEL Paul C. , GUEST John R.
Published: March 2005

The gene encoding the NAD+‐dependent glutamate dehydrogenase (GDH) of Clostridium symbiosum was cloned using the polymerase chain reaction (PCR) because it could not be recovered by standard techniques. The nucleotide sequence of the gdh gene was determined and it was over‐expressed from the controllable tac promoter in Escherichia coli so that active clostridial GDH represented 20% of total cell protein. The recombinant plasmid complemented the nutritional lesion of an E. coli glutamate auxotroph. There was a marked difference between the nucleotide compositions of the coding region (G + C = 52%) and the flanking sequences (G + C = 30% and 37%). The structural gene encoded a polypeptide of 450 amino acid residues and relative molecular mass (Mr) 49295 which corresponds to a single subunit of the hexameric enzyme. The DNA‐derived amino acid sequence was consistent with a partial sequence from tryptic and cyanogen bromide peptides of the clostridial enzyme. The N‐terminal amino acid sequence matched that of the purified protein, indicating that the initiating methionine is removed post‐translationally, as in the natural host. The amino acid sequence is similar to those of other bacterial GDHs although it has a Gly‐Xaa‐Gly‐Xaa‐Xaa‐Ala motif in the NAD+‐binding domain, which is more typical of the NADP+‐dependent enzymes. The sequence data now permit a detailed interpretation of the X‐ray crystallographic structure of the enzyme and the cloning and expression of the clostridial gene will facilitate site‐directed mutagenesis.
The interaction of thrombin with fibrinogen
Authors: STUBBS Milton T. , OSCHKINAT Hartmut , MAYR Irmgard , HUBER Robert , ANGLIKER Herbert , STONE Stuart R. , BODE Wolfram
Published: March 2005

The structure of the ternary complex of human α‐thrombin with a covalently bound analogue of fibrinopeptide A and a C‐terminal hirudin peptide has been determined by X‐ray diffraction methods at 0.25 nm resolution. Fibrinopeptide A folds in a compact manner, bringing together hydrophobic residues that slot into the apolar binding site of human α‐thrombin. Fibrinogen residue Phe8 occupies the aryl‐binding site of thrombin, adjacent to fibrinogen residues Leu9 and Val15 in the S2 subsite. The species diversity of fibrinopeptide A is analysed with respect to its conformation and its interaction with thrombin. The non‐covalently attached peptide fragment hirudin(54–65) exhibits an identical conformation to that observed in the hirudin‐thrombin complex. The occupancy of the secondary fibrinogen‐recognition exosites by this peptide imposes restrictions on the manner of fibrinogen binding. The surface topology of the thrombin molecule indicates positions P1′–P3′, differ from those of the canonical serine‐proteinase inhibitors, suggesting a mechanical model for the switching of thrombin activity from fibrinogen cleavage to protein‐C activation on thrombomodulin complex formation. The multiple interactions between thrombin and fibrinogen provide an explanation for the narrow specificity of thrombin. Structural grounds can be put forward for certain congenital clotting disorders.
Identification of the photosystem I antenna polypeptides in barley
Authors: KNOETZEL Jürgen , SVENDSEN Ib , SIMPSON David John
Published: March 2005

The light‐harvesting antenna of barley photosystem I (LHCI) was isolated from native photosystem I (PSI) complexes and fractionated into three pigment‐protein subcomplexes using two consecutive rounds of green gel electrophoresis. Each complex showed a characteristic polypeptide composition and low‐temperature fluorescence emission spectrum; they were designated as LHCI‐730, LHCI‐680A and LHCI‐680B. Their four apoproteins of 21, 22, 23 and 25 kDa were purified and NH2‐terminal sequences were determined; in the case of the NH2‐terminally blocked 25‐kDa protein, an internal sequence was obtained after cleavage with endoproteinase Lys‐C. This made possible an assignment of the four proteins to the four types (I–IV) of genes coding for chlorophyll a/b proteins of PSI (cab or lha genes). The LHCI‐730 complex was isolated as a heterodimer composed of the 21‐kDa (LHCI type IV) and the 22‐kDa (LHCI type I) polypeptides. Each LHCI‐680 complex had a single apoprotein. LHCI‐680A consisted of the 25‐kDa (LHCI type III) and LHCI‐680B of the 23‐kDa (LHCI type II) polypeptides. LHCI‐680B was associated with the non‐pigmented PSI‐E subunit, indicating that this protein may function in the binding of this antenna to the reaction centre.
Purification and characterization of human salivary‐gland prokallikrein from recombinant baculovirus‐infected insect cells
Authors: ANGERMANN Axel , RAHN Hans‐Peter , HEKTOR Thomas , FERTIG Georg , KEMME Michael
Published: March 2005

A full‐length cDNA encoding human salivary‐gland preprokallikrein was inserted into the baculovirus Autographa californica nuclear polyhedrosis virus downstream of the polyhedrin promoter. The gene was expressed in transfected Spodoptera frugiperda cells and the recombinant product secreted into the culture medium. By alternating anion‐exchange chromatography and gel‐filtration steps, twice repeated, prokallikrein was purified to homogeneity, which was confirmed by amino acid analysis and N‐terminal sequence determination. The prepropeptide was processed correctly, including the removal of the signal peptide. The resulting proenzyme was found to be glycosylated, had a molecular mass of 35 kDa and an isoelectric point of 4.6. The yield of purified recombinant protein reached a level of 5 mg/1 insect cell culture. After trypsin digestion of prokallikrein, the biological activity of the released kallikrein was demonstrated by its specific amidase, esterase and kininogenase activity. The expression and purification of prokallikrein, as described here, offers the opportunity to study the proenzyme activation through protein engineering techniques in detail.
Red‐edge excitation fluorescence measurements of several two‐tryptophan‐containing proteins
Authors: WASYLEWSKI Zygmunt , KOŁOCZEK Henryk , WAŚNIOWSKA Alicja , ŚIZOWSKA Krystyna
Published: March 2005

The dependence of the fluorescence emission maximum of the tryptophan residues in several two‐tryptophan‐containing proteins (horse liver alcohol dehydrogenase, yeast 3‐phosphoglycerate kinase, Staphylococcus aureus metalloprotease and bee venom phospholipase A2) on the excitation wavelengths has been studied. Using fluorescence‐resolved spectroscopy, we have dissected the contributions of particular tryptophan residues located in different parts of the protein molecule. The results demonstrate that dipolar structural relaxation can occur in the environment of tryptophan residues buried within protein molecules. The observed spectral shifts upon red‐edge excitation of these residues can depend on temperature or ligand binding, as demonstrated in case of metalloprotease and alcohol dehydrogenase. No spectral shifts upon red‐edge excitation have been observed for tryptophan residues totally exposed to the rapidly relaxing aqueous solvent.
Primary structure of hemocyanin subunit c from Panulirus interruptus
Authors: NEUTEBOOM Ben , JEKEL Peter A. , BEINTEMA Jaap J.
Published: March 2005

The amino acid sequence of the hemocyanin subunit c from the spiny lobster, Panulirus interruptus, has been determined. The elucidation was mainly based on three digests, with CNBr, trypsin and endoproteinase Glu‐C, respectively. Additional evidence was obtained by sequencing of peptides from an endoproteinase Lys‐C digest. Subunit c is a polypeptide with 661 amino acid residues and with a carbohydrate group attached to residue 476 in the third domain. No heterogeneity was observed. The degree of identity with subunit a is 59%. Some differences with subunit a are an N‐terminal extension of six residues, a one‐residue C‐terminal extension, and a three‐residue deletion. Furthermore, carbohydrate attachment is in a different position, as are most half‐cystine residues. Limited trypsinolysis resulted in cleavage at the same site as in subunits a and b.
Interaction in vitro of scallop muscle arginine kinase with filamentous actin
Authors: REDDY S. Raghupathi Rami , HOUMEIDA Ahmed , BENYAMIN Yves , ROUSTAN Claude
Published: March 2005

Scallop muscle arginine kinase binds to F‐actin from mollusc and rabbit muscle in vitro. One site of interaction appears to be located in residues 305–325 of a C‐terminal fragment (residues 285–375) of actin. The binding is hindered in the presence of arginine, Mg2+‐ADP and NO3−, which form a dead‐end complex with the enzyme. F‐actin inhibits the enzyme activity non‐competitively with respect to Mg2+‐ATP. As a function of arginine concentration, the inhibition is of the mixed type, where Km is affected more than Vmax.
Properties of yeast debranching enzyme and its specificity toward branched cyclodextrins
Authors: TABATA Shiro , HIZUKURI Susumu
Published: March 2005

Debranching enzyme was purified from Saccharomyces cerevisiae by DEAE‐cellulose, ω‐aminobutyl agarose and hydroxyapatite column chromatography. The activity of the eluent was monitored by the iodine‐staining method which detects both the direct and indirect debranching enzymes. The elution profiles at every step showed a single peak with no shoulder. The crude and the purified enzyme preparations gave a single activity band with the same mobility on PAGE. The crude product produced 80% glucose compared to reducing sugar from glycogen‐phosphorylase‐limited dextrin while the partially purified and purified preparations produced 100% glucose.

The activity of the purified enzyme was characterized and compared with that of the rabbit muscle enzyme by using various branched cyclodextrins as substrates. Both enzymes hydrolyzed 6‐O‐α‐d‐glucosyl cyclodextrins to glucose and cyclodextrins, but did not act on 6‐O‐α‐maltosyl cyclomaltoheptaose. The yeast enzyme gave rise to glucose as a sole reducing sugar from 6‐O‐α‐maltotriosyl cyclomaltoheptaose and 6‐O‐α‐maltotraosyl cyclomaltoheptaose, indicating that maltosyl and maltotriosyl transfers, respectively, had occurred, prior to the action of amylo‐1,6‐glucosidase.

6‐O‐α‐d‐Glucosyl cyclomaltoheptaose and 6‐O‐α‐d‐glucosyl cyclomalto‐octaose, respectively, were better substrates than glycogen‐phosphorylase‐limited dextrin for the yeast and muscle enzymes. The yeast enzyme released glucose at a similar rate from 6‐O‐α‐maltotriosyl cyclomaltoheptaose as from 6‐O‐α‐maltotetraosyl cyclomaltoheptaose, but considerably lower rates than that from limit dextrin.

The yeast debranching enzyme appears to be exclusively oligo‐1,4→1,4‐glucantransferase‐amylo‐1,6‐glucosidase and does not have isoamylase.
Attenuation in the rph‐pyrE operon of Escherichia coli and processing of the dicistronic mRNA
Authors: ANDERSEN Jens Tønne , POULSEN Peter , JENSEN Kaj Frank
Published: March 2005

We have substituted on a plasmid the native promoter of the Escherichia coli rph‐pyrE operon with an inducible transcription‐initiation signal. The plasmid was used to study the mRNA chains derived from the operon at different intracellular concentrations of UTP and as a function of time following induction of transcription. The results showed that dicistronic rph‐pyrE mRNA was formed when the UTP pool was low, and that a monocistronic rph mRNA was the major transcription product in high‐UTP pools, thus supporting an UTP‐controlled attenuation mechanism for regulation of pyrE gene expression. However, the dicistronic rph‐pyrE transcript was rapidly processed into two monocistronic mRNA units, and a cleavage site was mapped near the attenuator in the intercistronic region, close to the site where transcription was terminated in high‐UTP pools. Furthermore, the major 3′ end of the pyrE mRNA was mapped near a palindromic structure of similarity to the family of repetitive extragenic palindromic sequences, 35 nucleotide residues after the stop codon of the pyrE gene.
Determination of the nucleotide sequences in mouse U14 small nuclear RNA and 18S ribosomal RNA responsible for in vitro intermolecular base‐pairing
Authors: SHANAB Gamila M. , MAXWELL E. Stuart
Published: March 2005

U14 small nuclear RNA (snRNA) is an evolutionarily conserved RNA species that plays a role in rRNA processing. The conserved ability of fungal, amphibian and mammalian U14 snRNAs to hybridize with both homolous and heterologous eukaryotic 18S rRNAs indicates a potential role for this intermolecular RNA/RNA interaction in U14 snRNA function. To understand better the possible role of this intermolecular base‐pairing in rRNA processing, we have defined those nucleotide sequences in mouse U14 snRNA and 18S rRNA responsible for the observed in vitro hybridization. We have constructed, using synthetic DNA oligonucleotides, a U14 snRNA gene which has been positioned behind a T7 RNA polymerase promoter site and then inserted into a plasmid. The presence of natural or engineered restriction endonuclease sites within this construct has permitted the in vitro transcription of full‐length mouse U14 snRNA transcripts (an 87‐nucleotide mouse U14 snRNA minus 5′ or 3′ leader sequences) or 3′ terminally truncated U14 snRNA fragments. Hybridization of full‐length or truncated fragments of U14 snRNA to mouse 18S rRNA demonstrated the utilization of a previously proposed 18S rRNA complementary sequence located near the 3′ end of mouse U14 snRNA (nucleotides 65–78) for intermolecular hybridization. Conversely, RNase‐T1‐generated fragments of 18S rRNA capable of hybrid‐selection by U14 snRNA have been isolated and sequenced. A nested set of hybrid‐selected 18S rRNA fragments define a mouse 18S rRNA sequence (nucleotides 459–472) which exhibits perfect complementarity to the defined U14 snRNA sequence 65–78. Primer‐extension/chain‐termination mapping of mouse U14‐snRNA · 18S‐rRNA hybrids has confirmed the formation of the proposed hybrid structure. A second set of observed complementary sequences in mouse U14 snRNA (nucleotides 25–38) and mouse 18S rRNA (nucleotides 82–95) are not used for the in vitro hybridization of these two RNAs. Presumably the involvement of this prevents its base‐pairing with 18S rRNA. However, the strong evolutionary conservation of both U14‐snRNA · 18S‐rRNA hybrid structures and their juxtapositioning within the folded secondary structure of 18S rRNAs argues for a biological role for each in U14 snRNA function.
Multiple isoforms of a protein kinase C inhibitor (KCIP‐1/14‐3‐3) from sheep brain
Authors: TOKER Alex , SELLERS Lynda A. , AMESS Bob , PATEL Yasmina , HARRIS Alan , AITKEN Alastair
Published: March 2005

A potent inhibitor of protein kinase C (PKC), inhibitor protein‐1 (KCIP‐1), isolated form sheep brain has been shown to consist of eight isoforms by reverse‐phase HPLC. Direct protein sequence analysis has revealed these to be the same as those of 14‐3‐3 protein, described as an activator of tyrosine and tryptophan hydroxylases involved in neurotransmitter biosynthesis. The N‐termini of KCIP‐1 isoforms were shown to be acetylated, and secondary structure predictions revealed a high degree of α‐helix with an amphipathic nature. KCIP‐1 showed no inhibitory activity towards protein kinase M (the catalytic fragment of PKC) and had no effect on the activities of three other protein kinases, cAMP‐dependent protein kinase, Ca2+/calmodulin‐dependent protein kinase II and casein kinase 2. Four forms of KCIP‐1 were shown to be substrates for PKC in vitro, but none were phosphorylated by the other protein kinases mentioned above.
Frontier orbital study on the 4‐hydroxybenzoate‐3‐hydroxylase‐dependent activity with benzoate derivatives
Authors: VERVOORT Jacques , RIETJENS Ivonne M. C. M. , van BERKEL Willem J. H. , VEEGER Cees
Published: March 2005

Based on molecular orbital computer calculations the present paper provides a new hypothesis for catalytic characteristics of 4‐hydroxybenzoate‐3‐hydroxylase (EC 1.14.13.2). A clear correlation between In kcat for the conversion of a series of 4‐hydroxylated substrates and their E(HOMO) leads to the hypothesis that Frontier orbital HOMO characteristics [E(HOMO) and HOMO density on C3] of the substrates are the predominant factor in regulating the fate of a benzoate derivative at the active site of the enzyme. The HOMO characteristics can be used to explain whether a compound will be converted by the enzyme or merely acts as an effector. Furthermore the hypothesis provides quantitative theoretical support for a catalytic mechanism in which the substrate reacts in its dianionic form and for a mechanism in which the electrophilic attack of the C(4a)‐peroxyflavin, or of the hydroxyl radical derived from it, on the benzoate dianion is the rate limiting step in catalysis at pH 8, 25°C.

Finally, it is demonstrated that the hypothesis can be used as a basis for the formulation of working hypotheses in future research, investigating the conversion and regioselective orientation of the various possible substrates in the active site of the wild‐type 4‐hydroxybenzoate‐3‐hydroxylase, its mutants as well as of various other flavin‐dependent aromatic hydroxylases, such as for example 3‐hydroxybenzoate‐4‐hydroxylase (EC 1.14.13.23), 3‐hydroxybenzoate‐6‐hydroxylase (EC 1.14.13.24) and phenol hydroxylase (EC 1.14.13.7).
Mammalian thymidine kinase 2
Authors: JANSSON Olof , BOHMAN Christina , MUNCH‐PETERSEN Birgitte , ERIKSSON Staffan
Published: March 2005

Thymidine kinase 2 (TK2), also called mitochondrial thymidine kinase, is a pyrimidine deoxyribonucleoside kinase expressed in all cells and tissues. It was recently purified to apparent homogeneity from human leukemic spleen and the active enzyme was shown to be a monomer of a 29‐kDa polypeptide. The enzyme is feedback‐inhibited by both end products, dCTP and dTTP. Here we show that TK2 purified from several different sources, including purified beef heart mitochondria, could be directly photoaffinity labeled with radioactive dTTP (approximately 18% of all TK2 molecules were cross‐linked to dTTP after 20 min of ultraviolet irradiation) or to a lower extent with dCTP.

Photo‐incorporation was inhibited by the presence of the other effector but also the phosphate donor ATP blocked photolabeling, with dTTP. Addition of nucleoside substrates gave only a marginal inhibition of photo‐incorporation. There were no detectable difference in the molecular size of photolabeled TK2 isolated from human spleen, brain or placenta, monkey liver, beef heart and beef heart mitochondria. Nor was there any significant differences in the enzyme kinetic properties of these enzymes. Cleavage of labeled TK2 with cyanogen bromide showed that dTTP was incorporated into a single 3‐kDa peptide.

TK2 was the only pyrimidine deoxynucleoside kinase expressed in liver, heart and brain. A detailed characterization of the subunit structure and substrate specificity of this enzyme is of importance for the design of new antiviral and cytostatic therapies based on nucleoside analogs.
Substrate specificity of purified rabbit liver esterase ES‐1
Authors: Van LITH Hein A. , HALLER Manuela , Van ZUTPHEN Bert F. M. , BEYNEN Anton C.
Published: March 2005

1. Substrate hydrolysis by two purified rabbit liver esterase‐1 allozymes (ES‐1A and ES‐1B) was compared under conditions differing in substrate, pH and temperature. ES‐1A and ES‐1B activities had a similar pH and temperature dependency and similar thermal stability profile. 2. There were marked differences in specific activity of ES‐1A and ES‐1B. ES‐1A hydrolysed procaine more rapidly than ES‐1B, but was less active towards aspirin. The acetate and propionate esters of p‐nitrophenyl were hydrolysed slower by ES‐1A than by ES‐1B. 3. The effect of substrate concentration on ES‐1A activity did not comply with the Michaelis‐Menten kinetics, which may be due to so‐called substrate activation. 4. At identical substrate concentration, pH and temperature, selected artificial esters were better substrates for ES‐1A than selected physiological substrates. β‐Naphthyloctanoate was found to be a suitable substrate for ES‐1A. 1,3‐Dioctanoylglycerol was hydrolysed at a rate of only 2% of that of β‐naphthyloctanoate. 5. With methyl, p‐nitrophenyl, β‐naphthyl and 4‐methylumbelliferyl esters as substrates, ES‐1A activity is influenced by length and structure of the acyl moiety. Likewise, ES‐1A activity is influenced by the nature of the alkyl moiety of acetate esters. With acetate and methyl esters, branched chains when compared with unbranched chains reduced the esterase activity of ES‐1A. Elongation of the acyl moiety up to four or five C atoms gradually raised the velocity of methyl, p‐nitrophenyl, and 4‐methylumbelliferyl ester hydrolysis by ES‐1A. A similar pattern was found for the length of the alkyl moiety of acetate esters. 6. The high degree of similarity between the observed substrate specificity of rabbit ES‐1A and that reported earlier for rat ES‐10, suggests that these two esterases have a common evolutionary origin.
Presence of a sodium‐translocating ATPase in membrane vesicles of the homoacetogenic bacterium Acetobacterium woodii
Authors: HEISE Reno , MÜLLER Volker , GOTTSCHALK Gerhard
Published: March 2005

Inverted membrane vesicles of the homoacetogenic bacterium Acetobacterium woodii catalyzed the hydrolysis of ATP with a rate of 100–150 nmol · min−1· mg protein−1. The ATPase was stimulated 1.4–1.6‐fold by NaCl and inhibited by N,N′‐dicyclohexylcarbodiimide, tributyltin or azide. The degree of inhibition caused by F0‐directed but not F1‐directed inhibitors was affected by the Na+ concentration in the medium. These experiments indicated the presence of a sodium‐translocating ATPase. This was verified by transport studies. Upon addition of ATP to inverted vesicles, 22Na+ was actively transported into the intravesicular space up to a 24‐fold accumulation. Na+ transport was inhibited by the sodium ionophore N,N,N′,N′,‐tetracyclohexyl‐1,2‐phenyl‐enedioxydiacetamide but stimulated by valinomycin with potassium whereas the protonophore 3,5,‐di‐tert‐butyl‐4‐hydroxybenzylidenemalonitrile was without effect. N,N′‐dicyclohexylcarbodiimide and tributyltin inhibited 22Na+ transport. These experiments are in accordance with a primary electrogenic Na+ transport as catalyzed by a F1F0‐ATPase.
DNA‐activated protein kinase in Raji Burkitt's lymphoma cells
Authors: IIJIMA Shigeyuki , TERAOKA Hirobumi , DATE Takayasu , TSUKADA Kinji
Published: March 2005

Autophosphorylation of a DNA‐activated protein kinase (DNA‐PK) in Raji Burkitt's lymphoma cells generated a band that corresponded to a phosphoprotein of about 300 kDa on SDS/PAGE. This band corresponds to a 300–350‐kDa DNA‐PK found previously in HeLa cells. In addition to the 300‐kDa phosphoprotein, the band of a highly phosphorylated 58‐kDa protein was detected by SDS/PAGE of partially purified DNA‐PK preparations after the phosphorylation reaction in the presence of double‐stranded DNA. this phosphoprotein was specifically immunoprecipitated by mAb against c‐Myc. More highly purfied preparations of DNA‐PK, containing neither a 58‐kDa phosphoprotein nor detectable activities of other kinases, phosphorylated recombinant c‐Myc proteins in the presence of DNA. the c‐Myc phosphorylation by DNA‐PK was markedly stimulated by relxed, double‐stranded DNA, but neither by single‐stranded DNA nor by RNA. Phosphopeptide mapping and phosphoamino acid analysis indicated that DNA‐PK phosphorylates c‐Myc in vitro at several serine residues.
Purification, primary structure, bacterial expression and subcellular distribution of an oocyte‐specific protein in Xenopus
Authors: ROTHER Russell P. , FRANK Mark B. , THOMAS Patricia S.
Published: March 2005

This study defines a novel Xenopus laevis protein (P100) that has recently been shown to be recognized by scleroderma patient sera. Using a combination of differential solubility in detergents, hydroxyapatite chromatography and one‐dimensional PAGE, P100 was purified to apparent homogeneity and the amino acid sequence was obtained. An oligonucleotide derived from this sequence was used to clone P100 cDNA through a polymerase‐chain‐reaction cloning strategy. The entire P100 cDNA sequence was determined, identifying a novel 83000‐Da protein. Two alleles for P100 were transcribed in the oocyte, with only one predicted amino acid change between them. Bacterial expression of a clone containing the entire P100 coding region produced a protein that migrated at a mass 15% greater than that predicted from the amino acid sequence, indicating an aberrant electrophoretic mobility. The mRNA transcript for P100 was only expressed during the previtellogenic stages of oogenesis (stages I and II) and was absent from other Xenopus tissues. Similarly, the P100 protein was found only in Xenopus oocytes and was localized to the cytoplasm of these cells. P100 irreversibly bound single‐stranded‐DNA–cellulose but not double‐stranded‐DNA–cellulose. These data demonstrate the presence of a novel oocyte‐specific protein in Xenopus.
C‐terminal sequencing of protein
Authors: TSUGITA Akira , TAKAMOTO Keiji , KAMO Masaharu , IWADATE Hiromoto
Published: March 2005

Peptides or proteins were hydrolyzed by vapors of 90% pentafluoropropionic acid or heptafluorobutyric acid at 90°C for various time periods. The hydrolyzate mixtures analyzed by both fast‐atom‐bombardment and electrospray ionization mass spectrometry showed a series of C‐terminal successive degradation molecular ions. The degradation reaction may be due to the selective formation of an oxazolone ring at the C‐terminal amino acid, followed by hydrolytic removal of the C‐terminal amino acid. The major side reactions were cleavages of the peptide bonds at the C side of the internal aspartic acid residue and the N side of serine residue.
Ascorbate‐mediated transmembrane electron transport and ascorbate uptake in leukemic cell lines are two different processes
Authors: SCHWEINZER Esther , GOLDENBERG Hans
Published: March 2005

Transmembrane reduction of extracellular oxidants by K562 and U937 leukemic cells was stimulated by catalytic amounts of ascorbate or dehydroascorbate. This stimulation was not due to transport of ascorbate in different redox states in and out of the cells. The membrane redox cycle was strictly dependent on the presence of the cells at every stage, and showed high affinity for ascorbate with simple linear kinetics. Metabolic inhibitors and sulfhydryl reagents inhibited this stimulation. Ascorbate uptake was also dependent on oxidation, but in a very different manner and with much lower affinity for ascorbate. The uptake was non‐saturable in the concentration range used. There was some release of ascorbate from the cells, which cannot account for an appreciable part of the reduction of extracellular electron acceptors.
Changes in membrane fluidity and Na+/K+‐ATPase activity during human trophoblast cell culture
Authors: MAZZANTI Laura , RABINI Rosa Anna , BIAGINI Graziella , PUGNALONI Armanda , DE PIRRO Roberto , FALOIA Emanuela , MANCINI Valentina , ROMANINI Carlo , CESTER Nelvio
Published: March 2005

The human placenta plays an essential role in embryo development, in particular regulating the transport of ions, nutrients and immunoglobulins from the maternal to the fetal circulation. Trophoblast organization into a syncytial layer involves structural and functional steps that may be monitored and elucidated by in vitro studies. The structural stages by which the syncytial trophoblast is formed are not yet understood. In order to clarify the mechanism of trophoblast development, we studied the morphological characteristics of the syncytial trophoblast formation in culture and the functional changes (transport properties and membrane microviscosity) accompanying the structural modifications.

By using both 5‐nitroxystearate and 16‐nitroxystearate as spin labels, we observed an initial increase in membrane order over 0–24 h of culture, which can be associated with two events: recovery of cell membranes from trypsin and initial aggregation of cytotrophoblasts. The similar behaviour of the order parameters determined with both probes indicates that membrane order changes both inside and in the outer part of the lipid bilayer. The subsequent decrease in membrane order observed at 36–48 h might be related to the process of cellular fusion.

The increase in sodium/potassium pump activity in the first 24 h of culture might be an expression of cell recovery following trypsin treatment. The subsequent decrease might represent an adaptive mechanism by which metabolic energy is mainly used for morphogenetic changes.
Regulation of gene expression by cytokines and virus in human cells lacking the type‐I interferon locus
Authors: WATHELET Marc G. , BERR Pascale M. , HUEZ Georges A.
Published: March 2005

A number of genes that are induced by type‐I interferons are also activated by one or more other inducers, including double‐stranded RNA, viruses, interferon‐γ, interleukin‐1 and tumor necrosis factor. However, these inducers can also activate the expression of type‐I interferons. Thus, the activation of type‐I interferon‐inducible genes by these other inducers could be direct, or a secondary consequence of the induction of interferon. To distinguish between these possibilities, we have used cell lines lacking all type‐I interferon genes to study the direct effect of potential inducers on the expression of 14 interferon‐inducible human genes. We show that double‐stranded RNA, virus, interferon‐γ or tumor necrosis factor‐α can act directly to induce specific subsets of type‐I interferoninducible genes in the absence of any possible type‐I interferon involvement. The cis‐acting element which confers inducibility by type‐I interferon has been shown in some cases to confer inducibility by interferon‐γ, double‐stranded RNA or virus as well. However, not all promoters containing such an element respond to both interferon and other inducers. Thus, the ability of a given gene to respond to different inducers most likely depends on the exact nature and specific combination of cis‐acting elements present in its promoter.
Activation of ATPase activity of 14S dynein from Tetrahymena cilia by microtubules
Authors: SHIMIZU Takashi , HOSOYA Natsumi , HISANAGA Shin‐ichi , MARCHESE‐RAGONA Silvio P. , PRATT Melanie M.
Published: March 2005

The ATPase activity of 14S dynein was activated by the presence of microtubule‐associated‐protein‐free microtubules. The activation was 2.5–3.5 fold at 10 mg microtubule/ml, and the activity increased further with increasing microtubule concentration. The microtubule–14S‐dynein complex, microtubule bundles with 14S dynein, was treated with a zero‐length chemical cross‐linker, 1‐ethyl‐3‐[3‐(dimethylamino)propyl]carbodiimide (EDC). The ATPase activity of the complex responded to EDC in a biphasic, concentration‐dependent manner and, at most, it was enhanced 5–10 fold. The complex treated with EDC was no longer unbundled by addition of ATP, as revealed by electron‐microscopic observation.

Several ATP analogues, which support in vitro microtubule translocation mediated by 14S dynein, were turned over faster by this mechanochemical enzyme in the presence of microtubules than in their absence. However, some ATP analogues which do not support the translocation were also turned over faster in the presence of microtubules. Thus, microtubule‐dynein motility and substrate‐turnover activation are not tightly coupled, which indicates that all three major motor systems, actin–heavy‐meromyosin, microtubule‐kinesin [Shimizu, T., Furusawa, K., Ohashi, S., Toyoshima, Y. Y., Okuno, M., Malik, F. & Vale, R. D. (1991) J. Cell Biol. 112, 1189–1197] and microtubule‐dynein, have this characteristic property in common.
Requirement of N‐ and C‐terminal regions for enzymatic activity of human T‐cell leukemia virus type I protease
Authors: HAYAKAWA Takaki , MISUMI Yuko , KOBAYASHI Makoto , YAMAMOTO Yoshio , FUJISAWA Yukio
Published: March 2005

The requirement of N‐ and C‐terminal regions for the enzymatic activity of human T‐cell leukemia virus type I (HTLV‐I) protease was investigated using a series of deletion mutants. The activity was analyzed by autoprocessing of the protease itself or by processing of the gag p53 precursor. The deletional analyses indicated that Asp38–Gly152 with an additional Met‐Pro sequence at the N‐terminus was probably sufficient for the enzymatic activity, although the mature HTLV‐I protease consists of Pro33–Leu157. A molecular model of HTLV‐I protease, which was constructed by comparison with the structure of Rous sarcoma virus protease, predicted that Pro33–Leu37 and Gly143–Leu147 would form a β‐sheet. Our experimental results and the model structure suggest that (a) five amino acids in the N‐terminal region (Pro33–Leu37), which are thought to be involved in the β‐sheet, are not crucial for the enzymatic activity; (b) Pro153–Leu157 is not necessary but Pro148–Gly152 is important for the enzymatic activity, in addition to Gly143–Leu147 involved in the β‐sheet.
Structural and functional characterization of Escherichia coli peptidyl‐prolyl cis‐trans isomerases
Authors: COMPTON Larry A. , DAVIS Janice M. , MACDONALD J. Randy , BÄCHINGER Hans Peter
Published: March 2005

Peptidyl‐prolyl cis‐trans isomerases (PPIases), enzymes that catalyze the cis‐trans isomerization of peptide bonds to which proline contributes the nitrogen, were purified from Escherichia coli. In this organism, at least two PPIases are present. Both the cationic (periplasmic) and anionic (cytoplasmic) PPIases are inhibited by cyclosporin A with a Ki of 25–50 μM, a concentration 1000‐fold higher than that required for eukaryotic PPIases. Although isoelectric focusing indicates that the two enzymes differ in isoelectric point by at least 4.0 pH units, the specific activities of the enzymes toward the tetrapeptide substrate succinyl‐Ala‐Ala‐Pro‐Phe‐methyl‐coumarylamide are equivalent. The activity of both enzymes for a series of substituted succinyl‐Ala‐Xaa‐Pro‐Phe‐para‐nitroanilide tetrapeptides suggests that the structure and function of the active site of the prokaryotic proteins is similar to that of eukaryotic cyclophilins. Both enzymes are capable of catalyzing the refolding of thermally denatured type III collagen. Antibodies against the periplasmic PPIase do not recognize the cytoplasmic enzyme, indicating significant differences in epitopes between the two forms. Circular dichroism spectroscopy indicates that the secondary structure of the cationic protein consists of 17%α‐helix, 34%β‐sheet, 17% turns, 33% random coil and is very similar to human cytosolic PPIase.
Yeast actin is relatively well behaved
Authors: NEFSKY Bradley , BRETSCHER Anthony
Published: March 2005

Actin from yeast has been reported previously to have unusual polymerization properties. Here we report a simple sensitive spot assay for actin and use it to develop a high‐yield procedure for the purification of actin from the yeast Saccharomyces cerevisiae. The polymerization properties of purified yeast actin are quantitatively similar to all other characterized actins. We have characterized this actin with respect to its ability to interact with yeast profilin and tropomyosin, the only yeast actin‐binding proteins so far purified and characterized. Yeast profilin can sequester yeast actin monomers and thereby reduce the ability of yeast actin to polymerize, whereas it has little effect on the degree of polymerization of rabbit skeletal muscle actin. By contrast, there is no apparent difference between the binding of yeast or smooth muscle tropomyosin to yeast or rabbit skeletal muscle actin. The availability of purified yeast actin should facilitate a detailed examination of its interaction with recently discovered yeast actin‐binding proteins.

Greer and Schekman (1982) [Greer, C. & Scheckman, R. (1982), Mol. Cell Biol. 2, 1279–1286] reported that an intrinsic property of yeast actin is a Ca2+ dependent increase in critical concentration with the formation of 15–50‐nm particles. Our purified actin does not have this property. By modifying the purification protocol, we can obtain a preparation having a Ca2+‐dependent change in polymerization properties. The Ca2+‐dependent effect results in a slower polymerization rate as well as the formation of shorter filaments. Since this effect could be mediated by a protein present at a very low stoichiometry to actin, and we do not see any contaminating peptides, we have not pursued this effect further. We suggest that the Ca2+‐dependent properties of the Greer and Schekman preparation are most likely due to a minor contaminant.
Defining the role of protein kinase c in calcium‐ionophore‐(A23187)‐mediated activation of phospholipase A2 in pulmonary endothelium
Authors: CHAKRABORTI Sajal , MICHAEL John R. , SANYAL Tapati
Published: March 2005

We sought to investigate the mechanisms by which the calcium ionophore A23187 triggers arachidonic acid release in bovine pulmonary endothelial cells and to test the hypothesis that protein kinase C is involved in this process. Our results indicate that the mechanism by which A23187 increases phospholipase A2 activity and arachidonic acid release in bovine pulmonary arterial endothelial cells depends upon the concentration studied. At concentrations of 1 μM and 2.5 μM, A23187 increases phospholipase A2 activity and arachidonic acid release without stimulating protein kinase C. At concentrations of 5–12.5 μM, A23187 increases arachidonic acid release and phospholipase A2 activity in conjunction with a dose‐dependent activation of membrane‐bound protein kinase C. To test the hypothesis that these doses of A23187 increase phospholipase A2 activity by stimulating protein kinase C, we studied the effect of prior treatment with the protein kinase C inhibitor sphingosine. Sphingosine inhibits the increase in phospholipase A2 activity and arachidonic acid release caused by A23187 over the range 5–12.5 μM. To investigate further the potential role of protein kinase C, we studied the effects of the inactive phorbol ester 4α‐phorbol 12β‐myristate 13α‐acetate (4α‐PMA) and an active phorbol ester 4β‐phorbol 12β‐myristate 13α‐acetate (4β PMA). Neither 4α‐PMA nor 4β‐PMA affected basal arachidonic acid release. 4α‐PMA also did not augment the effects of A23187. In contrast, 4β‐PMA significantly augments the increase in phospholipase A2 activity and arachidonic acid release caused by lower doses of A23187. Under these conditions, sphingosine completely inhibits the stimulatory effects of 4β‐PMA on protein kinase C translocation, phospholipase A2 and arachidonic acid release. Thus, at low doses (1 μM and 2.5 μM) A23187 increases phospholipase A2 activity and arachidonic acid release by a mechanism that does not involve protein kinase C. At these A23187 doses, activating membrane‐bound protein kinase C with 4β‐PMA causes a synergistic increase in phospholipase A2 activity and arachidonic acid release. At higher doses (5–12.5 μM), A23187 acts in large part by stimulating protein kinase C translocation. Overall, our results indicate that activating membrane‐bound protein kinase C by itself is an insufficient stimulus to increase phospholipase A2 activity and arachidonic acid release in pulmonary endothelial cells, but activating protein kinase C can substantially augment the increase in phospholipase A2 activity and arachidonic acid caused by a small increase in intracellular calcium.
Aprotinin can inhibit the proteolytic activity of thrombin
Authors: PINTIGNY Dominique , DACHARY‐PRIGENT Jeanne
Published: March 2005

Aprotinin has been shown to reduce blood loss and blood requirement when administered prior to surgery and this therapeutic benefit appears to be related to its specificity as a protease inhibitor. The inhibition of plasmin by aprotinin is well characterized, but little is known of its effect on thrombin. In preliminary experiments, we showed that aprotinin can prevent platelet aggregation induced by thrombin. Follow‐up studies have now been performed in order to clarify the effect of aprotinin on thrombin. A fluorescence study of the direct binding of aprotinin to human α‐thrombin was analysed according to the Michaelis‐Menten model and a dissociation constant of 30 × 10−6 mol · l−1 was determined. Aprotinin can displace p‐aminobenzamidine, a fluorescent‐probe molecule which binds to the active site of serine proteases, showing that the active site of thrombin was involved. Aprotinin also inhibited the ability of thrombin to induce a fibrin clot from purified fibrinogen and to induce the hydrolysis of the chromogenic substrate H‐D‐phenylalanylpipecolylarginine‐p‐nitroanalidehydrochloride (S‐2238). With S‐2238, double‐reciprocal plots show that the inhibition is competitive with a Ki of 61 μM and a Km of 1.72 μM. Aprotinin was a potent inhibitor of thrombin‐induced aggregation. A Schild plot of the aggregation data yielded a slope of 0.97 ± 0.12 and an apparent dissociation constant of 57.0 ± 13.1 μM (mean ± SEM). Thus, the inhibition of thrombin‐induced platelet aggregation by aprotinin fits a model of competitive inhibition. Conclusions are that, in addition to a possible direct effect of aprotinin on platelets, the inhibition of thrombin‐induced platelet activation by aprotinin can be also explained, in part, by a direct effect of the inhibitor on the thrombin molecule itself. This supports the concept that a proteolytic step is involved in the platelet response to thrombin. Finally, evidence is in favour of the participation of Trp245 in the fluorescence response of thrombin on binding to aprotinin.
Purification and partial characterization of a β‐1,3‐glucan‐binding‐protein membrane receptor from blood cells of the crayfish Pacifastacus leniusculus
Authors: DUVIC Bernard , SÖDERHÄLL Kenneth
Published: March 2005

A receptor for the 100 kDa β‐1,3‐glucan‐binding protein [Duvic, B. and Söderhäll, K. (1990) J. Biol. Chem. 265, 9327–9332] has been purified from hemocyte membranes of the crayfish Pacifastacus leniusculus. The purification was achieved by DEAE‐cellulose chromatography of detergentsolubilized membranes. The receptor had an apparent molecular mass of 350 kDa when subjected to native polyacrylamide‐gel electrophoresis and was composed of two non‐covalently associated subunits of about 230 kDa and 90 kDa, as judged by SDS/polyacrylamide‐gel electrophoresis or two‐dimensional electrophoresis. The receptor could only bind the β‐1,3‐glucan‐binding protein if this protein had previously reacted with a β‐1,3‐glucan, laminarin, and the binding site was located on the 230 kDa subunit. The binding of laminarin‐treated β‐1,3‐glucan‐binding protein to its receptor was a saturable process and binding data indicated a single high‐affinity‐binding site with a Kd of 0.35 ± 0.15 μM as determined by Scatchard analysis. The receptor had a requirement for divalent cations and a pH optimum of 6.5 for binding the laminarin‐treated β‐1,3‐glucan‐binding protein. Laminarin, as well as oligosaccharides such as D‐glucose, sialic acid, N‐acetyl glucosamine or methyl‐α‐D‐mannoside, could not affect the binding of the β‐1,3‐glucan‐binding protein to its receptor.
Transcript levels for nuclear‐encoded mammalian mitochondrial respiratory‐chain components are regulated by thyroid hormone in an uncoordinated fashion
Authors: LUCIAKOVA Katarina , NELSON B. Dean
Published: March 2005

Thyroid hormone is one of the few known physiological regulators of mammalian mitochondrial biogenesis. Although it exerts a global effect on biogenesis, it does so by regulating the expression of a limited number of unidentified mitochondrial proteins. We have investigated these hormone‐regulated proteins in rat liver. Hormone injection induced a 30‐fold increase in the levels of cytochrome‐c1 mRNA after 3 d. In addition, the mRNA for the growth‐activated adenine‐nucleotide translocator, ANT2, was increased 13‐fold and that for the ATPase N,N′‐dicyclohexylcarbodiimidebinding protein increased 4–5‐fold. Mitochondrial transcripts of cytochrome‐oxidase subunit I also increased. No changes were found in the mRNA levels for the F1‐ATPase β‐subunit or cytochrome oxidase IV. A single low dose of triiodothyronine induces rapid increases in cytochrome‐c1 and ANT2 mRNA species which parallel changes in the activity of the hormone‐responsive malic enzyme, but are earlier than other mitochondrial biogenetic events. These data strengthen the view that thyroid hormone regulates synthesis of specific components within each respiratory‐chain complex and that these products apparently play key roles in inner‐membrane biogenesis and assembly. The significance of ANT2 induction is also discussed with respect to the rapid respiratory response induced by thyroid hormone.