bluuebunny/crossref_metadata_2025 huggingface dataset:
The ω‐hydroxylation of arachidonic acid by human polymorphonuclear leukocytes
Authors: HATZELMANN Armin , ULLRICH Volker
Published: March 2005

Incubations of [1‐14C]arachidonic acid with unstimulated human polymorphonuclear leukocytes resulted in the formation of four new metabolites in a previously described reverse‐phase HPLC system. Three of these metabolites were largely suppressed in a CO/O2 (80/20, by vol.) atmosphere indicating a cytochrome‐P450‐dependent monooxygenase reaction. In agreement with this assumption is their NADPH/O2‐dependent formation in the microsomal fraction. One metabolite was identified by gas chromatography/mass spectrometry analysis as ω‐hydroxy‐arachidonic acid and the two others were secondary products identified as ω‐carboxy‐arachidonic acid and 5,20‐dihydroxy‐E,Z,Z,Z‐6,8,11,14‐eicosatetraenoic acid. Since the affinity for arachidonate of the ω‐monooxygenase was quite low and the presence of LTB4 suppressed the ω‐hydroxylation of arachidonate, we conclude that the known LTB4ω‐monooxygenase is responsible for the formation of ω‐hydroxy‐arachidonate. It is unlikely, however, that significant concentrations of these metabolites are formed by activated polymorphonuclear leukocytes in vivo. The fourth metabolite remains tightly associated with the leukocytes but has not been further characterized.
Nucleotide sequence of cDNA and predicted amino acid sequence of rat liver uricase
Authors: ITO Masaki , SUZUKI Masami , TAKAGI Yasuyuki
Published: March 2005

A cDNA clone for rat liver uricase (EC 1.7.3.3), which is localized in the core of peroxisomes, was isolated from a rat liver cDNA library in λgt11. The clone, referred to as λURC‐1, induced formation of a fusion protein (molecular mass 140 kDa) in the presence of isopropyl β‐d‐thiogalactoside. Immunoglobulin G reactive with the fusion protein recognized only a protein with molecular mass of 33 kDa, corresponding to the molecular mass of uricase. The nucleotide sequence of the isolated cDNA was determined and the amino acid sequence was predicted. An open reading frame was identified and found to encode a polypeptide of 280 amino acids with a molecular mass of 32226 Da. The cDNA contained 14 base pairs of 5′‐untranslated sequence and 192 base pairs of 3′‐untranslated sequence. The sequences of four internal peptide fragments, determined by Edman degradation, were identical to parts of the sequence predicted from the cDNA. The complete amino acid sequence predicted for rat liver uricase was compared with that of soybean nodulin uricase and nine highly homologous regions of the two enzymes were found.
Nuclease hypersensitivity in the β‐globin gene region of K562 cells
Authors: CAO Shi‐Xian , SCHECHTER Alan N.
Published: March 2005

We have investigated chromatin structure in the β‐globin gene region of the K562 human erythroleukemic cell line by using S1 and DNase I nuclease sensitivity assays. Despite the lack of β‐globin gene expression in these cells, we find nuclease‐hypersensitive sites to these enzymes in its 5′ and 3′ flanking regions in K562 chromatin. This result is in contrast to previous reports in which no hypersensitive sites were found in the immediate vicinity of this gene. In the 3′ region, one major hypersensitive site at 0.9 kpb 3′ and three minor hypersensitive sites at 0.7 kbp, 0.5 kbp 3′ and 0.2 kbp 5′ of the polyadenylation site were observed; these sites are very similar to those found in fetal liver and adult bone marrow cells in which the β‐globin gene is expressed. We find hypersensitive sites to both enzymes in the 5′ region of the β‐globin gene: a major site 0.8 kbp 5′ to the cap site, and two minor sites 1.2 and 1.5 kbp 5′ to the cap site. The –0.8 kbp site is also present in plasmids containing the β‐globin gene. Our results suggest that the lack of β‐globin gene expression may be related to the lack of hypersensitivity sites in the immediate (150 bp) 5′ flanking region of the β‐globin gene, as occurs in other active globin genes.
The amino acid sequence of wheat histone H2B(2)
Authors: BRANDT Wolf Friedrich , de ANDRADE RODRIGUES Jeronimo , VON HOLT Claus
Published: March 2005

Two of the four electrophoretic histone H2B variants present in wheat embryos have been isolated. The complete primary structure of the H2B(2) variant has been deduced from sets of overlapping peptides generated by CNBr cleavage, Staphyloccocus aureus V8 protease, endoproteinase Arg‐C, the post‐proline cleaving enzyme, chymotrypsin and cleavage in dilute acid. A minimum of 17 peptides were required to establish the sequence. This variant has a blocked N terminus and comprises a total of 149 amino acids. The C‐terminal two‐thirds of the protein are highly homologous to vertebrate H2B. In contrast, the N‐terminal third is entirely different and contains an N‐terminal extension of 23 residues in which the sequence Ala‐Glu‐Lys or variants are repeated several times. This region is also highly homologous to the H2B from Tetrahymena pyriformis. It shows in addition similarities to wheat H2A(1), and bovine H1.
The primary structure of the histone H2A(2) type from wheat germ
Authors: de ANDRADE RODRIGUES Jeronimo , BRANDT Wolf Friedrich , VON HOLT Claus
Published: March 2005

The histone H2A(2) type from wheat germ comprises at least two highly homologous isohistones with 151 amino acid residues. Microheterogeneity occurs mainly at the N‐terminal and C‐terminal regions, These isohistones have both N‐terminal (7 amino acid residues) and C‐terminal (15 amino acid residues) extensions relative to calf thymus histone H2A.
Temperature‐dependent binding to the thylakoid membranes of nuclear‐coded chloroplast heat‐shock proteins
Authors: GLACZINSKI Heike , KLOPPSTECH Klaus
Published: March 2005

Two nuclear‐coded heat‐shock proteins (HSP) of pea (Pisum sativum) are synthesized as larger precursors of 26 kDa and 30 kDa in vitro. They are transported post‐translationally into isolated, homologous chloroplasts where they are processed to mature proteins of 22 kDa and 25 kDa, respectively. When the chloroplasts used for the transport are isolated from control plants grown at 25°C the 22‐kDa and 25‐kDa HSPs are located in the stroma of the chloroplasts. However, when chloroplasts are prepared from heat‐shocked plants both proteins are found bound to the thylakoid membranes. The transition of the non‐binding to the binding status is comparatively sharp and occurs between 36°C and 40°C in the variety ‘Rosa Krone’. The transition temperature has been determined at 38°C for ‘Rosa Krone’ and at 40°C for the variety ‘Golf’. At 42°C, 15‐min treatment of the plants is sufficient to induce membrane binding, which persists for at least 4–6 h (but not for 24 h) after return to the ambient temperature. Once lost, membrane binding can be reinduced by a second heat‐shock treatment in vivo. High light intensities during the heat shock interfere with the binding capacity for heat‐shock proteins.
Aminopeptidase yscII of yeast
Authors: HIRSCH Hans H. , RENDUELES Paz SUÁREZ , ACHSTETTER Tilman , WOLF Dieter H.
Published: March 2005

Mutant strains of the yeast Saccharomyces cerevisiae defective in aminopeptidase yscII were isolated by screening for reduced external activity against the chromogenic substrate lysine β‐naphthylamide. One of the selected mutant strains analyzed in detail showed wild‐type staining activity when tested at 23°C but mutant activity after exposure to 37°C, suggesting a temperature‐sensitive mutation. Electrophoretic separation of mutant crude extracts on non‐denaturing polyacrylamide gels and subsequent activity staining using lysine and leucine β‐naphthylamides as substrates revealed that in all strains isolated the same distinct activity band was affected, which corresponded to the aminopeptidase activity identified previously as aminopeptidase yscII [Achstetter, T., Ehmann, C. & Wolf, D. H. (1983) Arch. Biochem. Biophys. 226, 292–305].

All mutants strains isolated fell into the same complementation group. Tetrad dissection of sporulated diploids heterozygous for the wild‐type and mutant allele resulted in a 2:2 segregation of mutant and wild‐type phenotype indicating a single gene mutation. The characteristics of the mutations analyzed point to the gene which we called APE2 as the structural gene of aminopeptidase yscII. No vital consequences of aminopeptidase yscII deficiency on cell life and differentiation could be detected. However, the enzyme seems to be involved in the cellular supply of leucine from externally offered leucine‐containing dipeptide substrates.
Ion binding to cytochrome c
Authors: AREAN Carlos Otero , MOORE Geoffrey R. , WILLIAMS Glyn , WILLIAMS Robert J. P.
Published: March 2005

This paper is a further study of ion binding to protein surfaces and builds on the studies of the binding of [Cr(CN)6]3− and [Fe(edta)(H2O)]− previously reported [Williams et al. (1982) FEBS Lett. 15, 293–299; Eley et al. (1982) Eur. J. Biochem. 124, 295–303]. In the present paper the binding of polyaminocarboxylate complexes of gadolinium have been studied. Eight ion‐binding sites have been identified on the surface of cytochrome c. These exhibit different binding specificities which, in some cases, are not fully understood. However it is clear that simple outer‐sphere interactions are not the sole determining factor for the association of metal ion complexes with proteins. The NMR paramagnetic difference spectrum method has been shown to be good at locating binding sites and revealing qualitative differences in their relative affinities for a range of complex types. However the use of relaxation probes is not a good method for the quantitative determination of binding constants; for this, isostructural shift probes must be sought.
Membrane‐potential‐dependent changes in the stoichiometry of charge translocation by the mitochondrial electron transport chain
Authors: MURPHY Michael P. , BRAND Martin D.
Published: March 2005

The charge/oxygen (q+/O) stoichiometry of mitochondria respiring on succinate was measured under conditions of high membrane potential (ΔΨ). The technique used was a variation of the steady‐state method of Al‐Shawi and Brand [(1981) Biochem. J. 200, 539–546]. We show that q+/O was about 2.7 at high values of ΔΨ (170 mV). As ΔΨ was lowered from 170 mV to 85 mV with the respiratory inhibitor malonate the q+/O stoichiometry increased to 6.0.

A number of artefacts which could have led to an underestimation of the q+/O stoichiometry were eliminated. These included effects of any rapid change in mitochondrial volume, internal pH, activity of the endogenous K+/H+ exchanger or in H+ conductance due to changes in ΔΨ after the addition of inhibitor. The experiments presented here are the first direct demonstration that the stoichiometry of proton pumping by the mitochondrial respiratory chain changes as ΔΨ is varied.
Isolation and characterization of two proteoheparan sulfate species of calf arterial tissue
Authors: SCHMIDT Annette , SCHÄFER Edgar , BUDDECKE Eckhart
Published: March 2005

When calf aortic tissue, preincubated under organ culture conditions in the presence of [35S]sulfate, was submitted to a sequential collagenase and elastase digestion and guanidinium chloride extraction, the bulk of proteoheparan sulfate was obtained in the elastase fraction. Ion‐exchange chromatography on DEAE‐cellulose of the elastase digest under dissociative conditions yielded a proteoglycan fraction that contained heparan sulfate as the sole glycosaminoglycan. The proteoheparan sulfate fraction was resolved into a high‐molecular‐mass (P‐HS 1) and a low‐molecular‐mass (P‐HS 2) fraction by gel filtration on Sephacryl S‐400. P‐HS 1 has a Mr of 175000 and possesses four heparan sulfate side‐chains (Mr 32000) covalently bound to the protein core via a galactose‐and xylose‐containing polysaccharide‐protein binding region. The protein core (Mr 38000), which was obtained after deglycosylation of PG‐HS 1 with trifluormethane sulfonic acid, contained in addition a few N‐glycosidically linked oligosaccharide units representing a complex type with terminal neuraminic acid residues. P‐HS 2 is a single‐chain peptidoheparan sulfate of Mr of 38000 containing one heparan sulfate chain (Mr 32000) linked to a polypeptide (Mr 6000). The ratio of specific radioactivities of P‐HS 1 and P‐HS 2 was 1:0.66.
Purification and characterization of arginine kinase from sea‐urchin eggs
Authors: RATTO Anne , CHRISTEN Richard
Published: March 2005

In most invertebrates, creatine kinase is replaced by arginine kinase, which catalyzes reversibly the transfer of a phosphate group between adenosine triphosphate and arginine. In sea‐urchin larvae, arginine kinase only is expressed whereas in adult sea‐urchins both arginine kinase and creatine kinase can be found in the same tissue. In order to study their developmental regulation and properties, we have purified arginine kinase to homogeneity from the eggs of the sea‐urchin Paracentrotus lividus. The purification involves ethanol and ammonium sulfate precipitations, followed by an anion‐exchange chromatography, an affinity chromatography and a gel filtration. A 500‐fold increase in specific activity leads to a specific activity of 360 IU/mg protein at 25°C. Arginine kinase (pI = 5.7) is rapidly and irreversibly inactivated at 45°C. Amino acid composition and Km values (2.08 mM for phospho‐l‐arginine and 1.25 mM for ADP) are also given. Determination of molecular mass by gel filtration and separation by SDS/polyacrylamide gel electrophoresis indicate that the enzyme is an 81‐kDa dimer of two subunits of42kDa.
The effect of poly(ADP‐ribose) on interactions of DNA with histones H1, H3 and H4
Authors: WĘSIERSKA‐GA̧DEK Józefa , SAUERMANN Georg
Published: March 2005

The competition between poly(ADP‐ribose) and DNA for binding of the histones H1, H3 and H4 was studied, using a membrane filter‐binding test. Poly(ADP‐ribose) differently affected the interaction between DNA and the individual histones. While poly(ADP‐ribose) effectively competed with DNA for binding of histone H4, it equally competed with DNA for binding of histone H3 and only inefficiently competed with DNA for binding of histone H1. Moreover, preformed complexes were correspondingly affected by the addition of competing polynucleotides, thereby also indicating the reversibility of complex formation. The competition capacity of DNA for histone H4 binding did not depend on DNA size. Competition experiments with poly(A) also indicated that poly(ADP‐ribose) preferentially affected DNA‐histone H4 interaction.

The significance of the differing binding properties is discussed with regard to the possible molecular function of poly(ADP‐ribose), especially with regard to its potential effect on nucleosome structure.
Purification and characterization of a ketimine‐reducing enzyme
Authors: NARDINI Mirella , RICCI Giorgio , CACCURI Anna Maria , SOLINAS Sandro Paolo , VESCI Loredana , CAVALLINI Doriano
Published: March 2005

An NAD(P)H‐dependent reductase able to reduce a new class of cyclic unsaturated compounds named ketimines has been detected and purified 2500‐fold from pig kidney. Some molecular and kinetic properties of this enzyme have been determined. The enzymatic reduction proceeds with a classical ping‐pong mechanism and some results suggest that the true substrate has the ketiminic structure and is in equilibrium with the enaminic and keto‐open forms. As previously described, ketimines arise from the deamination of a number of sulfur‐containing amino acids, i.e.l‐cystathionine, l‐lanthionine and s‐aminoethyl‐l‐cysteine, catalyzed by a widespread mammalian transaminase. The enzymatic reduction products of ketimines have been identified as cyclothionine, 1,4‐thiomorpholine 3,5‐dicarboxylic acid and 1,4‐thiomorpholine 3‐carboxylic acid. Some of these compounds have been detected in mammals, thus suggesting a possible role of this enzyme in their biosynthesis.
Bacteriorhodopsin precursor is processed in two steps
Authors: WÖLFER Ulrich , DENCHER Norbert A. , BÜLDT Georg , WREDE Paul
Published: March 2005

Besides the Coomassie‐blue‐stained band corresponding to mature bacterioopsin two additional bands of slightly higher apparent molecular masses were observed in purple membrane preparations from Halobacterium halobium SDS‐PAGE. The staining intensity within the triple band pattern varied with the age of the cell culture. For cells in the stationary growth phase the lower band, corresponding to mature bacterioopsin, is the predominant one. Immunodetection and site‐specific proteolysis with papain identified the upper band as originating from the previously described precursor of bacterioopsin with its 13‐amino‐acid‐long N‐terminal presequence. Our results suggest that the intermediate band is due to a modified precursor of bacterioopsin with a truncated presequence of about eight amino acids. A two‐step mechanism for the processing of pre‐bacterioopsin to the mature protein in this archaebacterium is proposed.
Major pertussis‐toxin‐sensitive GTP‐binding protein of bovine lung
Authors: MORISHITA Rika , KATO Kanefusa , ASANO Tomiko
Published: March 2005

Two GTP‐binding proteins which can be ADP‐ribosylated by islet‐activating protein, pertussis toxin, were purified from the cholate extract of bovine lung membranes. Both proteins had the same heterotrimeric structure (αβγ), but the α subunits were dissociated from the βγ when they were purified in the presence of AlCl3, MgCl2 and NaF. The molecular mass of the α subunit of the major protein (designated GLu, with βγ) was 40 kDa and that of the minor one was 41 kDa. The results of peptide mapping analysis of α subunits with a limited proteolysis indicated that GLuα was entirely different from the α of brain Gi or Go, while the 41‐kDa polypeptide was identical with the α of bovine brain Gi. The kinetics of guanosine 5′‐[3‐O‐thio]triphosphate (GTP[γS]) binding to GLu was similar to that to lung Gi but quite different from that to brain Go. On the other hand, incubation of GLuα at 30°C caused a rapid decrease of GTP[γS] binding, the inactivation curve being similar to that of Goα but different from that of Giα. The α subunits of lung Gi and GLu did not react with the antibodies against the α subunit of bovine brain Go. The antibodies were raised in rabbits against GLuα and were purified with a GLuα‐Sepharose column. The purified antibodies reacted not only with GLuα but also with the 41‐kDa protein and purified brain Giα. However, the antibodies adsorbed with brain Giα reacted only with GLuα, indicating antisera raised with GLuα contained antibodies that recognize both Giα and GLuα, and those specific to GLuα. These results further indicate that GLu is different from Gi or Go. Anti‐GLuα antibodies reacted with the 40‐kDa proteins in the membranes of bovine brain and human leukemic (HL‐60) cells. The βγ subunits were also purified from bovine lung. The β subunit was the doublet of 36‐kDa and 35‐kDa polypeptides. The lung βγ could elicit the ADP‐ribosylation of GLuα by islet‐activating protein, increase the GTP[γS] binding to GLu and protect the thermal denaturation of GLuα. The antibodies raised against brain βγ cross‐reacted with lung β but not with lung γ.
Identification and sequence determination of a cDNA clone for tomato pectin esterase
Authors: RAY John , KNAPP Julie , GRIERSON Don , BIRD Colin , SCHUCH Wolfgang
Published: March 2005

Cell wall softening during tomato fruit ripening is brought about through the action of a number of pectolytic enzymes. We have reported previously the cloning and characterisation of the cDNA for the major cell wall softening (degrading) enzyme, polygalacturonase [Grierson, D., Tucker, G. A., Keen, J., Ray, J., Bird, C. R. and Schuch, W. (1986) Nucleic Acids Res. 14, 8595–8603]. We have now isolated a cDNA clone for tomato pectin esterase, an enzyme also implicated in cell wall softening. Here we report the structure of this cDNA and compare it with the structure of pectin esterase derived from amino acid sequence experiments [Markovic, O. and Jornvall, H. (1986) Eur. J. Biochem. 158, 455–462].

We have used the pectin esterase cDNA clone to analyse pectin esterase gene expression during development and ripening of normal and mutant fruit.
Three types of rat U1 small nuclear RNA genes with different flanking sequences are induced to express in vivo
Authors: WATANABE Naoko , OHSHIMA Yasumi
Published: March 2005

There are about 50 copies of U1 RNA genes/pseudogenes in the rat genome. To date, we have isolated so far 25 phage clones carrying a U1 RNA gene/pseudogene from two rat genomic libraries. The 12 clones were selected by hybridization with the U1 RNA coding region under a stringent condition, and were mapped and sequenced. Here, we report three types of U1 RNA genes with different flanking sequences, all of which were shown to be induced to express in vivo by transfection with their polylinker‐inserted maxi U1 RNA genes into cultured rat cells. Although these three classes of U1 RNA genes have few homologous flanking sequences, they provide both upstream and downstream of the genes two conserved blocks, which may possibly play an important role in U1 RNA expression.
Crystals of modified bovine neurophysin II
Authors: ROSE John P. , YANG Daniel , YOO Chung Soo , SAX Martin , BRESLOW Esther , WANG Bi‐Cheng
Published: March 2005

An enzymatically modified form of bovine neurophysin II has been crystallized in three unique crystal forms. The orthorhombic form crystallizes in space group P21212 with a= 15.33 nm, b= 6.92 nm, c= 3.63 nm, with four molecules in the asymmetric unit. The monoclinic form crystallizes in space group P21 with a= 6.22 nm, b= 9.55 nm, c= 5.45 nm and β= 110.2°, with eight molecules in the asymmetric unit. The tetragonal form crystallizes in space group P41212 or P43212 with a= 14.1 nm and c= 14.2 nm, with twelve molecules in the asymmetric unit. We report here the crystallization conditions, as well as the crystal data.
The influence of fibrin(ogen) fragments on the kinetic parameters of the tissue‐type plasminogen‐activator‐mediated activation of different forms of plasminogen
Authors: NIEUWENHUIZEN Willem , VOSKUILEN Marijke , VERMOND Anton , HOEGEE‐de NOBEL Bep , TRAAS Daan W.
Published: March 2005

In the present work we have determined Km,app and kcat,app values for tissue‐type plasminogen‐activator‐catalyzed activation of Glu‐plasminogen, Lys‐plasminogen and mini‐plasminogen in the absence and in the presence of fibrinogen‐derived fragments. These were CNBr fragment 2, the Aα chain remnant of CNBr fragment 2 (Aα 148–207) and plasmin‐generated fragment D‐EGTA. The time course of plasmin formation from the various types of plasminogen (plg) was measured spectrophotometrically in a coupled assay system where d‐valyl‐l‐leucyl‐l‐lysine p‐nitroanilide served as a plasmin substrate. The kinetic constants are summarized as follows. (Values in parentheses are concentrations at which the minimum Km,app and maximum kcat,app value is reached.)

Type of plg
Added fragment

None
CNBr fragment 2
Aα 148–207
D‐EGTA

Km
kcat
Km
kcat
Km
kcat
Km
kcat

nM
min−1
nM
min−1 (nM)
nM
min−1 (nM)
nM
min−1 (nM)

Glu
53
0.12
3 (10)
3.5 (350)
16 (350)
8.6 (17000)
14 (500)
0.58 (750)

LYS
30
0.37
10 (10)
13 (700)
26 (170)
16.3 (17000)
13 (750)
1.15 (750)

Mini
61
0.21
no significant effects
no significant effects
13 (500)
0.36 (375)

In conclusion our results show that CNBr fragment 2, Aα 148–207 and to some extent D‐EGTA mimic the accelerating effect of fibrin. The first two of these fragments did not accelerate activation of mini‐plasminogen, lacking the kringle structures I–IV. This suggests that the stimulating effects of these two fragments were dependent on the presence of kringles I–IV of the plasminogen molecule.
Pore formation by pho‐controlled outer‐membrane proteins of various Enterobacteriaceae in lipid bilayers
Authors: BAUER Katharina , SCHMID Angela , BOOS Winfried , BENZ Roland , TOMMASSEN Jan
Published: March 2005

The structural genes of the PhoE porins of Klebsiella pneumonia, Enterobacter cloacae and Escherichia coli C, cloned in multicopy plasmids, were transfered into a porin‐deficient E. coli K‐12 strain, which was constitutive for the pho regulon, and the PhoE porins were isolated and purified. PhoE of Salmonella typhimurium could not be cloned but was isolated from a pho‐constitutive strain. Reconstitution experiments with artificial lipid bilayer membranes showed that the different PhoE proteins formed pores exhibiting a single‐channel conductance of about 200 pS at 0.1 M KCl. All PhoE porins formed anion‐selective channels in KCl at neutral pH. The degree of the selectivity was dependent on the PhoE species. The different PhoE porins formed general diffusion pores similar to the general porins but exhibited a considerable advantage for the permeation of phosphate through the outer membrane as compared to the constitutive OmpC and OmpF porins of E. coli K‐12.
Respiratory nitrate reductase from Paracoccus denitrificans
Authors: BALLARD Anna L. , FERGUSON Stuart J.
Published: March 2005

The b‐type haem centres of the three (α, β and γ) subunit nitrate reductase from Paracoccus denitrificans have been analysed by redox potentiometry. Two components were identified with mid‐point potentials +95 mV and +210 mV.

Washing, in the absence of Mg2+ ions, of cytoplasmic membrane vesicles from P. denitrificans promoted selective release of nitrate reductase activity. The released enzyme was purified by chromatography and shown to contain α and β but not γ polypeptides. A haem spectrum was absent, consistent with the lack of the α subunit. The α and β polypeptides of the water‐soluble nitrate reductase had molecular masses that were identical to those of the detergent‐purified enzyme and also of the nitrate reductase in cytoplasmic membranes. This observation, together with the failure of protease inhibitors to prevent release from the membrane, indicates that the release is not related to limited proteolysis of the α and/or β polypeptides. The relative molecular mass of the water‐soluble αβ enzyme was estimated to be approximately 200000.

The water‐soluble nitrate reductase was released from intact inverted cytoplasmic membrane vesicles as jugded by loss of NADH‐NO−3 reductase activity and retention by the vesicles after washing of uncoupler‐sensitive NADH‐oxidase activity. These observations show that α and β polypeptides, and therefore the active site for nitrate reduction, are located on the cytoplasmic side of the membrane.

Attempts to reverse the nitrate reductase activity of the enzyme, using nitrite as reductant plus ferricyanide or chlorate as tested oxidants, were unsuccessful. The implications for the mechanism of the enzyme are discussed.
Hormonal regulation of low‐density lipoprotein (LDL) receptor activity in human hepatoma Hep G2 cells
Authors: WADE David P. , KNIGHT Brian L. , SOUTAR Anne K.
Published: March 2005

Low‐density lipoprotein (LDL) receptors of approximate Mr 130000 on non‐reduced gels have been identified in Hep G2 cells by immuno‐ and ligand‐blotting of cell extracts. Measurement of LDL receptor protein by scanning ligand blots was correlated with the specific binding, uptake and degradation of 125I‐labelled LDL by intact cells, confirming that this is mediated by the LDL receptor. Cells incubated in medium with serum expressed significant LDL receptor activity. This increased when cells were transferred to medium containing lipoprotein‐deficient serum (LPDS) but was not maximal because a further increase occurred when compactin was included in the medium. Inclusion of 17α‐ethinyl estradiol or 17β‐estradiol in the medium at concentrations up to 500 ng/ml had no effect on LDL receptor activity in the cells as assayed by ligand blotting. Inclusion of insulin (100 mU/ml) in the preincubation medium containing LPDS resulted in a twofold increase in LDL‐receptor protein and of LDL binding and degradation by intact cells. Insulin also diminished the suppressive effect of LDL on LDL receptor activity. If insulin exerts this effect in vivo it may partly explain why the liver expresses LDL receptors despite high levels of LDL in plasma and interstitial fluid.
Characterization of covalently cross‐linked somatostatin receptors in hamster beta cell insulinoma
Authors: COTRONEO Patrizia , MARIE Jean‐Claude , ROSSELIN Gabriel
Published: March 2005

The selective binding of somatostatin‐28 (SS‐28) to beta cells of hamster insulinoma was characterized using HPLC‐purified 125I‐[Leu8,d‐Trp22, Tyr25]SS‐28 or 125I‐SS‐28. A single class of high‐affinity sites (Kd= 53 ± 5 pM) was observed with a binding capacity of 2.85 pmol/mg membrane protein. A large number of relatively low‐affinity sites was found also. The order of potency of different peptides to inhibit 125I‐SS‐28 binding is SS‐28 > SS‐14 > SMS‐201–995 and the respective half‐maximal inhibitory doses are 0.16 nM, 10 nM and 1000 nM. CCK8 and other active pancreatic peptides (glucagon, insulin, gastric inhibitory peptide, vasoactive intestinal peptide, oxyntomodulin) do not inhibit the SS‐28 receptor binding. 125I‐SS‐28‐labeled beta membranes were successfully cross‐linked using either the cleavable cross‐linker dithiobis(succinimidylpropionate) (1 mM) alone or with a heterobifunctional agent, N‐hydroxysuccinimidyl‐4‐azidobenzoate (HSAB). In both cases five molecular components were revealed, after polyacrylamide gel electrophoresis of the membrane proteins and autoradiography, with the following molecular mass: 196‐kDa, 132 kDa, 69 kDa, 45 kDa and 28 kDa. The labeling of 196‐kDa, 132‐kDa and 45‐kDa species was specific in that they could be inhibited by unlabeled SS‐28. The major labeled species corresponds to the 132‐kDa band and no change in the mobility of this HSAB covalently bound SS‐28 receptor was found after addition of dithiothreitol, suggesting that this specific receptor does not contain interchain disulphide bonds. The molecular mass of SS‐28 receptors differs markedly from that of guinea‐pig pancreatic acinar membranes, where a single 93‐kDa protein is identified as a 125I‐SS‐28 receptor site in comparative experiments. Both the binding kinetics and structural differences sustain the selective action of SS‐28 in the endocrine pancreas.
Existence of malfunctioning proα2(I) collagen genes in a patient with a proα2(I)‐chain‐defective variant of Ehlers‐Danlos syndrome
Authors: HATA Ryu‐Ichiro , KURATA Shun‐ichi , SHINKAI Hiroshi
Published: March 2005

Collagen synthesis was examined in skin fibroblasts from a patient with a variant of Ehlers‐Danlos syndrome.

The relative rate of collagen synthesis to total protein synthesis in the patient's fibroblasts was always one‐half of that in fibroblasts from normal controls. Total collagen synthesis, as assessed by quantification of total hydroxyproline, was also significantly lower than that of controls, indicating that the rate of collagen synthesis by the patient's fibroblasts was decreased compared with that by normal fibroblasts. Analysis of procollagen and collagen components showed the absence of the proα2(I) chain and its derivatives. Dot‐blot and Northern‐blot analyses showed the patient's fibroblasts to contain less than 10% of the mRNAs for proα2(I) found in control fibroblasts. In spite of these results, Southern blot analysis of genomic DNA indicated the presence of the same number of genes for the proα2(I) collagen chain in the patient's fibroblasts as in control fibroblasts, suggesting malfunctioning proα2(I) collagen genes as the cause for failure of the patient's fibroblasts to synthesize proα2(I) collagen chains.
Pyruvic‐acid‐containing polysaccharide in the cell wall of Bacillus polymyxa AHU 1385
Authors: KOJIMA Naoya , KAYA Shunji , ARAKI Yoshio , ITO Eiji
Published: March 2005

Three acidic polymer fractions with molecular masses of about 16 kDa, 35 kDa and 70 kDa were isolated from lysozyme digests of N‐acetylated cell walls of Bacillus polymyxa AHU 1385 by ion‐exchange chromatography and gel chromatography. These fractions, containing mannosamine, glucosamine and pyruvic acid in a molar ratio of about 1:1:1 together with glycopeptide components, were characterized as polysaccharide‐linked glycopeptides with one, two and more polysaccharide chains. On the other hand, treatment of the cell walls with glycine/HCl buffer, pH 2.5, at 100°C for 10 min followed by separation of water‐soluble products on ion‐exchange chromatography gave three polysaccharide fractions, PS‐I–III, which contained different amounts of pyruvic acid (0, 0.6 and 0.9 residue/mannosamine residue) along with equimolar amounts of mannosamine and glucosamine. Pyruvate‐free polysaccharides similar to PS‐I were also obtained from PS‐II, PS‐III and polysaccharide‐linked glycopeptides by treatment with 10 mM HCl at 100°C for 1 h. Results of analyses of these polysaccharide preparations by 1H‐NMR and 13C‐NMR measurement and methylation, together with data from characterization of fragments obtained by hydrogen fluoride hydrolysis, lead to the most likely structure, →3)[4,6‐O‐(1‐carboxyethylidene)]ManNAc(β1→4)GlcNAc(β1 →, for the acidic polysaccharide of this strain.
Plastid‐localised seed acyl‐carrier protein of Brassica napus is encoded by a distinct, nuclear multigene family
Authors: SAFFORD Richard , WINDUST John H. C. , LUCAS Clare , DE SILVA Jacquie , JAMES Christopher M. , HELLYER Amanda , SMITH Colin G. , SLABAS Antoni R. , HUGHES Stephen G.
Published: March 2005

Acyl‐carrier protein (ACP) is a key component involved in the regulation of fatty acid biosynthesis in plants. cDNA clones encoding ACP from Brassica napus (oil seed rape) embryos have been isolated using oligonucleotide probes derived from heterologous ACPs. Analysis of the DNA sequence data, in conjunction with N‐terminal amino acid sequence data, revealed ACP to be synthesized from nuclear DNA as a precursor containing a 51‐amino‐acid N‐terminal extension.

Immunocytochemical studies showed ACP to be localised solely within the plastids of B. napus seed tissue and it would therefore appear that the N‐terminal extension functions as a transit peptide to direct ACP into these organelles. Analysis of several cDNA clones revealed sequence heterogeneity and thus evidence for an ACP multigene family. From ten cDNA clones, six unique genes, encoding five different mature ACP polypeptides, were identified. Northern blot hybridisation studies provide evidence that the seed and leaf forms of rape ACP are encoded by structurally distinct gene sets.
Purification of an insulin‐sensitive cyclic AMP phosphodiesterase from rat liver
Authors: BOYES Stewart , LOTEN Ernest G.
Published: March 2005

A low‐Km cyclic nucleotide phosphodiesterase solubilised from rat liver membranes by mild proteolysis with chymotrypsin has been purified to apparent homogeneity. The purification included chromatography on cellulose phosphate, Ecteola‐cellulose, hydroxyapatite, a theophylline affinity matrix and HPLC on a DEAE‐substituted column. The purified enzyme has linear kinetic plots with a Km of 0.24 μM and a Vmax of 6.2 μmol mg−1 min−l with cyclic AMP as a substrate. It also hydrolyses cyclic GMP with a Km of 0.17 μM and a Vmax which is about a third of that with cyclic AMP. Cyclic GMP is also a competitive inhibitor of cyclic AMP hydrolysis with a Ki of 0.18 μM. The proteolytically solubilised enzyme has a subunit molecular mass of 73 kDa by SDS gel electrophoresis and of 130 kDa by HPLC size‐exclusion chromatography, suggesting that it exists as a dimer.

A partially purified preparation of this enzyme was used to raise antiserum in a sheep. The antiserum immunoprecipitated activity from liver and adipose tissue of rat and mouse. It had little activity against phosphodiesterase from other rat tissues or other species. Insulin‐activated phosphodiesterase from both adipocytes and hepatocytes was immunoprecipitated by the antiserum suggesting that the purified enzyme was an insulin‐sensitive phosphodiesterase.
Variations in guanine‐binding proteins (Gs, Gi) in cultured bovine adrenal cells
Authors: BEGEOT Martine , LANGLOIS Dominique , PENHOAT Armelle , SAEZ José M.
Published: March 2005

The corticotropin (ACTH) or cholera‐toxin‐induced cAMP production by cultured bovine adrenal cells increased progressively between days 0 and 7 of culture. Angiotensin II (A‐II), which inhibited both basal and ACTH‐stimulated adenylate cyclase of crude adrenal membranes, had no effect on ACTH‐induced or cholera‐toxin‐induced cAMP production by fresh isolated cells (day 0) but progressively potentiated the stimulatory action of both effectors from day 0 → 1 to day 7 of culture. In contrast, phorbol ester had a potentiating effect on fresh isolated cells. Pretreatment of cells with pertussis toxin enhanced the potentiating effect of A‐II on cells between 0 and 3 days of culture, but not after 7 days. ADP‐ribosylation by cholera toxin (ribosylating αs proteins) or pertussis toxin (αi proteins), of adrenal membranes prepared from fresh isolated or cultured cells revealed an increase in αs and a dramatic decrease in αi, the ratios αi/αs on days 0, 3 and 7 of culture were 4, 0.6 and 0.1 respectively. These results indicate that (a) A‐II had a double effect on ACTH‐induced or cholera‐toxin‐induced cAMP production: one inhibitory mediated by Gi, the other stimulatory mediated by protein kinase C activation; this could explain the lack of apparent effect of A‐II on fresh cells; (b) the progressive decrease of αi might be responsible for the appearance of the potentiating effect of A‐II whereas the progressive increase of αs could explain the enhanced responsiveness to ACTH or cholera toxin of cultured cells.
The disaggregation of calcium‐depleted casein micelles
Authors: GRIFFIN Mary C. A. , LYSTER Richard L. J. , PRICE John C.
Published: March 2005

The effect of depletion of Ca2+ on the composition and size distribution of casein micelles in milk has been examined using chemical analysis, size exclusion chromatography, fast protein liquid chromatography, turbidimetry and photon correlation spectroscopy. Partial removal of Ca2+ by EDTA and subsequent dialysis resulted in disaggregation of some of the casein micelles; as the EDTA concentration increased, the proportions of Ca2+ and phosphate relative to protein in the micelles remaining intact decreased. However, the composition of the intact micelles, with respect to the different caseins, and the number‐frequency size distribution were essentially unchanged.
Expression of atrial natriuretic factor as a cleavable fusion protein with chloramphenicol acetyltransferase in Escherichia coli
Authors: DYKES Colin W. , BOOKLESS A. Brett , COOMBER Barry A. , NOBLE Stewart A. , HUMBER David C. , HOBDEN Adrian N.
Published: March 2005

Recombinant fusion proteins containing human atrial natriuretic factor, ANF(1–28) joined to chloramphenicol acetyltransferase (CAT) via cleavable linker sequences have been produced in Escherichia coli. The linker sequences were designed to allow the release of authentic ANF(1–28) following proteolytic cleavage by enterokinase or thrombin, or chemical cleavage with 2‐(2‐nitrophenylsulphenyl)‐3‐methyl‐3′‐bromoindolenine. Proteins, containing ANF(1–28) fused to the carboxyl‐terminal region of CAT (using the ScaI restriction site in the cat gene), were largely soluble in E. coli and were obtained in higher yield than analogues containing ANF(1–28) linked to shorter CAT sequences. The longer derivatives also retained CAT activity allowing subsequent purification by affinity chromatography.
Immunoelectron microscopic localisation of ribosomal proteins from Bacillus stearothermophilus that are homologous to Escherichia coli L1, L6, L23 and L29
Authors: HACKL Wolfgang , STÖFFLER‐MEILICKE Marina
Published: March 2005

The locations of proteins BL1, BL6, BL23 and BL29 from Bacillus stearothermophilus have been determined on the ribosomal surface by immunoelectron microscopy. All four proteins were localized in the same region of the 50s subunit as their homologous counterparts from Escherichia coli, indicating that the ribosomal architecture is the same in both species. This finding is of great importance as it allows structural data obtained on ribosomes from either organism to be incorporated into a unique ribosome model.
The 20α‐hydroxysteroid dehydrogenase of Streptomyces hydrogenans
Authors: RIMSAY Robert L. , MURPHY Gordon W. , MARTIN Curtis J. , ORR James C.
Published: March 2005

In addition to the well‐known 3α,20β‐hydroxysteroid dehydrogenase (‘cortisone reductase’), Streptomyces hydrogenans produces a relatively stable, NAD‐dependent 20α‐hydroxysteroid dehydrogenase of molecular mass approximately 48 kDa. This enzyme catalyzes the transfer of hydrogen from the 4‐pro‐S position of NADH.
Human aldolase A gene
Authors: IZZO Paola , COSTANZO Paola , LUPO Angelo , RIPPA Emilia , PAOLELLA Giovanni , SALVATORE Francesco
Published: March 2005

The complete nucleotide sequence of the human aldolase A isoenzyme gene is reported. The cloned gene sequence, spanning 7530 bp, includes twelve exons and occurs as a single copy per haploid human genome. The structural organization of the gene is quite complex: eight exons containing the coding sequence are common to all mRNAs extracted from human and other mammalian sources; four additional exons are present in the 5′ untranslated region, of these one is contained in the ubiquitous type of mRNA, the second is in the muscle‐specific type of mRNA and the third and fourth are in a minor species of mRNA found in human liver tissue. Furthermore, the determined sequence includes 1000 nucleotides upstream from the first exon (exon I) in the 5′ flanking region, and 400 nucleotides, which include the polyadenylation signal, downstream from the termination codon.

S1‐nuclease‐protection analysis of the 5′ end of mRNA extracted from human cultured fibroblasts, muscle and hepatoma cell lines indicates the existence of four different transcription‐initiation sites. The latter are also supported by the presence of conventional sequences for eukaryotic promoters. Therefore, the four promoters on the same gene generate different tissue‐specific transcripts, which share the translated sequence, but each has a unique 5′ untranslated region as a result of differential mRNA processing.

The nucleotide homology at the coding region and the intron‐exon organization of the three human and mammalian aldolase A, B and C genes confirm that they arose from a common ancestral gene, and that aldolase B diverged first.
Maitotoxin triggers the cortical reaction and phosphatidylinositol‐4,5‐bisphosphate breakdown in amphibian oocytes
Authors: BERNARD Véronique , LAURENT Anne , DERANCOURT Jean , CLÉMENT‐DURAND Monique , PICARD André , Le PEUCH Christian , BERTA Philippe , DORÉE Marcel
Published: March 2005

Maitotoxin, a potent marine toxin extracted from peredinians, was found to mimic fertilization in Xenopus oocytes and to trigger the breakdown of phosphatidylinositol 4,5‐bisphosphate [PtdIns(4,5)P2, the precursor of inositol 1,4,5‐trisphosphate], an increase of intracellular pCa and the cortical reaction, including the exocytosis of cortical granules and a wave‐like propagation of contraction in the animal hemisphere.

All these effects of maitotoxin required the presence of external calcium. Moreover, the toxin considerably increased Ca2+ influx in amphibian oocytes arrested at first meiotic prophase, due to the permanent activation of voltage‐dependent Ca2+ channels. Nevertheless it is doubtful that maitotoxin acts primarily as a Ca2+ ionophore or at the level of Ca2+ channels. Indeed no stimulation of Ca2+ uptake was observed in metaphase‐II‐arrested oocytes, although maitotoxin readily triggered the breakdown of PtdIns(4,5)P2 as well as the cortical reaction in such cells. On the other hand, PtdIns(4,5)P2 breakdown was not reduced in oocytes microinjected with EGTA, although the calcium chelator prevented the oocytes from undergoing the cortical reaction. Taken together, these findings support the view that the toxin might act primarily by increasing PtdIns(4,5)P2 phosphodiesterase activity.
New insights into maitotoxin action
Authors: SLADECZEK Fritz , SCHMIDT Bernhard Hubert , ALONSO Richard , VIAN Laurence , TEP Aline , YASUMOTO Takeshi , CORY Robert Nassim , BOCKAERT Joël
Published: March 2005

Maitotoxin (3 ng/mol) induced a massive uptake of 45Ca2+ into BC3H1 cells. This effect exhibits a lag phase of 3 min. Inositol diphosphate formation occurred concomittantly with the 45Ca2+ uptake but inositol monophosphate formation was found only after a 5‐min delay following toxin addition. Maitotoxin‐induced 45Ca2+ influxes could not be blocked by either 1 μM verapamil, 1 μM nifedipine or 1 mM La3+ but was blocked by Zn2+ (IC50= 41 μM). In addition to inositol phosphate formation and 45Ca2+ uptake, maitotoxin stimulated a large uptake of Na+ and a great loss of K+ in BC3H1 cells. In the absence of Ca2+ (1 mM EGTA) none of the four maitotoxin effects could be detected. After restoration of Ca2+ the maitotoxin effects reappeared even when the toxin itself was no longer present. The divalent cation, Co2+ (1 mM), inhibited ion movements induced by maitotoxin and also digitonin (8.1 μM). The toxin action showed a very pronounced pH dependence. At low pH, maitotoxin was inactive. The dose‐response curves for H+ ion inhibition of maitotoxin‐induced Ca2+ uptake showed a shift to the right when determined in the absence of HCO3‐ and HCO3‐/Cl‐ ions.

It was concluded that the primary action of maitotoxin in BC3H1 cells was a pore‐forming or channel‐forming activity of a non‐classical type. Some properties of maitotoxin resemble those of α‐latrotoxin, others those of pore‐forming agents such as melittin or α‐toxin of Staphylococcus aureus.
The herpes simplex virus type I DNA polymerase
Authors: WEISSHART Klaus , KNOPF Charles W.
Published: March 2005

Polyclonal antibodies responding specifically to the N‐terminal, central and C‐terminal polypeptide domains of the herpes simplex virus type I (HSV‐1) DNA polymerase of strain Angelotti were generated. Each of the five different rabbit antisera reacted specifically with a viral 132 ± 5‐kDa polypeptide as shown by immunoblot analysis. Enzyme binding and inhibition studies revealed that antibodies raised to the central and the C‐terminal domains of the protein inhibited the polymerizing activity by 70–90%, respectively, which is well in line with the proposed site of the catalytic center of the enzyme and with the possible involvement of these polypeptide chains in DNA–protein interactions. In agreement with this, antibodies directed towards the N‐terminal domain bound to the enzyme without effecting the enzymatic activity. The strong binding but low inhibitory properties of antibodies directed to the polypeptide region between residues 1072 and 1146 confirms previous suggestions that these C‐terminal sequences, which share no homology to the Epstein‐Barr virus DNA polymerase, are less likely involved in the building of the polymerase catalytic site. Antibodies, raised to the very C terminus of the polymerase (EX3), were successfully used to identify a single 132 ± 5‐kDa polypeptide, which coeluted with the HSV DNA polymerase activity during DEAE‐cellulose chromatography, and were further shown to precipitate a major viral polypeptide of identical size. From the presented data it can be concluded that the native enzyme consists of a single polypeptide with a size predicted from the long open reading frame of the HSV‐1 DNA polymerase gene.