bluuebunny/crossref_metadata_2025 huggingface dataset:
Ubiquinone‐5 is reduced by Superoxide in the aerobic state by NADPH oxidase of guinea pig macrophages
Authors: Nakamura Michio , Murakami Masakazu , Umei Toshihiko , Minakami Shigeki
Published: July 2002

The reduction of ubiquinone‐5 (Q1) by the phagocytosis‐specific NADPH oxidase of guinea pig macrophages was not inhibited by Superoxide dismutase (SOD) at concentrations usually used for O2
− assay but was inhibited at about 100‐times higher concentrations. Titration of the reaction with SOD and a comparison with that of xanthine oxidase showed that the inhibition was not due to the semiquinone oxidation accelerated by a removal of O2
− but due to the accelerated dismutation of O2
− which otherwise reduces the quinone. Molecular oxygens are therefore preferential electron acceptors in the NADPH oxidase even in the presence of Q1.
Water permeability of chloroplast envelope membranes
Authors: McCain Douglas C. , Markley John L.
Published: July 2002

In tulip tree (Liriodendron tulipifera) leaves, the proton NMR signal from chloroplast water is resolved from that of water in other leaf compartments. We used the saturation‐transfer NMR method to measure the mean water molecule residence time within a chloroplast, (88 ± 17) ms at 20°C. From the measured chloroplast dimensions, we calculate an effective permeability coefficient of (9 ± 2) × 10−4
for the chloroplast envelope membrane. This is the first in vivo measurement of chloroplast water permeability.
Membranal tyrosine protein kinase activity (but not cAMP‐dependent protein kinase activity) is associated with growth of rat mammary tumors
Authors: Sharoni Yoav , Radian Sara , Levy Joseph
Published: July 2002

Tyrosine protein kinase
Mammary tumor
DMBA
Tumor growth
The complete amino‐acid sequence of the small bacteriochlorophyll‐binding polypeptide B800‐850β from light‐harvesting complex B800‐850 of Rhodopseudomonas capsulata
Authors: Tadros Monier Habib , Frank Rainer , Drews Gerhart
Published: July 2002

Bacteriochlorophyll‐binding polypeptide
Primary structure
Rhodopseudomonas capsulata
B800‐850 antenna pigment complex
Novel structure of heat shock messenger ribonucleoproteins in Drosophila Kc cells
Authors: Setyono-Han Buddy , Köhler Kurt
Published: July 2002

Poly(A)+ polyribosomal mRNA is normally complexed with a specific set of proteins forming ribonucleoprotein particles, termed polyribosomal mRNPs. We have found that in Drosophila during the heat shock response, their structure is profoundly altered as shown by their greatly decreased density in Cs2SO4 gradients. RNA‐protein cross‐linking, conducted in vivo, revealed that polyribosomal mRNA is associated with only two polypeptides of 70 and 38 kDa; while those from cells raised at 25°C revealed a more complex protein pattern. The 38 kDa protein is located on the poly(A) sequences.
The nucleotide binding site of F1‐ATPase which carries out uni‐site catalysis is one of the alternating active sites of the enzyme
Authors: Kozlov I.A. , Milgrom Ya.M. , Murataliev M.B. , Vulfson E.N.
Published: July 2002

Nucleotide‐depleted mitochondrial F1‐ATPase binds 3'‐(2')‐O‐(2‐nitro‐4‐azidobenzoyl)‐derivatives of ATP (NAB‐ATP) and GTP (NAB‐GTP) when these nucleotide analogues are added to the enzyme in equimolar quantities in the presence of Mg2+ (uni‐site catalysis conditions). The binding of NAB‐ATP is accompanied by its hydrolysis and inorganic phosphate dissociation from the enzyme; NAB‐ADP remains bound to F1‐ATPase. The F1‐ATPase·NAB‐ADP complex has no ATPase activity and its reactivation in the presence of an excess of ATP is accompanied by NAB‐ADP release. The illumination of the F1‐ATPase complexes with NAB‐ADP or NAB‐GDP leads to the covalent binding of one nucleotide analogue molecule to the enzyme and to the irreversible inactivation of F1‐ATPase. It follows from the results obtained that the modification of just one of the F1‐ATPase catalytic sites is sufficient to complete the inhibition of ATPase activity.
Antibodies directed against guanylic acid and guanosine are nucleolus specific probes
Authors: Iswari S. , Krishnamurthy D.S. , Jacob T.M.
Published: July 2002

Purified antibodies against guanylic acid and guanosine binding to RNA at guanosine residues were used to probe human lymphocyte preparations by indirect immunofluorescence. Neither antibody gave any banding pattern with metaphase chromosomes but both showed binding to specific sites in the interphase nuclei. Evidence presented indicates that these sites are guanosine residues on rDNA transcripts at the nucleolar organizer regions.
ADP‐ribosylation of metaphase and interphase nonhistones using [3H]adenosine as a radioactive label
Authors: Adolph Kenneth W. , Song Min-Kyung H.
Published: July 2002

ADP‐ribosylation
Nonhistone
Metaphase chromosome
Interphase nucleus
[3H]Adenosine
HeLa cell
Photolabelling of D‐β‐hydroxybutyrate apodehydrogenase with azidoaryl phospholipids
Authors: El Kebbaj M'Hamed S. , Berrez Jean-Marc , Lakhlifi Tahar , Morpain Claude , Latruffe Norbert
Published: July 2002

Two synthetic photoactive azidoarylphosphatidylcholines were used to investigate the level of interaction between D‐β‐hydroxybutyrate apodehydrogenase (apoBDH), an amphipathic membrane protein, with the hydrophobic domain of phospholipids. The two synthetic lecithins, PL I (1‐myristoyl‐2‐12‐N‐(4‐azido‐2‐nitrophenyl) aminododecanoyl‐sn‐glycero‐3‐phosphocholine) and PL II (1‐myristoyl‐2‐(2‐azido‐4‐nitrobenzoyl)‐sn‐glycero‐3‐phosphocholine), are able to reactivate the non‐active purified apoBDH as well as the non‐photoactive homologs, indicating that the photoreactive chemical groups are without effect on the cofactor properties of phosphatidylcholine. Photoirradiation of reconstituted complexes between phospholipid containing azidoaryllecithin and apoBDH leads to a covalent binding of some synthetic lecithin molecules on the protein. The labelling, about 3 times higher with PL II than with PL I, suggests that the area of interacting domain of BDH with the hydrophobic moiety of phospholipid is more important at or near the surface of the lipid bilayer than in the inner part. This approach is further demonstration that BDH is an integral protein.
Subclass‐specific antibody‐dependent binding of macrophages to supported planar lipid monolayer membranes
Authors: Kimura Kazuko , Nakanishi Mamoru
Published: July 2002

Subclass‐specific antibody‐dependent binding of macrophages to supported planar lipid monolayers has been studied. The binding of the P388D1, macrophage‐like cell line to planar DMPC or DPPC monolayers was dependent on IgC subclasses and hapten concentrations. The binding efficiencies were as follows on both ‘solid’ and ‘fluid’ lipid monolayer membranes; IgGI = IgG2a>IgG2b>IgG3 = IgA. These results suggest that the present system is very useful for studying the mechanisms of the transmembrane signal triggered by Fc receptors of macrophages.
A role for adrenaline and calmodulin in modulating cyclic AMP levels during the lactogenic cycle
Authors: Muñoz Aira , Lavandero Sergio , Donoso Eduardo , Puente Javier , Sapag-Hagar Mario
Published: July 2002

The effect on lactose production of several external modulators of intracellular cyclic AMP was studied in rat mammary gland tissue slices and explants. Adrenaline, a β‐adrenergic receptor effector, forskolin, a direct adenylate cyclase activator and fluphenazine, a calmodulin inhibitor, all produced an increase in the intracellular level of cyclic AMP and a concomitant inhibition of lactose production. These results suggest a role for adrenaline and calmodulin in modulating cyclic AMP levels in mammary tissue during the lactogenic cycle.
Age‐dependent changes in the level of a 34 kDa DNA‐binding protein in developing chick embryo liver
Authors: David E. , Shanmugam G.
Published: July 2002

The relative amounts of a DNA‐binding protein of 34 kDa increased during the early stages of development of chick embryo liver. The content of this protein reached a maximum in 18‐19‐day‐old embryonic livers and decreased afterwards in older embryonic and post‐natal chick livers. The 34 kDa polypeptide is the major DNA‐binding protein (DBP) of embryonic liver and it preferentially binds to single‐stranded DNA. The quantity of the 34 kDa DBP was relatively very low in embryonic muscle, heart and brain.
Splicing pathways of the chicken apo very low density lipoprotein II (pre)messenger RNA
Authors: Noteborn Mathieu , Arnberg Annika , de Jonge Martinus , Geert A.B. , Gruber Max
Published: July 2002

The precursor‐mRNA transcribed from the chicken apo very low density lipoprotein II gene was identified. This gene which is under full estrogen control and only expressed in the liver, possesses three introns. Splicing intermediates were characterized by hybridization with intron‐specific probes, and by electron microscopy of R‐loops. The introns appear to be excised in a non‐obligatory order, but at different rates.
A DNA binding protein from the extreme thermophile Thermus thermophilus
Authors: Zierer Rainer , Grote Mathias , Dijk Jan , Wilson Keith
Published: July 2002

A DNA binding protein was extracted and purified from Thermus thermophilus HB8 grown at 75°C. The protein has an M
r of 10 000 and from the partial data available shows a weak but clear sequence homology to the DNA binding protein II family. The protein is able to bind to DNA, and can also bind to RNA and to ribosomes. It affects the melting profile of the nucleic acids. From circular dichroic spectra an α‐helix content of 32% and β‐sheet of 40% are estimated. The proton NMR spectrum indicates a well folded tertiary structure. Upon binding of oligonucleotides pronounced changes in the NMR spectrum of the protein can be seen. Comparable changes are observed in circular dichroic spectra of protein‐oligonucleotide complexes. Hexagonal crystals, which diffract to at least 3 Å, of the DNA binding protein have been obtained using ammonium sulphate as precipitant. The space group is P6222, with cell dimensions a = b = 62.9 Å and c = 110.6 Å.
Kinetics of the membrane‐bound inorganic pyrophosphatase from Rhodospirillum rubrum chromatophores
Authors: Strid Åke , Nyrén Pål , Boork Jeff , Baltscheffsky Margareta
Published: July 2002

The behaviour of the membrane‐bound proton‐translocating pyrophosphatase (H+‐PPase) in Rhodospirillum rubrum chromatophores upon application of an electrochemical potential is studied. The rate constants are shown to be affected in an asymmetric fashion. The forward rate constant (PPi synthesis) is shown to be at least 45‐times larger during illumination than when there is no proton‐motive force. The hydrolysis rate is increased maximally 8‐times when the potential is dissipated. The effect of the electrical field gradient is thus mainly to increase the forward rate of the reaction. The H+‐PPase also seems to be a functionally simpler enzyme than the H+‐ATPase, lacking the hydrolysis activation step during energization found in the latter.
Increased basal gluconeogenesis in the aged rat
Authors: Satrústegui Jorgina , Cuezva José M. , Machado Alberto
Published: July 2002

Post‐absorptive gluconeogenesis from lactate measured in vivo increases 3‐fold in 24‐month‐old rats compared to 3‐month‐old animals. Fractional lactate turnover rates showed no significant differences between the two groups of animals. Lower plasma glucose concentrations and insulin‐glucagon ratios may explain the increase in gluconeogenesis observed in aged rats.
Crystallization and spectroscopic investigation with polarized light of the reaction center‐B875 light‐harvesting complex of Rhodopseudomonas palustris
Authors: Wacker Thomas , Gad'on Nasser , Becker Andreas , Mäntele Werner , Kreutz Werner , Drews Gerhart , Welte Wolfram
Published: July 2002

A pigment‐protein complex containing a reaction center and the B875 light‐harvesting complex from Rhodopseudomonas palustris was purified and crystallized in the presence of detergent. Thin, rectangular crystals were obtained and used for optical spectroscopy. The protein pattern as well as the spectrum of the solubilized and crystallized complex show that the purified complex was crystallized without a major change in its composition and conformation. The absorption band intensities in the plane of the crystal as well as the spectra of the tilted crystal indicate that the 590 nm Qx transitions are aligned along the long axis of the crystal, while the 875 nm Qy transitions are aligned along the short axis of the crystal plane. The results favor a model where the antenna bacteriochlorophylls associated with one reaction center have a common Qx direction and perpendicular to it a distribution of 875 nm Qy transitions exhibiting a maximum along one direction.
Microsomal membrane subfractionation by a lectin affinity method
Authors: Sehmi D. , Williams L.G. , Williams D.J.
Published: July 2002

Concanavalin A‐agarose treatment of rat liver post‐mitochondrial supernatant removes a fraction rich in cholesterol and 5'‐nucleotidase activity but low in glucose‐6‐phosphatase. At the same time, radiolabel associated with the cell surface is removed. We interpret these findings as evidence that concanavalin A binds to, and under these circumstances will remove, fragments of plasma membrane present in the microsomal fraction and believe that this may be of use in the gentle, and rapid subfractionation of microsomal membranes.
Effects of cholesterol on the kinetics of mitochondrial ATPase
Authors: Rindina F.Calanni , Baracca A. , Solaini G. , Rabbi A. , Castelli G.Parenti
Published: July 2002

Enrichment of the inner mitochondrial membrane with cholesterol induces an increase in ATPase activity with a decrease in the K
m for ATP. Cholesterol also abolishes the discontinuity normally found in the Arrhenius plot of ATPase activity. Since no change is detected in the rate of proton translocation through the ATPase membrane sector, it is concluded that cholesterol incorporation induces changes in the hydrolytic step of ATPase via a conformational change transmitted from the membrane sector to the catalytic sector F1.
Stable binding interactions among subunits of the chloroplast ATP synthase (CF1‐CF0 as examined by solid support (nitrocellulose)‐subunit reconstitution‐immunoblotting
Authors: Süss Karl-Heinz
Published: July 2002

The technique termed solid support‐subunit reconstitution‐immunoblotting has been applied to examine stable binding interactions among renatured subunits of the chloroplast ATP synthase. Reconstitution of the CF1 subunits α, β and γ and subunit III of CF0 with thylakoid membrane polypeptides separated electrophoretically and transferred to nitrocellulose filters followed by immunodetection of hybrid subunit complexes revealed interactions between α‐ϵ, α‐CF0‐I, α‐CF0‐II, α‐δ, β‐γ, β‐CF0‐II β‐ϵ, β‐CF0‐III, γ‐CF0‐II, γ‐ϵ, γ‐CF0‐III, CF0‐III‐ϵ. Thus, the data confirm results on neighbour relationships of ATP synthase subunits obtained by cross‐linking and suggest that the method employed is generally applicable to examine protein‐protein binding interactions.
Analysis of folding intermediates from the main toxin of Naja naja samarensis containing two blocked cysteines
Authors: Gacond J.J. , Rovelli G. , Bargetzi J.P. , Juillerat M.A.
Published: July 2002

The formation of folding intermediates blocked by iodoacetate from the main toxin of Naja naja samarensis was monitored by FPLC and the populations were identified by amino acid analyses. We have determined conditions allowing for the highest yield in the populations with two blocked cysteines. The free cysteines remaining under these conditions were labeled with iodo[14C]acetate and localized by peptide mapping in one of the products isolated by ion‐exchange chromatography (NT3III). We have also investigated the effects on the native‐like characteristics of such molecules of an incubation at equilibrium with a mixture of cysteine and cystine. We find that many different molecular populations are present during the folding process and that disulfide exchanges allow for the reconstitution of native‐like products having open disulfides even under strongly denaturing conditions.
Isolation and characterization of variant IGF‐1 as well as IGF‐2 from adult human brain
Authors: Carlsson-Skwirut Christine , Jörnvall Hans , Holmgren Arne , Andersson Charlotte , Bergman Tomas , Lundquist Gunilla , Sjögren Barbro , Sara Vicki R.
Published: July 2002

The forms of somatomedin present in the adult human brain have been characterized in this study. Two peptides were purified by acidification, size exclusion chromatography, affinity chromatography, FPLC and HPLC. structural analysis identified these peptides as the variant form of IGF‐1 with a truncated N‐terminal region earlier isolated from human fetal brain and IGF‐2. The presence of the truncated IGF‐1 variant and IGF‐2 in the human CNS suggests their role as neuropeptides.
Binding inhibition by monoclonal antibodies of ovine placental lactogen to growth hormone receptors in human liver
Authors: Chan John S.D. , Nie Z.-R. , Seidah N.G. , Chrétien M.
Published: July 2002

In the radioreceptor assay for growth hormone (RRA‐GH) using [125I]iodo‐hGH, hGH and human liver membrane particulate fractions as tracer, hormone standard and receptors, respectively, ovine placental lactogen (oPL) is capable of inhibiting the binding of [125I]iodo‐hGH in a parallel manner with hGH and in equipotency. Similarly, in the RRA‐GH by employing [125I]iodo‐oPL, oPL and human liver membrane particulate fractions as tracer, hormone standard and receptors, respectively, hGH is also equipotent as oPL in inhibiting the binding of [125I]iodo‐oPL in a parallel fashion. The addition of monoclonal antibodies against oPL in the assay was effective in inhibiting the binding of [125I]iodo‐oPL to human liver, but could not, however, inhibit the binding of [125I]iodo‐hGH to human liver. Furthermore, the addition of the monoclonal antibodies in the RRA‐GH did not affect the parallelism of the oPL standard but lowered the total binding of oPL. Our studies indicate that the structure of the binding sequence in oPL which binds to the GH receptor of human liver is not identical to the equivalent sequence of hGH and that the monoclonal antibodies compete with GH receptors in human liver for the binding of oPL.
Chilling followed by incubation at 37°C causes a reduction in NAD+ levels which can be prevented by the poly(ADP‐ribose)transferase inhibitor 3‐aminobenzamide
Authors: Cantoni Orazio , Sestili Piero , Cattabeni Flaminio , Stocchi Vilberto
Published: July 2002

The exposure of cells for 60 min to a serum free medium at ice temperature followed by a return to normal culture conditions (30 min at 37°C) caused a dramatic decrease in NAD+ levels. This decrease in NAD+ was prevented by 3‐aminobenzamide. Alkaline elution analysis of DNA from cultures that were sisters to the ones utilized for measuring cellular NAD+ content revealed an absence of DNA breakage. These data suggest that poly(ADP‐ribose)transferase may be induced in conditions not involving DNA fragmentation. The induction of this enzyme could therefore represent a cellular emergency reaction and not just a response to DNA damage.
Early modifications of gene expression induced in liver by azo‐dye diet
Authors: Lamas Eugénia , Schweighoffer Fabien , Kahn Axel
Published: July 2002

The expression and regulation of the phosphoenolpyruvate carboxykinase gene were not grossly modified by feeding rats a 3'‐methyl‐4‐(dimethylamino)azobenzene‐containing diet despite maximum expression of the L‐type pyruvate kinase gene being dramatically reduced as early as the 24th hour of the carcinogenic diet. Inhibition of aldolase B mRNA synthesis occurred more slowly, being maximum at the 3rd day. After stopping administration of the carcinogen, a very rapid, but transient increase of the L‐type pyruvate kinase mRNA was observed at the 24th hour, whereas aldolase B mRNA increased only slowly. The amount of aldolase A mRNA fell quickly after termination of carcinogen administration, levels being normal at the 2nd–3rd day. At this time, the histological structure of the liver was indistinguishable from that of animals still receiving the azo‐dye diet. It appears, therefore, that in the rat both administration and withdrawal of the azo‐dye carcinogen induce rapid modifications of the expression of some genes, before any cellular modification is distinguishable.
Characterization of nucleotide‐binding sites on the chloroplast coupling factor 1 using two photolabile analogs
Authors: Abbott Marilyn S. , Shavit Noun , Selman-Reimer Susanne , Selman Bruce R.
Published: July 2002

Nucleotide‐binding sites on the chloroplast coupling factor 1 (CF1) have been probed using two photoreactive ADP analogs: 2‐azido‐ADP (2‐N3‐ADP) and 2',3'‐O‐(4‐benzoyl)benzoyl‐ADP (Bz‐ADP). Photolabeling of the isolated CF1 with 2‐N3‐ADP results in incorporation of the analog exclusively into the β‐subunit of the enzyme. The location of the nucleotide‐binding site(s) within the β‐subunit of the CF1 was investigated using peptide mapping. Within the discrimination limits of this technique, it is concluded that the azidoand benzoyl‐modified analogs both bind to the same conformation of the nucleotide‐binding site(s) of soluble CF1. Bz‐ADP, however, labels the binding site(s) on membrane‐bound CF1 in a slightly different manner.
Specificity of prolyl endopeptidase
Authors: Nomura Kohji
Published: July 2002

A series of tetrapeptides, Cbz(Bz)‐Gly‐X‐Leu‐Gly, were synthesized and the kinetic parameters, k
cat and , determined for their hydrolyses by prolyl endopeptidase from Flavobacterium. The peptides with X = N‐Me‐Ala, Sar and Ala as well as the standard substrate (X = Pro) were found to be good substrates, while those with X = α‐aminobutyryl, Hyp, Ser and Gly were poor substrates, and those with X = pipecolyl, α‐aminoisobutyryl, N‐Me‐Val, N‐Me‐Leu, Hyp(O‐Bzl) and Ser(O‐Bzl) were not cleaved at all. These results suggest that the specificity‐determining site or Sl subsite of the enzyme is designed to fit exactly the proline residue of the substrate with allowance for the residues carrying substituents at the N and/or Cα which must not exceed the size of the pyrrolidine ring of proline.
Modulation of cytosolic protein kinase C activity by ferricyanide: priming event seems transmembrane redox signalling
Authors: Malviya A.N. , Anglard P.
Published: July 2002

Transformed 3T3/10T1/2 cultured cells incubated with ferricyanide caused a decrease of 2 mM EDTA extractable cytosolic protein kinase C activity in 2 min, whereas 5 or 20 min ferricyanide treatment reverted the enzyme activity to that observed without ferricyanide. The ferricyanide effect in 2 min was abolished by amiloride and sustained by ouabain. Thus, deactivation‐activation of cytosolic protein kinase C is attributed to an unknown signal generation during H+ accumulation coupled with the Na+/H+ exchange phase. In this mechanism the priming event concerns the transmembrane redox process shedding H+ into the cell interior while impermeant ferricyanide acts as a unique electron acceptor.
A novel 40 000 Da Ca2+‐dependent actin modulator from bovine brain
Authors: Hurny A.C. , Wnuk W.
Published: July 2002

A monomeric protein of M
r 40000 that modulates the polymer state of actin has been isolated from bovine brain. When added either to preformed actin filaments or to monomeric actin, prior to polymerization, the modulator reduces the low‐shear viscosity of F‐actin provided that Ca2+ is present. The 40 kDa protein also inhibits the rate of actin polymerization. The inhibition is fully suppressed by removal of Ca2+ and restored by subsequent readdition of Ca2+, suggesting that the Ca2+‐controlled interaction of actin with the 40 kDa modulator is freely reversible.
Mapping of HLA genes using pulsed‐field gradient electrophoresis
Authors: Ragoussis Jiannis , van der Bliek Alexander , Trowsdale John , Ziegler Andreas
Published: July 2002

The technique of pulsed‐field gradient electrophoresis (PFGE) allows the determination of gene linkage relationships since DNA fragments up to 2 Mb can be separated. PFGE was employed to study linkage of class I, II and III genes belonging to the human major histocompatibility (HLA) complex. The results establish that the class II DOβ and DZα genes are linked with the DP Subregion, centromeric to the DQ/DX‐DR‐C4 chromosomal segment, and allow us to estimate the minimal length of the entire HLA complex.
Activation of guanylate cyclase by E. coli heat‐stable enterotoxin (STa)
Authors: Epstein Susan A. , Giannella Ralph A. , Brandwein Harvey J.
Published: July 2002

Bacterial toxin
Guanylate cyclase
cyclic GMP
ADP‐ribosylation
GTP‐binding protein
Secretory diarrhea
A commentary on the inhibition by retinoids of leukotriene B4 production in leukocytes
Authors: Randall R.W. , Tateson J.E. , Dawson J. , Garland L.G.
Published: July 2002

The order of potency of retinoids as inhibitors of A23187‐induced production of leukotriene B4 (LTB4) in human polymorphonuclear leukocytes (PMN) was retinoic acid > retinal > retinol. However, the conversion of exogenous arachidonate (AA) to LTB4 by PMN homogenates was inhibited in the rank order retinol > retinal ⪢ retinoic acid. The agreement between active concentrations of retinol in these two systems is consistent with this compound acting directly to inhibit AA metabolism: this is not so for the other retinoids. The order of potency for inhibition of phorbol dibutyrate (PDBu)‐stimulated superoxide (O−
2) production in HL60 granulocytes was retinol > retinoic acid ⪢ retinal (inactive); neither retinol nor retinal displaced [3H]PDBu from HL60 cells. We conclude that inhibition of LTB4 production by retinoic acid and retinal is neither through inhibition of AA metabolism nor through inhibition of protein kinase C.
Na+ transport by reconstituted Na+,K+‐ATPase in the presence of various nucleotides
Authors: Svinukhova Irina A. , Boldyrev Alexander A.
Published: July 2002

The ability of ATP, CTP, ITP, GTP, UTP and two synthetic ATP analogs to provide for ouabain‐sensitive Na+ accumulation into proteoliposomes with a reconstituted Na+,K+‐ATPase (ATP phosphohydrolase, EC 3.6.1.37) was investigated. A correlation between the proton‐accepting properties of the nucleotides and their ability to provide for active transport was found. The proton‐accepting properties of the substrate seem to be a necessary condition for the shift from the K‐form to Na form of Na+,K+‐ ATPase — an immutable step in the active translocation of Na+ and K+ through the Na+ pump.
Fragment of an endogenous inhibitor produced in Escherichia coli for calcium‐activated neutral protease (CANP) retains an inhibitory activity
Authors: Imajoh Shinobu , Kawasaki Hiroshi , Emori Yasufumi , Ishiura Shoichi , Minami Yasufumi , Sugita Hideo , Imahori Kazutomo , Suzuki Koichi
Published: July 2002

A C‐terminal fragment of an endogenous rabbit liver inhibitor for calcium‐activated neutral protease (CANP) was produced in Escherichia coli and its inhibitory activity was examined after purification. The truncated inhibitor (373 amino acid residues), which contains two internal repeat structures, inhibits 2 mol CANP whereas the native liver inhibitor (639 residues), containing four internal repeat structures, inhibits 4 mol CANP. This supports the hypothesis that the repeating unit is the functional unit of inhibition. The results also indicate that post‐translational modification of the inhibitor is not essential for inhibition.
Adenylate cyclase stimulating agents and mitogens raise fructose 2,6‐bisphosphate levels in human fibroblasts Evidence for a dual control of the metabolite
Authors: Bruni Paola , Vasta Valeria , Farnararo Marta
Published: July 2002

Fructose 2,6‐bisphosphate, the most potent activator of 6‐phosphofructo‐1‐kinase, has been demonstrated to mediate the increase of glycolytic flux induced by mitogens human fibroblasts. In the present work the molecular basis of transmembrane control of fructose 2,6‐bisphosphate has been investigated. Prostacyclin and isoprenaline, known to activate adenylate cyclase, are able to increase fructose 2,6‐bisphosphate levels, indicating that in human fibroblasts cyclic AMP plays a positive role in the control of the metabolite concentration, opposite to that exerted in hepatocytes. Substances known to activate protein kinase C such as phorbol 12‐myristate 13‐acetate, or to stimulate phosphoinositide turnover such as thrombin and bradykinin are also effective in raising fructose 2,6‐bisphosphate. Therefore, we conclude that cyclic AMP and protein kinase C are likely involved in the control of fructose 2,6‐bisphosphate levels in human fibroblasts.
On the rate of F1‐ATPase turnover during ATP hydrolysis by the single catalytic site Evidence that hydrolysis with a slow rate of product release does not occur at the alternating active site
Authors: Milgrom Ya.M. , Murataliev M.B.
Published: July 2002

Under conditions of molar excess of enzyme, isolated F1‐ATPase from beef heart mitochondria catalyses ATP hydrolysis biphasically. The rate constants for product release are ∼10−1 and 10−4−10−3 s−1, respectively. The slow phase of ATP hydrolysis is insensitive to EDTA. [γ‐32P]ATP splitting in the slow phase cannot be chased from the enzyme during several catalytic turnovers. It follows from these results that the slow single‐site hydrolysis of ATP (k
cat∼10−4s−1), initially observed by Grubmeyer et al. [(1982) J. Biol. Chem. 257, 12092–12100], is not carried out by the normal catalytic site. In contrast, the phase of rapid ATP hydrolysis (k
cat∼10−1s−1) is completely prevented by EDTA and is believed to be the normal function of the normal catalytic site of F1‐ATPase.
Identification and purification of a novel G protein from neutrophils
Authors: Dickey Burton F. , Pyun Hae Yung , Williamson Kim C. , Navarro Javier
Published: July 2002

A novel G protein which appears to couple chemotactic peptide receptors to a polyphosphoinositide phospholipase C has been purified from rabbit neutrophils. Neutrophil membranes were solubilized with sodium cholate and fractionated by successive anion exchange, gel filtration and hydrophobic chromatography. Guanosine‐5′‐(3‐O‐thio)triphosphate binding activity was purified 170‐fold from the soluble extract. The α‐subunit of the purified G protein was identified by pertussis toxin‐catalyzed ADP‐ribosylation, and found to have an M
r of 40 000. The β‐subunit (M
r 36 000) comigrated on SDS‐polyacrylamide gel electrophoresis with the β‐subunits of bovine brain Gi and Go. The neutrophil pertussis toxin substrate is highly unstable in cholate solution unless 30% ethylene glycol is added. Structural and functional analysis of this novel G protein will advance our understanding of the molecular mechanisms of coupling of receptors to phospholipase C.
Detection of Ca2+‐dependent cyclic GMP binding protein in frog rod outer segments
Authors: Shinozawa Takao , Terada Shoichi , Matsusaka Hiroshi , Yamashita Shinsuke
Published: July 2002

For the identification of the cGMP‐sensitive ion channel protein of frog rod outer segments (ROS), we analyzed cGMP binding proteins in the ROS by photoaffinity labeling with [3H]cGMP. We found three cGMP binding polypeptides (66 kDa, 92 kDa and 100 kDa) in the membrane protein fraction of ROS. cGMP binding to the 66 kDa polypeptide required the addition of 2 mM CaCl2. We propose that this polypeptide corresponds to the cGMP‐activated channel protein reported by Cook et al. [(1987) Proc. Natl. Acad. Sci. USA 84, 585–589]. The 100 kDa and 92 kDa polypeptides are subunits of the cGMP phosphodiesterase.
Activity of human, bovine and porcine platelet‐derived growth factor in a radioreceptor assay with human placental membrane protein
Authors: Czyrski Józef A. , Gawlikowski Wojciech
Published: July 2002

Radioiodinated human platelet‐derived growth factor (125I‐PDGF), and unlabeled human (Hu), bovine (Bo), and porcine (Po) PDGF were used to study PDGF receptors in human term placental membrane preparations. The binding of 125I‐Hu‐PDGF was inhibited by unlabeled preparations. Half‐maximal occupancy of the binding sites occurred at 7–9 nM of Hu‐, Bo‐ or Po‐PDGF. Scatchard analysis of the binding data indicated a single class of receptors having a K
d value of about 1.1–1.2 × 10−10 M. Thus, the sensitive radioreceptor assay does not detect any species specificity of PDGF.
Kinetic measurements of electron transfer in coupled chromatophores from photosynthetic bacteria A method of correction for the electrochromic effects
Authors: Venturoli Giovanni , Virgili Marco , Melandri B.Andrea , Crofts Antony R.
Published: July 2002

A quantitative study of the kinetics of electron transfer under coupled conditions in photosynthetic bacteria has so far been prevented by overlap of the electrochromic signals of carotenoids and bacteriochlorophyll with the absorbance changes of cytochromes and reaction centers. In this paper a method is presented by which the electrochromic contribution at any wavelength can be calculated from the electrochromic signal recorded at 505 nm, using a set of empirically determined polynomial functions. The electrochromic contribution to kinetic changes at any wavelength can then be subtracted to leave the true kinetics of the redox changes. The corrected redox changes of the reaction center measured at 542 and 605 nm mutually agree, thus providing an excellent test of self‐consistency of the method. The corrected traces for reaction center and of cytochrome b‐566 demonstrate large effects of the membrane potential on the rate and poise of electron transfer. It will now be possible to study the interrelation between proton gradient and individual electron transfer reactions under flash or steady‐state illumination.
A new redox loop formality involving metal‐catalysed hydroxide‐ion translocation A hypothetical Cu loop mechanism for cytochrome oxidase
Authors: Mitchell Peter
Published: July 2002

A new hypothetical type of redox loop is described, which translocates hydroxide ions instead of protons. Conventional protonmotive redox loops use carriers of protons with electrons (e.g. QH2/Q systems) to couple electron transfer to the translocation of protons. The putative hydroxidemotive redox loop uses carriers of hydroxide ions against electrons (e.g. transition‐metal centres) to couple electron transfer to the translocation of hydroxide ions. This simple idea leads to the proposal of a hydroxidemotive Cu loop mechanism that may possibly be applicable to the CuA or CuB centre of cytochrome oxidase, and might thus account for the coupling of electron transfer to net proton translocation in that osmoenzyme.
Effect of phase transitions in hydrated 1,2‐dipalmitoylphosphatidylethanolamine bilayers on the spin probe order parameter
Authors: Horniak L. , Kutejová E. , Balgavý P.
Published: July 2002

The ‘main’ phase transition Lβ→Lα of hydrated 1,2‐dipalmitoylphosphatidylethanolamine (DPPE) bilayers in excess water affects the ESR order parameter S
33 of N‐cetyl‐N,N‐dimethyl‐N‐tempoylammonium bromide (CAT‐16), 5‐doxylstearic acid (5‐DSA) and 16‐doxylstearic acid (16‐DSA) spin probes. The ‘pretransition’ and ‘subtransition’ suggested to occur in hydrated DPPE by Chowdhry et al. [(1984) Biophys. J. 45, 901–904] and Silvius et al. [(1986) Biochemistry 25, 4249–4258], respectively, affect exclusively the S
33 of CAT‐16, but not that of 5‐DSA and 16‐DSA spin probes. The subtransition occurs about 15 ± 1°C below the main transition.
Adenosine derivatives with N6‐alkyl, ‐alkylamine or ‐alkyladenosine substituents as probes for the A1‐receptor
Authors: van Galen P.J.M. , Ijzerman A.P. , Soudijn W.
Published: July 2002

Three series of N
6‐substituted adenosine derivatives were synthesized, having in common an unbranched alkyl chain with lengths varying from 2 to 12 methylene units, but differing in their ω‐alkyl substituents: N
6‐n‐alkyladenosines (I), N
6‐ω‐amino‐alkyladenosines (II) and α ω,di‐(adenosin‐N
6‐yl)alkanes (III). The compounds of the latter series combine two functional groups in one molecule. A1‐receptor affinity of these compounds was measured as inhibition of [3H]PIA binding to calf brain membranes. With relatively short chain lengths, compounds in series I are the most potent. In this series, optimum acitivity is reached with N
6‐n‐pentyladenosine (K
i = 0.50 nM). With short chain lengths, compounds in series II and III are 6–20‐fold less potent than their congeners in series I. The potency order however is reversed with higher chain lengths. While affinity in series II and III increases strongly, reaching an optimum with the nonyl derivatives, affinity in series I decreases sharply with alkyl chains larger than 8 methylene units. Highest affinity is found with 9‐amino‐nonyladenosine (K
i = 0.32 nM). In general, the ω‐aminoalkyl derivatives are somewhat more potent than the corresponding di‐adenosinyl derivatives. Implications for the possible topography of the N
6 region of the A1‐receptor and the area further removed from N
6 are dicussed.
The mechanism of cytochrome oxidase and other reaction centres for electron/proton pumping
Authors: Williams R.J.P.
Published: July 2002

The functional significance of the metal centres of cytochrome oxidase is deduced from the ways in which the centres are bound into its peptides. To this end use is made of structural knowledge of other metallo‐proteins for dioxygen binding, haemocyanin and haemoglobin, and for electron transfer, cytochromes b and azurin. The order and manner in which the motions of helical sections of the oxidase are linked to proton pumping are suggested and a comparison is made with other proton pumps, for example that of ATP synthetases.
Inhibitory effects of pertussis toxin on a depolarization‐evoked Ca2+ influx in NG108‐15 cells
Authors: Nomura Yasuyuki , Tohda Michihisa
Published: July 2002

Depolarized stimulation 1.5‐fold increased Ca2+ influx which was inhibited by pretreatment with verapamil or LaCl3. Treatment with pertussis toxin, islet‐activating protein (IAP), induced a reduction in 50 mM K+‐ induced Ca2+ influx and stimulated adenylate cyclase (AC) activity in NG108‐15 cells. However, addition of dibutyryl cAMP or forskolin treatment elevating cAMP level exerted no effects on a depolarization‐induced Ca2+ influx. Dissociated B‐oligomer of IAP after treatment with dithiothreitol and ATP increased a depolarization‐evoked Ca2+ influx. It is suggested that inhibitory GTP‐binding protein (Gi) or other IAP substrate proteins could directly be involved in Ca2+ influx via voltage‐sensitive Ca2+ channel.
Effect of DFP on loading of fura 2/AM and quin 2/AM into single smooth muscle cells prepared from guinea pig taenia coli
Authors: Maruyama Ikuo , Oyamada Hideto , Hasegawa Tomohiko , Ohtsuka Kazuko , Momose Kazutaka
Published: July 2002

The effect of diisopropyl fluorophosphate (DFP), a potent cholinesterase (ChE) inhibitor, on loading quin 2 acetoxymethyl ester (quin 2/AM) and fura 2/AM into smooth muscle cells isolated from guinea pig taenia coli was investigated spectrofluorometrically. The presence of DFP during the loading permitted the incorporation of quin 2 into the cells, so that it became possible to measure intracellular Ca2+ concentrations using the ester of this dye. Also, DFP significantly enhanced the incorporation of fura 2 into the cells. These results indicate that loading of quin 2/AM and fura 2/AM into the smooth muscle cells may depend on the suppression of ChE or various serine protease activities outside cells.
A cDNA coding for human sex hormone binding globulin Homology to vitamin K‐dependent protein S
Authors: Gershagen Sten , Fernlund Per , Lundwall Åke
Published: July 2002

Affinity purified antibodies to human sex hormone binding globulin (SHBG) were used in screening a human liver cDNA library, constructed in the expression vector λgt 11. One clone, identified as producing recombinant SHBG, carried a cDNA insert of 1.1 kb. The nucleotide sequence of the insert had an open reading frame coding for 356 amino acid residues. The coding sequence was followed by a short 3′‐region of 19 non‐translated nucleotides and a poly(A) tail. Confirmation that the cDNA clone represented human SHBG was obtained by the finding of a complete agreement in amino acid sequence with several peptide fragments generated from purified SHBG by proteolytic cleavage. The primary structure of SHBG shows a considerable homology to that of protein S, a vitamin K‐dependent protein with functions in the coagulation system.
Partial characterization of type X collagen from bovine growth‐plate cartilage Evidence that type X collagen is processed in vivo
Authors: Ayad Shirley , Kwan Alvin P.L. , Grant Michael E.
Published: July 2002

Sequential extraction of bovine growth‐plate cartilage with 4 M guanidinium chloride and pepsin was used to identify the intact and pepsinized forms respectively of type X collagen. This collagen occurs predominantly as the processed [α1(X)]3 form in vivo, although the procollagen [proα1(X)]3 form can also be detected. The bovine proα1 (X) and α1(X) chains have M
r, values identical to the corresponding chick species (M
r 59 000 and 49 000). However, the pepsinized α1(X)p chains (M
r 47 000) are larger than those of the chick (M
r 45 000), and the bovine collagen type X is further distinguished by being disulphide‐bonded within the triple‐helical domain.
GTP‐dependent ADP‐ribosylation of a 22 kDa protein in the endoplasmic reticulum membrane
Authors: Robinson Andrew , Austen Brian
Published: July 2002

Treatment of salt‐stripped rough microsomal membranes from pancreas or liver with NAD and cholera toxin in the presence of GTP yields an ADP‐ribosylated non‐ribosomal 22 kDa protein. Membranes containing the modified protein are less active in the co‐translational processing of secretory preproteins translated from isolated mRNA in a reticulocyte translation system, but signal peptidase activity is unchanged, suggesting that the 22 kDa protein is involved in the targetting or translocation of secretory proteins at the membrane of the endoplasmic reticulum.
Electron paramagnetic resonance and magnetic circular dichroism studies of a hexa‐heme nitrite reductase from Wolinella succinogenes
Authors: Blackmore Richard S. , Brittain Thomas , Gadsby Paul M.A. , Greenwood Colin , Thomson Andrew J.
Published: July 2002

The nature of the heme centers in the hexa‐heme dissimilatory nitrite reductase from the bacterium Wolinella succinogenes has been investigated with EPR and magnetic circular dichroism spectroscopy. The EPR spectrum of the ferric enzyme is complex showing, in addition to magnetically isolated low‐spin ferric hemes with g values of 2.93, 2.3 and 1.48, two sets of signals at g = 10.3, 3.7 and 4.8, 3.21, which we assign to two pairs of exchange coupled hemes. The MCD spectra show that the isolated hemes are bis‐histidine coordinated and that there is one high‐spin ferric heme. The exchange coupling is lost on treatment with SDS.
ras proteins enhance the phosphorylation of a 38 kDa protein (p38) in rat liver plasma membrane
Authors: Hegde Ashok N. , Das M.R.
Published: July 2002

Phosphorylation of a 38 kDa protein (p38) present in rat liver plasma membrane has been shown for the first time to be enhanced by ras proteins. This increase in phosphorylation is about 3–16‐fold depending on the incubation time and the type of ras protein used. Acid treatment removes phosphate from this protein suggesting that the phosphorylation involves phosphoramidate derivatives of basic amino acids. Experiments carried out in the presence of diethylpyrocarbonate suggest that the phosphorylation occurs on (a) histidine residue(s). It is probable that the function of p38 in the cell is modulated by ras proteins through phosphorylation. The significance of phosphorylation of p38 in terms of malignant transformation is presently unknown.
15N and 1H NMR evidence for multiple conformations of the complex of dihydrofolate reductase with its substrate, folate
Authors: Birdsall B. , De Graw J. , Feeney J. , Hammond S. , Searle M.S. , Roberts G.C.K. , Colwell W.T. , Crase J.
Published: July 2002

The binding of folate to Lactobacillus casei dihydrofolate reductase in the presence and absence of NADP+ has been studied by 15N NMR, using [5‐15N]folate. In the presence of NADP+, three separate signals were observed for the single 15N atom, in agreement with our earlier evidence from 1H and 13C NMR for multiple conformations of this complex [(1982) Biochemistry 21, 5831–5838]. The 15N spectra of the binary enzymefolate complex provide evidence for the first time that this complex also exists in at least two conformational states. This is confirmed by the observation of two separate resonances for the 7‐proton of bound folate, located by two‐dimensional exchange spectroscopy.
Accessibility of Trypanosoma brucei procyclic glycosomal enzymes to labeling agents of various sizes and charges
Authors: Patthey J.-P. , Deshusses J.
Published: July 2002

The high efficiency of glycolysis in Trypanosoma brucei has been attributed to impermeability of the glycosomal membrane to most metabolites. However, the strong stimulation of the glycolytic rate by exogenous metabolites and coenzymes in intact glycosomes is only compatible with their accessibility to the internal space. The accessibility of glycosomal enzymes to protein labeling agents of varying charge and size has been investigated. The results show that the glycosomal membrane is permeable to small molecules of the size of metabolites, but impermeable to larger molecules.
Increased chloride efflux in fibroblasts from X‐linked muscular dystrophies and clones from Duchenne carriers
Authors: Rugolo Michela , Rocchi Mariano , Lenaz Giorgio , Romeo Giovanni
Published: July 2002

Previous studies have suggested an increased chloride membrane permeability in Duchenne muscular dystrophy (DMD) fibroblasts. We report that an increased chloride efflux with respect to controls is present not only in fibroblasts from DMD, but also from two other X‐linked muscular dystrophies, Becker and Emery‐Dreifuss, as well as in clones from DMD carrier females. The latter observation suggests that, at least in DMD, the increased chloride efflux phenotype might be subject to lyonization.
The leader peptides from bacteriorhodopsin and halorhodopsin are potential membrane‐spanning amphipathic helices
Authors: von Heijne Gunnar , Segrest Jere P.
Published: July 2002

We show that the N‐terminal leader peptides from the bacterial membrane proteins bacteriorhodopsin and halorhodopsin can be expected to form amphipathic α‐helices with a highly hydrophobic nonpolar face and a narrow, negatively charged polar face. This finding is discussed in terms of a model for the integration of these proteins into the bacterial membrane.
Spinach leaf 6‐phosphofructo‐2‐kinase
Authors: Walker Griffin H. , Huber Steven C.
Published: July 2002

A novel form of 6‐phosphofructo‐2‐kinase was partially purified from spinach (Spinacia oleracea L.) leaves. As isolated, the new enzyme form possessed little or no enzymatic activity. However, pretreatment with 2 mM Mg‐ATP dramatically increased the activities of 6‐phosphofructo‐2‐kinase and fructose‐2,6‐bisphosphatase. Activation only occurred in the presence of magnesium plus ATP; either alone was ineffective. The ATP activation was reversed by treatment with alkaline phosphatase and could be completely restored by subsequent incubation with Mg‐ATP. Thus, protein phosphorylation appears to be the mechanism involved. This is the first evidence that higher plants contain an interconvertible form of the enzyme.
Phosphorylation affects the DNA affinity of the nuclear protein 24/7 from human tumor cells
Authors: Radtke Janna , Unteregger Gerhard
Published: July 2002

In the present study we investigated the binding behavior of nuclear proteins from human tumor cells to human placental DNA coupled on CNBr‐activated Sepharose. When nuclear proteins soluble in 5 M urea prepared from serum‐stimulated cells and containing the majority of the nonhistone proteins were applied onto a dsDNA column, next to several other proteins one prominent group consisting of at least 2 distinct proteins with a pI at 7 and a molecular mass near 24 kDa bound to DNA. The DNA‐binding ability of one of them is lost on phosphorylation and is recovered after dephosphorylation using alkaline phosphatase. Additionally, normal human fibroblasts taken as controls exhibit comparatively low levels of these 24/7 proteins, indicating a particular function in tumor cells.