bluuebunny/crossref_metadata_2025 huggingface dataset:
Chemical modification of microsomal cytochrome P450: role of lysyl residues in hydroxylation activity
Authors: Kunz Barbara C. , Richter Christoph
Published: July 2002

Cytochrome P450 purified from phenobarbital‐induced rat liver microsomes was acetylated at 3 lysyl residues. When reconstituted with purified NADPH‐cytochrome P450 reductase, the modified cytochrome showed full activity and substrate‐induced spectral changes with d‐benzphetamine. With 7‐ethoxycoumarin, neither enzymic activity nor binding was detected. It is concluded that the positively charged lysine residues of cytochrome P450 are important for metabolism of 7‐ethoxycoumarin by cytochrome P450.
Does the lactose carrier of Escherichia coli function as a monomer?
Authors: Keith Wright J. , Weigel Ulrich , Lustig Ariel , Bocklage Hubertus , Mieschendahl Martin , Müller-Hill Benno , Overath Peter
Published: July 2002

The purified lactose carrier of Escherichia coli (product of the lacY gene) is shown to be a monomer in detergent micelles of dodecyl‐O‐β‐D‐maltoside. The negative‐dominant phenotype of mutant carriers (lacY
−d mutants) could not be verified by measurements of the rate of galactoside transport in lacY
+/Y
−d diploid strains. It is proposed that the membrane‐embedded carrier functions as a monomer in galactoside‐H+ symport.
Phosphoenolpyruvate‐dependent protein kinase activity in rat skeletal muscle
Authors: Khandelwal Ramji L. , Mattoo Roshan L. , Bruce Waygood E.
Published: July 2002

Phosphoenolpyruvate‐dependent protein kinase activity has been demonstrated in the soluble fraction of rat skeletal muscle. The reaction was not due to the formation of ATP in the incubation mixture. Cyclic AMP, calcium, ATP and a number of phosphate acceptor proteins did not stimulate the reaction. One 32P‐labelled protein (M
r 25 000) was observed on SDS gels. The phosphorylated protein contained acid stable phosphoserine as a major phosphorylated amino acid. The phosphorylation reaction in crude extracts was not directly proportional to the amount of protein, but typical of a two‐component system; i.e., kinase and substrate. The chromatography of soluble proteins on Ultrogel AcA44 separated the phosphate acceptor protein(s) from the phosphoenolpyruvate‐dependent protein kinase activity.
Non‐invasive, kinetic measurements of [3H]nitrendipine binding to isolated rat myocytes by condensed phase radioluminescence
Authors: von Tscharner Vinzenz , Bailey Ian A.
Published: July 2002

The binding of 3H‐labelled drug molecules to membranes of living cells gives rise to photon emission from tryptophan residues at proteinaceous binding sites. This phenomenon, called condensed phase radioluminescence, has been used to measure non‐invasively the kinetics of [3H]nitrendipine binding and dissociation on the same samples of cultured beating cardiac myocytes. Signal arose only from bound drug molecules. Binding was monoexponential (τ = 5.5 min) as was dissociation (14.3 min). Preincubating cells with non‐radioactive nifedipine reduced the amplitude and rate of [3H]nitrendipine but not of [3H]dihydroalprenolol binding. The potential uses of this phenomenon are discussed.
Accumulation of glyceride‐modified pre‐penicillinase of Bacillus licheniformis in Escherichia coli treated with globomycin
Authors: Hussain Musaddeq , Oliver Lampen J.
Published: July 2002

The membrane penicillinase of Bacillus licheniformis is a glyceride‐cysteine lipoprotein whose NH2 terminus is analogous to the major outer membrane lipoprotein of Escherichia coli. When E. coli cells producing B. licheniformis penicillinase were treated with the antibiotic, globomycin, a precursor of the penicillinase, pre‐penicillinase, accumulated in the cell. It could be immunoprecipitated with anti‐penicillinase antibodies; it contained palmitate; and one of its two cysteine residues was modified by glycerol. The action of globomycin, probably indirectly, also activates protease which acts differently on the pre‐penicillinase than does the signal peptidase. The results strongly indicate that the pre‐penicillinase is processed by the globomycin‐sensitive signal peptidase in E. coli, and the modification of precursor by lipid precedes removal of the signal peptide as it does with the membrane lipoproteins of E. coli.
76‐kDa poly(A)—protein is involved in the formation of 48 S initiation complexes
Authors: Schmid Hans-Peter , Schönfelder Max , Setyono Buddy , Köhler Kurt
Published: July 2002

In erythropoietic mouse cells induced by Friend leukemia virus, ≈50% of non‐polyribosomal globin mRNA is found in 48 S initiation complexes ready to be translated. EDTA releases 15 S globin mRNPs, homologous to polyribosomal globin mRNPs. The 76‐kDa poly(A)—protein is one of its main protein components. The other 50% of non‐polyribosomal message can be separated as 20 S ‘free’ mRNPs. Its protein composition is different, especially the 76‐kDa protein is lacking. The role of this protein is discussed.
Identification and isolation of vinculin from platelets
Authors: Koteliansky V.E. , Gneushev G.N. , Glukhova M.A. , Venyaminov S.Y. , Muszbek L.
Published: July 2002

A vinculin‐like protein was identified in chicken as well as in bovine platelets by ELISA competitive binding assay using antibodies against vinculin from chicken gizzard. By a modified procedure (J. Biol. Chem. (1980) 255, 1194–1199) we succeeded in isolating bovine platelet vinculin to apparent homogeneity. The structural identity of platelet and chicken gizzard vinculin was demonstrated by circular dichroism analysis. It was also shown that platelet vinculin induces a significant decrease in the low shear viscosity of F‐actin. Vinculin, in all probability, plays an important role in the organization of actin filaments in platelets, especially in the linkages of microfilaments to the membrane.
Three‐dimensional structure of lectin from pea (Pisum sativum) at 5 Å resolution
Authors: Riskulov R.R. , Dobrokhotova Z.D. , Kuzev S.V. , Lobsanov Yu.D. , Lubnin M.Yu. , Mokulskaya T.D. , Myshko G.E. , Proskudina L.T. , Rogacheva M.M. , Saprykina L.F. , Khrenov A.A. , Mokulskii M.A.
Published: July 2002

The protein lectin from pea (M
r 49 000) crystallizes in space group P212121 with cell dimensions a = 51.0 Å, b = 61.7 Å, c = 137.6 Å and z = 4. The three‐dimensional structure of pea lectin at 5 Å resolution was determined by multiple isomorphous replacement method. The data were collected on an ARGUS multichannel diffractometer. The pea lectin molecule can be described as a dimer with approximate dimensions 85 × 55 × 40 Å. The borderline between the globules appears in the three‐dimensional model as a shallow groove on its surface. Both globules have two dense layers. The molecule proved to be quite similar to the dimer of concanavalin A.
Expression of human α1‐antitrypsin in Escherichia coli
Authors: Bollen Alex , Loriau Rosette , Herzog Albert , Hérion Pascal
Published: July 2002

Complementary DNA coding for human α1‐antitrypsin has been placed under the control of the λPR promotor carrier by the expression vector pCQV2 [1]. In conditions which allow transcription from this promotor (thermoinactivation of the repressor), Escherichia coli cells harbouring the recombinant plasmid pULB1114 express human α1‐antitrypsin (± 9000 molecules/cell). The product has a M
r of 44 000, corresponding to mature unglycosylated α1‐antitrypsin.
Regulation of 2′,5′‐oligo(A) synthetase activity in theophylline‐treated NIH 3T3 cells
Authors: Itkes A.V. , Turpaev K.T. , Kartasheva O.N. , Tunitskaya V.L. , Kafiani C.A. , Severin E.S.
Published: July 2002

Sequential treatment of NIH 3T3 cells with theophylline and actinomycin D results in an enhancement of 2′,5′‐oligo(A) synthetase activity 2–3‐fold exceeding that induced by treatment with theophylline only. This ‘superinduction’ phenomenon suggests the existence of a negative control of the enzyme that involves a labile, transcription‐dependent factor.
Vacuoles are not the sole compartments of proteolytic enzymes in yeast
Authors: Emter Othniel , Wolf Dieter H.
Published: July 2002

Localization in vacuoles, the lysosome‐like organelle of yeast, was checked for several newly detected proteolytic enzymes. While aminopeptidase Co and carboxypeptidase S were found in vacuoles, proteinase D and proteinase E as well as a variety of other proteolytic activities detectable with the aid of chromogenic peptide substrates do not reside in this cell compartment.
A nuclear Overhauser enhancement study on the imino proton resonances of a DNA pentadecamer comprising the specific target site of the cyclic AMP receptor protein in the ara BAD operon
Authors: Gronenborn Angela M. , Clore G.Marius , Jones Michael B. , Jiricny Joseph
Published: July 2002

A 500 MHz 1H‐NMR study on a synthetic DNA pentadecamer comprising the specific target site of the cAMP receptor protein in the ara BAD operon is presented. Using pre‐steady state NOE measurements, unambiguous assignments of all the imino proton resonances and associated adenine (H2) resonances are obtained. From the NOE data interbase pair interproton distances involving the imino and adenine (H2) protons are determined. It is shown that these distances are very similar to those expected for classical B DNA (RMS difference of 0.5 Å), but are significantly different from those expected for classical A DNA (RMS difference of 1.1 Å)
A comparison of the phosphorylated and unphosphorylated forms of isocitrate dehydrogenase from Escherichia coli ML308
Authors: Garland Donita , Nimmo H.G.
Published: July 2002

NADP+ can protect active isocitrate dehydrogenase against attack by several proteases. Inactive phosphorylated isocitrate dehydrogenase is much less susceptible to proteolysis than the active enzyme, and it is not protected by NADP+. The results suggest that binding of NADP+ to, or phosphorylation of, active isocitrate dehydrogenase induces similar conformational states. Fluorescence titration experiments show that NADPH can bind to active but not to inactive isocitrate dehydrogenase. It is suggested that the phosphorylation of isocitrate dehydrogenase may occur close to its coenzyme binding site.
A three‐step purification of human α1‐acid glycoprotein
Authors: Laurent Philippe , Miribel Laurent , Bienvenu Jacques , Vallve Charles , Arnaud Philippe
Published: July 2002

α1‐Acid glycoprotein (AGP) was purified to homogeneity by a 3‐step procedure using pseudo‐ligand affinity chromatography on immobilized Cibacron blue F3GA, Procion red HE3B, and preparative column isoelectric focusing. The overall yield of the combined techniques was 88%. Analysis of the purified AGP by lectin affinity chromatography on immobilized Con A and immunoaffinoelectrophoresis indicated that the most acidic form did not interact with the lectin, while the two more basic fractions possessed different affinities for Con A. In addition, 3 different populations of AGP were clearly separated by Con A affinity chromatography.
Abnormal proteins of shortened length are preferentially degraded in the cytosol of cultured MRC5 fibroblasts
Authors: Wharton S.A. , Hipkiss A.R.
Published: July 2002

Puromycyl peptides were degraded in MRC5 fibroblasts more rapidly than normal proteins labelled for the corresponding length of time for both long and short labelling periods. The degradation of the puromycyl peptides occurred almost exclusively in the cytosol of the cells. Even when the half‐lives of normal and puromycyl peptides were manipulated to be similar, proportionally more of the normal proteins were degraded in the lysosomes. The rapid degradation of the puromycyl peptides was not due to the inhibition of protein synthesis brought about by puromycin but was due to the structure of the substrates themselves. The degree and intracellular site of degradation of puromycyl peptides closely mimic those of abnormal (missense) proteins containing amino acid analogues.
Does the DNA methylase Eco dam pair nucleotide sequences to form site‐specific duplexes?
Authors: Buryanov Ya.I. , Zinoviev V.V. , Vienozhinskis M.T. , Malygin E.G. , Nesterenko V.F. , Popov S.G. , Gorbunov Yu.A.
Published: July 2002

The Eco dam methylase is active on denatured DNA and single‐stranded synthetic oligonucleotides containing GATC sites. The results suggest that on interaction with single‐stranded oligonucleotides the Eco dam methylase is able to form a duplex structure within the GATC site, and that this duplex site is a substrate for enzyme.
Effect of the DNA‐binding domain of poly(ADP‐ribose)synthetase on accurate transcription initiation in a HeLa cell lysate
Authors: Ohtsuki Masahiko , Sekimizu Kazuhisa , Agemori Michiko , Shizuta Yutaka , Natori Shunji
Published: July 2002

The effect of papain‐digested fragments of poly(ADP‐ribose)synthetase on accurate transcription initiation by RNA polymerase II was studied using a HeLa cell lysate. It was found that the DNA‐binding domain of the enzyme, like the intact enzyme, preferentially suppressed random transcription initiation, whereas the automodification domain had no effect on transcription.
The effect of glucagon‐induced adenosine 3',5'‐monophosphate concentrations on bile acid synthesis in isolated rat liver cells
Authors: Botham Kathleen M. , Suckling Keith E. , Boyd George S.
Published: July 2002

The effect of increased intracellular adenosine 3',5'‐monophosphate (cAMP) concentrations on bile acid synthesis in isolated rat hepatocytes was investigated. When the cells were incubated in the presence of glucagon (0.2 μM) and theophylline (1 mM) the observed rise in the level of cAMP was accompanied by an increase in bile acid production. Hepatocyte cAMP concentrations after 1 h of incubation showed a highly significant positive linear correlation with the amounts of bile acid synthesised by the cells during this time. These results suggest that bile acid production is related to the concentration of cAMP in isolated hepatocytes and provide evidence for a role for the cyclic nucleotide in the regulation of bile acid synthesis.
Inhibition by somatostatin of the vasopressin‐stimulated adenylate cyclase in a kidney‐derived line of cells grown in defined medium
Authors: Roy Christian
Published: July 2002

LLC‐PK1L cells, a kidney‐derived cell line grown in defined medium, possess a vasopressin‐sensitive adenylate cyclase. Somatostatin was able to inhibit the vasopressin‐induced increase in adenylate cyclase activity, without affecting the basal enzyme activity. This inhibition was competitive. No effect of somatostatin could be detected on [3H]vasopressin binding suggesting an interaction of somatostatin with the vasopressin‐sensitive system distal to the hormone—receptor interaction. At variance with N6‐L‐2‐phenylisopropyladenosine (PIA), GTP did not potentiate the inhibition by somatostatin. The inhibition of the vasopressin stimulation by somatostatin and that by PIA were additive. Changing the composition of the cell growth medium increased the number of vasopressin receptors per cell. Cells with a high number of vasopressin receptors were less sensitive to inhibition by somatostatin. Such results suggested that somatostatin and vasopressin receptors and/or the inhibitory (Ni) and stimulatory (Ns) regulatory transducing components are regulated by different mechanisms.
Differences in C‐terminal amino acid sequences between erythrocyte and liver cytochrome b5 isolated from pig and human
Authors: Kimura Sadao , Abe Kiyoshi , Sugita Yoshiki
Published: July 2002

Two forms of cytochrome b
5, a soluble erythrocyte form and a membrane‐bound liver form, were purified from pig and human, and structural differences between them were analyzed. Porcine and human erythrocyte cytochrome b
5 consisted of 97 amino acid residues and contained the same catalytic domain structure (residue 1–96) as that of the corresponding liver cytochrome b
5, but had one amino acid replacement at the C‐terminus (residue 97). These results suggest that erythrocyte cytochrome b
5 is not derived from the liver protein by proteolysis but a translational product from another distinct mRNA of cytochrome b
5.
Separation of endo‐ and exo‐type cellulases using a new affinity chromatography method
Authors: van Tilbeurgh Herman , Bhikhabhai Ramagauri , Göran Pettersson L. , Claeyssens Marc
Published: July 2002

A new efficient biospecific adsorbent for the purification of cellulases was prepared from p‐aminobenzyl 1‐thio‐β‐D‐cellobioside and Affigel‐10 (Biorad). The two exocellobiohydrolases, CBH I and CBH II, from Trichoderma reesei QM 9414 were adsorbed, whereas the endoglucanase activities were unretarded. The adsorbed CBH I can be displaced selectively by elution with lactose, whereas cellobiose elutes the CBH II.
A cyclic AMP‐dependent phosphorylation of spectrin dimer
Authors: Lutz Hans U.
Published: July 2002

In contrast to the properties of spectrin obtained from [32P]phosphate‐labeled red cells, purified spectrin dimer could be phosphorylated by a cAMP‐dependent protein kinase from bovine heart. Both spectrin bands were phosphorylated. Spectrin band 2 contained in addition to autophosphorylated peptides several phosphopeptides that were distinct from autophosphorylated ones. The cAMP‐dependent phosphorylation of spectrin band 1 was modulated by reducing agent and the concentration of spectrin. At high concentrations spectrin band 2 was predominantly labeled. The cAMP‐dependent phosphoform of spectrin band 2 had a pI slightly higher than that of autophosphorylated spectrin band 2, but lower than that of ankyrin.
Characteristics of the cell surface antigen, p72, associated with a variety of human tumours and mitogen‐stimulated T‐lymphoblasts
Authors: Price M.R. , Campbell D.G. , Robins R.A. , Blecher T.E. , Baldwin R.W.
Published: July 2002

The monoclonal antibody 791T/36, prepared against a human osteogenic sarcoma cell line, 791T, reacts with a variety of human tumours and also mitogen‐stimulated PBMN cells. The target antigen as expressed upon 791T cells is a monomeric plasma membrane‐associated glycoprotein with an apparent M
r of 72000. By quantitative flow cytofluorimetry, approx. 105 antibody molecules bound per cell to T‐lymphoblasts induced with PHA or Con A, whereas only a few thousand antibody molecules bound per cell to unstimulated cells, so that the antigen may be classified as a lymphocyte activation antigen. On lymphoblasts, the 791T/36 again reacted with a protein with an apparent M
r of 72000. This antigen therefore has a dual role as a tumour marker and lymphocyte activation antigen which may be implicated in the regulation of cell proliferation.
The stereochemistry of the formation of the methyl group in the glutamate mutase‐catalysed reaction in Clostridium tetanomorphum
Authors: Hartrampf Günther , Buckel Wolfgang
Published: July 2002

The adenosylcobalamin‐dependent enzyme glutamate mutase from Clostridium tetanomorphum catalyses the reversible rearrangement of (2S)‐glutamate to (2S,3S)‐3‐methylaspartate. In this conversion 6 carbon centers are involved. The stereochemistry of 4 has been elucidated whereas the formation of the methyl group from the methylene group remains to be established. To solve this problem, (2S,3R)‐ and (2S,3S)‐[3,3‐2H1,3H]glutamates were prepared via the 2‐oxo[3,3‐2H2 or 3H]glutarates by incubation with isocitrate dehydrogenase in deuterium oxide or tritiated water. The labelled glutamates were fermented with growing cells of C. tetanomorphum to butyrate and acetate. Butyrate was further degraded to acetate in which methyl group over 90% of the tritium of the starting glutamate was retained. The chirality of the acetates was determined with malate synthase and fumarase. In both samples complete racemisation was found. This result confirms the rule that racemisation occurs in all adenosylcobalamin‐dependent rearrangements in which methyl groups are formed. A methylene radical as intermediate could explain these observations. In a control experiment inversion of configuration in the formation of the methine group of (2S,3S)‐3‐methylaspartate from the methylene group of (2S)‐glutamate was confirmed. Glutamates stereospecifically labelled at C‐4 were synthesized from chiral acetates via citrate.
On the dehydration of (R)‐lactate in the fermentation of alanine to propionate by Clostridium propionicum
Authors: Schweiger Georg , Buckel Wolfgang
Published: July 2002

All the enzymes of the pathway of (S)‐alanine fermentation to acetate and propionate were detected in cell‐free extracts of Clostridium propionicum. Among these (S)‐glutamate dehydrogenase (NAD), (R)‐lactate dehydrogenase (NAD) and propionate CoA‐transferase were purified to apparent homogeneity. Their structures were presumably α6, α2 and α4, respectively. The latter enzyme was specific for short‐chain monocarboxylic acids with a pronounced preference for (R)‐lactate over the (S)‐enantiomer. The key step of the pathway, the dehydration of (R)‐lactate required acetyl phosphate and CoASH under anaerobic conditions. It was inhibited by hydroxylamine, arsenate, azide (1 mM each) or by 0.1 mM 2,4‐dinitrophenol. Thus it closely resembled the dehydration of (R)‐2‐hydroxyglutarate in Acidaminococcus fermentans, although an activation was not necessary.
Structural similarities among concanavalin A, haptoglobin, and trypsin
Authors: Dobryszycka Wanda , Przysiecki Bernard
Published: July 2002

Comparison of the primary structure of concanavalin A with those of trypsin and β (heavy) chain of haptoglobin revealed a significant degree of chemical similarity. The amino acid sequence of concanavalin A was found to be 53.6% related to trypsin and haptoglobin. Certain analogy in the tertiary structure was shown by the use of thionine binding to the specific active site, characteristic for serine proteases.
Muscle and liver pyruvate kinases are closely related: amino acid sequence comparisons
Authors: Hoar Colin G. , Nicoll Gordon W. , Schiltz Emile , Schmitt Wilfried , Bloxham David P. , Byford Michael F. , Dunbar Bryan , Fothergill Linda A.
Published: July 2002

Previous evidence has shown that the M1 and L pyruvate kinase isozymes differ markedly in kinetic and immunological properties, amino acid compositions and peptide maps. However, the amino acid sequence results we present here for the N‐terminal region and for a region of the C domain show that the M1 and L isozymes are very similar. The variable length of the N‐terminal sequences also explains the difference in regulation by phosphorylation between the M1 and L isozymes. The M1 isozyme lacks the serine residue that has been shown to be phosphorylated in the L isozyme.
New cell fusion method using polymer membrane
Authors: Karube Isao , Tamiya Eiichi , Matsuoka Hideaki
Published: July 2002

A porous polymer membrane of nitrocellulose or tetrafluoroethylene (TFE) was employed for fusion of Saccharomyces cerevisiae (AH22 and D13‐1A) protoplasts. Protoplasts were adsorbed on the membrane with slight suction. Some part of the protoplasts was trapped in pores of the membrane as observed by electron microscopy. The membrane retaining protoplasts was placed on a selective medium. Several colonies appeared on the medium after 5–7 days incubation at 30°C. The fusion of the two strains was ascertained by DNA content and genetic markers. Fusion frequency was 1.2 × 10−6 in the case of the TFE membrane.
Two cholesterol pools in Acholeplasma laidlawii membranes
Authors: Davis P.J. , Efrati H. , Raxin S. , Rottem S.
Published: July 2002

Cholestrol exchange kinetics between [14C]cholesterol‐labeled Acholeplasma laidlawii and Mycoplasma gallisepticum cells and phosphatidylcholine‐cholesterol vesicles followed a biphasic curve, with faster exchange rates for A. laidlawii. The same biphasic curve was obtained with isolated membranes. Cholesterol exchange between lipid vesicles and A. laidlawii cells depleted of phospholipids by phospholipase A2, fitted a monophasic linear curve. The data support the hypothesis that the biphasic cholesterol exchange kinetics do not result from the transbilayer distribution of cholesterol, but reflect the presence in the membrane of two cholesterol pools associated with lipids of high and low affinity for cholesterol.
Immobilization of α‐chymotrypsin on sucrose stearate‐palmitate containing liposomes
Authors: Bogdanov A.A. , Klibanov A.L. , Torchilin V.P.
Published: July 2002

Stable liposomes have been prepared from lipid mixture containing sucrose stearate‐palmitate. 1.2·10−4 mol of model enzyme α‐chymotrypsin per mol of lipid have been coupled to prepared liposomes activated by periodate oxidation of sucrose units.
Glycosidic bond rearrangements in isomeric xylobioses by yeast xylan‐degrading enzymes
Authors: Biely Peter , Petráková Eva
Published: July 2002

The cells of Cryptococcus albidus induced for xylan‐degrading enzymes are capable of transforming 1,2‐β‐xylobiose and 1,3‐β‐xylobiose into 1,4‐β‐xylobiose, the natural inducer. The conversion involves transglyco‐sylation and hydrolysis catalyzed by β‐xylosidase and β‐xylanase. A probable intermediate of the conversion of 1,2‐β‐xylobiose was isolated and identified as a trisaccharide, 4‐O‐β‐xylopyranosyl‐2‐O‐β‐xylopyranosyl‐D‐xylopyranose. The trisaccharide is cleaved by purified endo‐1,4‐β‐xylanase of C. albidus mainly at the 1,2‐β‐linkage yielding xylose and 1,4‐β‐xylobiose.
Inactivation and reactivation of light‐triggered ATP hydrolysis on the chloroplast coupling factor
Authors: Komatsu-Takaki Mizuho
Published: July 2002

Decay of light‐triggered ATP hydrolysis in the dark was diminished with a decrease in chloroplast concentration. The enhancing effect of NH4Cl on ATP hydrolysis decreased with dark time. The decrease was much faster than that in ATP hydrolysis activity. The NH4Cl effect increased with ATP preincubation time. Reactivation of ATP hydrolysis occurred with the progress of ATP hydrolysis. Pi enhanced the activation remarkably. These results suggest that ATP hydrolysis produces some energized state, which stimulates NH4C1 effect and makes coupling factor active in the presence of Pi and that to keep coupling factor active, energy is not necessarily needed.
Utilization of membranous lipid substrates by membrane‐bound enzymes
Authors: Rousseau A. , Gatt S.
Published: July 2002

The membranous lipase of rat liver microsomes was used to hydrolyze diacylglycerol (DG), generated within the microsomal membrane by treatment with phospholipase C, in two separate interactions. For an intramembrane enzyme‐substrate interaction, the enzyme and DG were present in the same microsomes. For intermembrane interactions, native microsomes of rat liver were used as carriers of the enzyme, while heated and phospholipase C‐treated microsomes of rat liver or brain were employed as carriers of the substrate. The v vs S curves of the intermembrane interaction were hyperbolic while those of the intramembrane utilization were parabolic.
Sucrose effects on the fluidity of natural and synthetic phospholipid bilayers
Authors: Uso Thierry , Rossignol Michel
Published: July 2002

Fluorescence anisotropy of DPH embedded in bilayers prepared from horse bean‐ (Vicia faba L. var. minor) root phospholipids and synthetic phospholipids, respectively, is increased by sucrose. Sucrose has both non‐specific and specific effects. The non‐specific effect appears at sucrose concentrations above about 1 M and is correlated with the lowering of vesicle size. The specific effect depends on the phospholipid polar heads and probably implies modifications of the hydration shell. These results show that characterization of membranes in the presence of sugars must be done with caution.
Intermediates in 16‐androstene biosynthesis in neonatal porcine testis
Authors: Kwan T.K. , Honour J.W. , Gower D.B.
Published: July 2002

The metabolism of pregnenolone has been studied in microsomal preparations of testes from 3‐week old piglets. Metabolites identified after derivatization by capillary gas‐chromatography and further by combined gas chromatography‐mass spectrometry were: 17‐hydroxy‐ and 16α‐hydroxypregnenolone, dehydroepiandrosterone, 5‐androstenediols, 5‐pregnenediol and andien‐β. Pregnenolone and 16‐dehydropregnenolone, previously not separable by other methods, were successfully resolved as methyl oximetrimethylsilyl ethers using capillary column gas‐chromatography.
Regulation of oxygen consumption and microcirculation by α‐sympathetic nerves in isolated perfused rat liver
Authors: Ji Sungchul , Beckh Karlheinz , Jungermann Kurt
Published: July 2002

In isolated rat liver perfused at constant flow with erythrocyte‐free Krebs‐Henseleit bicarbonate buffer containing 5 mM glucose and 2 mM lactate, perivascular stimulation of the hepatic nerves caused a rapid decrease of oxygen uptake, a decrease of the periportal and, after a transient rise, of the perivenous tissue p
o2
of surface acini, an increase of portal pressure, and an enhancement of glucose output. Furthermore, nerve stimulation changed the intrahepatic distribution of the perfusate drastically. Infusion of trypan blue 20 s after nerve stimulation resulted in a heterogeneous staining of the liver both at the surface and in cross‐sections, while it led to a homogeneous distribution in non‐stimulated controls. It is concluded that the major component in the mechanism of the nerve‐dependent decrease of oxygen uptake is the microcirculatory change rather than a metabolic effect.
Localised chemiosmosis and active transport of lactose in Escherichia coli
Authors: Ahmed Sohail
Published: July 2002

The phosphorylation potential (δG
p) in mitochondria has been found to be relatively insensitive to changes in the protonmotive force (δp). Similar observations have been made for lactose transport in Escherichia coli. Localised chemiosmosis has been invoked to explain these data. Here a postulate of localised chemiosmosis was put to experimental test and found to be incorrect. Alternative explanations for the δpdependence of lactose transport are presented.
Isolation and characterization of 1‐O‐α‐2‐acetamido‐2‐deoxy‐D‐galactopyranosyl‐myo‐inositol from pregnancy urine
Authors: Meyer Denise M. , Lemonnier Marguerite , Derappe Christian , Sellier Nicole , Platzer Nicole
Published: July 2002

A new neutral glycoside of myo‐inositol was isolated from the pregnancy urine of a single donor. Its structure was investigated by 1H‐NMR spectroscopy and mass spectroscopy. It was identified as 1‐O‐α‐2‐acetamido‐2‐deoxy‐D‐galactopyranosyl‐myo‐inositol. No such structure or sequence has previously been reported in either myo‐inositol or glucose glycosides.
Ciglitazone reverses cAMP‐induced post‐insulin receptor resistance in rat adipocytes in vitro
Authors: Kirsch Diethelm M. , Bachmann Werner , Häring Hans U.
Published: July 2002

Ciglitazone (cig), a thiazolidine‐dione, lowers glucose and insulin levels in animal models of diabetes type II but not in controls. Since catecholamines given to rat adipocytes in vitro induce insulin resistance similar to that seen in type II diabetes in vivo, we measured the effect of cig on mono‐A14‐[125I]insulin binding and 3‐O‐methyl‐D‐glucose transport (GT) in isolated rat adipocytes treated with isoprenaline (iso, 10 μM). Cig (⩽ 5 μM) reversed (ED10 nM) the inhibitory effect of iso on insulin stimulation of GT. It had no effect on either basal or insulin stimulated GT. Furthermore, cig did not influence insulin binding either in the presence or absence of iso, which indicates that cig acts only on a post‐insulin receptor level. Cig also reversed the inhibiton of GT by both forskolin, a cyclase activator and RO20‐1724, an imidazolidine phosphodiesterase inhibitor but not that of db‐cAMP. It thus seems that cig does not act within the cAMP system but only neutralizes its inhibitory effect on the insulin stimulation of GT.
Is the induction of neuroblastoma differentiation by CCA mediated by its effects on the electrochemical gradient?
Authors: Vayssière J.L. , Berthelot F. , Gros F. , Croizat B.
Published: July 2002

Various mitochondrial inhibitors are tested in neuroblastoma cells. Their effects on the mit‐proteins and some cytoskeletal proteins are compared to those of CCA, a differentiation inducer. This comparison favours the hypothesis that the primary effect of CCA induction is an alteration of the electrochemical gradient.
The organisation of methanol dehydrogenase and c‐type cytochromes on the respiratory membrane of Methylophilus methylotrophus
Authors: Quilter Jacqueline A. , Jones Colin W.
Published: July 2002

The membrane‐impermeant, protein‐labelling reagent [14C]isethionyl acetimidate has been used to investigate the detailed topography of the membrane‐associated methanol oxidase system in the methylotrophic bacterium Methylophilus methylotrophus. The results show that methanol dehydrogenase and cytochrome c
1 are significantly less exposed on the periplasmic surface of the membrane than either cytochrome C
H or an as yet unidentified 12‐kDa protein. The partial crypticity of the methanol dehydrogenase has been confirmed by fingerprinting with V8 protease. The results are discussed in terms of the electron transfer functions of the various redox proteins.
Detection of intermediary Clr with complete active site, using a synthetic proteinase inhibitor
Authors: Niinobe Michio , Ueno Yoshihiko , Hitomi Yuji , Fujii Setsuro
Published: July 2002

The synthetic proteinase inhibitor, FUT‐175 (6‐amidino‐2‐naphthyl‐4‐guanidinobenzoate), strongly suppressed activation of Clr at 37°C, causing 50% inhibition at 0.03 mM. To clarify whether the inhibitor was incorporated into the active site of intermediary Clr formed during the incubation, determination of the active site was tried using this inhibitor. Consequently, release of amidinonaphthol equimolar with the amount of Clr used was observed in the early period of incubation, in which the activation to Clr was about 5%. These results indicate that intermediary Clr already has a complete active site.
Reconstitution of photosynthetic charge accumulation and oxygen evolution in CaCl2‐treated PS II particles
Authors: Toyoshima Yoshinori , Akabori Kozo , Imaoka Akiko , Nakayama Haruto , Ohkouchi Nozomu , Kawamori Asako
Published: July 2002

Extraction of PS II particles with 1 M CaCl2 caused complete disappearance of the light‐induced signal of the possible Kok S2 state of the water‐splitting complex and total loss of the O2, evolving activity, concomitant with perfect removal of the 17‐, 23‐ and 34‐kDa proteins from the particles. The recovery of the multiline signal in the CaCl2‐treated PS II was performed by reinserting the 34‐kDa protein, when CI− was present in the solution for the EPR measurement. However, in the absence of Cl−, besides the 34‐kDa protein, the 17‐ and 23‐kDa proteins were required for the recovery of the signal. These results are compared with the results on the recovery of the O2, evolution in the reconstituted PS II to examine the role of these three proteins on the water splitting.
4‐(N‐tert‐Butyloxycarbonylaminomethyl)‐phenylisothiocyanate: its synthesis and use in microsequencing
Authors: Palacz Zbigniew , Salnikow Johann , Ju Shan-Wei , Wittmann-Liebold Brigitte
Published: July 2002

The chemical synthesis of 4‐(N‐tertbutyloxycarbonylaminomethyl)‐phenylisothiocyanate starting from 4‐nitrobenzylamine is described. This derivative represents an Edman‐type reagent with a masked amino group which renders the thiohydantoin upon deblocking susceptible to fluorogenic detection. The coupling efficiency is determined in comparison to degradations with PITC, DABITC and FITC. The detection sensitivity on thin layer chromatograms is compared to the thiohydantoins derived from DABITC.
Studies on the one‐ and two‐electron FAD‐mediated reactions by ferrodoxin‐NADP+ oxidoreductase from Anabaena
Authors: Serrano A. , Rivas J. , Losada M.
Published: July 2002

The NADPH‐diaphorase activity of the flavoprotein ferredoxin‐NADP+ oxidoreductase from the cyanobacterium Anabaena sp. strain 7119 shows a very alkaline optimum pH value, about 9.5–10.0, when assayed with two‐electron acceptor dyes, such as dichlorophenolindophenol or iodonitrotetrazolium. In contrast, the enzyme reactions mediated by the physiological one‐electron carrier ferredoxin exhibit an almost neutral optimum pH value. EPR spectra and pH titration of the NADPH‐reduced enzyme indicate that whereas the one‐electron reduced form (semiquinone) of the flavin coenzyme is formed at neutral pH values, the fully reduced form (hydroquinone) arises at the alkaline range above pH 9.0. These findings are also in agreement with data obtained from redox titrations of the enzyme FAD by using an NADPH‐regenerating system and are interpreted on the basis of the hydroquinone and semiquinone forms of the flavin coenzyme being, respectively, intermediates in the diaphorase and ferredoxin‐mediated catalytic activities of the reductase.
1‐N6‐Etheno‐ADP‐ribosylation of elongation factor‐2 by diphtheria toxin
Authors: Giovane A. , Balestrieri C. , Quagliuolo L. , Servillo L.
Published: July 2002

Diphtheria toxin fragment A is able to inhibit protein synthesis in the eukaryotic cell by ADP‐ribosylating the diphthamide residue of elongation factor‐2 (EF‐2) [(1980) J. Biol. Chem. 255, 10710‐10720]. The reaction requires NAD as ADP‐ribose donor. This work reports on the capacity of an NAD analog, the nicotinamide 1‐N
6‐ethenoadenine dinucleotide (ϵNAD), to be a substrate of diphtheria toxin fragment A in the transferring reaction of the fluorescent moiety, the ϵADP‐ribose, to the EF‐2. As a consequence of the transfer of the ϵADP‐ribosyl moiety to the EF‐2, there is an increase in the emission intensity of the fluorophore and a blue shift in its emission maximum. The ϵADP‐ribosylated EF‐2, like ADP‐ribosylated EF‐2, retains the capacity to bind GTP and ribosome. The utility of introducing a fluorescent probe in a well defined point of the EF‐2 molecule for conformational or binding studies is discussed.
Direct observation of the ion distribution between charged lipid membranes
Authors: Hentschel Manfred P. , Mischel Maja , Oberthür Radulf C. , Büldt Georg
Published: July 2002

Lipid membrane
Ion distribution
Neutron diffraction
Halobacterium cutirubrum tRNA sequences
Authors: Nicoghosian K. , Gu Xiang-Rong , Cedergren Robert
Published: July 2002

The primary structures of nine Halobacterium cutirubrum tRNAs are presented. These tRNAs are compared with other archaebacterial tRNAs and in particular with their H. volcanii counterparts. Striking similarities are observed among isoacceptor tRNA species; observed differences between a given tRNA pair of the two halophiles having the same anticodon vary from 0 to 14 nucleotides. Particular features including modified nucleotides of each of the nine tRNAs are indicated.
Effects of growth hormone‐releasing factor and somatostatin on growth hormone secretion and cellular cyclic AMP levels
Authors: Law Greg J. , Ray Keith P. , Wallis Michael
Published: July 2002

Human pancreatic growth hormone‐releasing factor (GRF‐44‐NH2) stimulated growth hormone (GH) secretion and intracellular cyclic AMP levels in cultured pituitary cells from both sheep and rat. Somatostatin (SRIF), over a wide range of doses and time, showed no significant effect on the elevated cyclic AMP levels in sheep cells, but did block the GH release in a dose‐dependent manner. In rat cells, however, SRIF inhibited GRF‐stimulated cyclic AMP levels by 75% maximum (still 8‐fold greater than the basal levels) and GH release to almost half the basal value. We conclude that somatostatin inhibits GRF‐elevated cyclic AMP levels in rat pituitary cells but not in sheep cells.
SV40 chromatin structure is not essential for viral gene expression
Authors: Graessmann A. , Bumke-Vogt C. , Buschhausen G. , Bauer M. , Graessmann M.
Published: July 2002

The biological activity and the fate of SV40 DNA (minichromosomes, DNA I, DNA II, DNA III) were tested in culture cells by immunofluorescence staining and blot analysis. Following microinjection of 2–4 circular SV40 molecules (minichromosomes, DNA I, DNA II) into the cytoplasm or the nuclei of monkey and rat cells, T‐ and V‐antigen synthesis was demonstrable in nearly every recipient cell. Only linear DNA induced T‐antigen synthesis with a very low efficiency after cytoplasmic injection. This low activity correlates with a rapid degradation of DNA III in the recipient cells. Further modifications observed immediately after injection are relaxation of superhelical molecules and formation of high‐M
r DNA. Assembly of the injected DNA into SV40 chromatin‐like structure, however, occured only late after early viral gene expression.
Monoclonal antibodies to mitochondrial coupling factor B
Authors: Joshi Saroj , Kantham Lakshmi , Kaplay Suresh , Sanadi D.Rao
Published: July 2002

Two monoclonal antibodies (MAb 1 and IV) have been prepared which showed high and specific reactions towards bovine heart mitochondrial coupling factor B (FB). Both have been identified as sub‐type IgG1 of mouse immunoglobulins. MAb I reacts with purified and functionally active fB, alkylated or oxidized forms of FB and even with peptides formed on digestion of FB with trypsin. When used together, MAb I and IV reacted with FB in immunoblots of normal and urea treated samples of mitochondria, submitochondrial particles, ammonia‐EDTA extracted particles, and H+‐ATPase. Both MAbs inhibited FB‐stimulated ATP‐dependent reverse electron flow activity when fB was incubated with the antibody either before or after its addition to FB‐deficient AE‐particles. Reactivity of MAb I towards fB declined upon exposure of fb to guanidine HCl while reactivity of MAb IV remained unaltered.
The role of glutamine synthetase activity in ammonium and methylammonium transport in anacystis nidulans R‐2
Authors: Boussiba S. , Gibson J.
Published: July 2002

Ammonium
Methylammonium transport
Methionine sulfoximine
Glutamine synthetase
Cyanobacteria
Anacystis nidulans
Proteolysis of liver acetyl coenzyme A carboxylase by cathepsin B
Authors: Wada Kenji , Tanabe Tadashi
Published: July 2002

Proteolysis of acetyl‐CoA carboxylase was examined with cathepsin B. When chicken liver acetyl‐CoA carboxylase was incubated with cathepsin B at pH 6.3, the native 220‐kDa polypeptide was primarily cleaved into two polypeptides of 125 and 115 kDa, and further degraded to polypeptides of 100–50 kDa.
Inhibitory effect of atrial natriuretic factor on adenylate cyclase activity in adrenal cortical membranes
Authors: Anand-Srivastava Madhu B. , Genest Jacques , Cantin Marc
Published: July 2002

The effect of rat synthetic atrial natriuretic factor (ANF) on adenylate cyclase activity was studied in adrenal cortical membranes. Synthetic ANF (Arg 101‐Tyr 126) inhibited adenylate cyclase activity in a concentration‐dependent manner. The maximum inhibition observed was about 25% with an apparent K
i of 5 x 10−11 to 10−10 M. Various hormones such as isoproterenol, dopamine, prostaglandin (PGE1) and ACTH‐stimulated adenylate cyclase to various degrees and ANF inhibited the stimulatory effect of all these hormones to some degree but never abolished it. In addition, ANF was also able to inhibit the stimulation exerted by forskolin which activates adenylate cyclase by receptor‐independent mechanism. This is the first study demonstrating the inhibitory effect of ANF on adrenal cortical adenylate cyclase. From these results it can be suggested that the inhibition of adenylate cyclase may be one of the mechanisms through which ANF exerts the inhibitory effect on steroidogenesis stimulated by various hormones and agents.
The carbohydrate moiety of aminopeptidase N of rabbit intestinal brush‐border membrane
Authors: Massey Dominique , Maroux Suzanne
Published: July 2002

Endoglycosidase F was used to eliminate the N‐linked complex glycans from intestinal aminopeptidase N. The glycans which were probably O‐linked remaining after the endoglycosidase F treatment exhibited the human blood group A and H determinants expressed in enzymes from A+ or A− rabbits, respectively. The molecular mass estimation of the two types of glycans by SDS‐polyacrylamide gel electrophoresis and the sugar composition of aminopeptidase from A+ and A− rabbits strongly suggested the presence of eight N‐linked complex glycans and two O‐linked oligosaccharides bearing the human group antigenicity.
On the existence of receptors to the pheromonal steroid, 5α‐androst‐16‐en‐3‐one, in porcine nasal epithelium
Authors: Hancock M.R. , Gennings J.N. , Gower D.B.
Published: July 2002

The binding of the odorant, 5α‐androst‐16‐en‐3‐one, to porcine nasal tissues, has been investigated using methods normally employed for studying both cytosolic and membrane‐bound receptors. 5α‐Androst‐16‐en‐3‐one was generally taken up more avidly by homogenates of olfactory (nervous) tissue than by respiratory tissue, but binding to the former was only partially prevented by prior heating or by excess ligand, suggesting some degree of specific binding. At low protein concentration, saturable binding was noted but these data were not reproducible. The binding of a non‐odorant, DHA, was only 2% that of 5α‐androst‐16‐en‐3‐one. Using agarose gel electrophoresis, some evidence was obtained for binding protein(s) to the odorous 16‐adrostene in porcine respiratory tissues, that were absent from previously heated tissue. Experiments with SDS‐treated, or cell‐membrane‐enriched preparations, of nasal epithelium did not show improved binding of 5α‐androst‐16‐en‐3‐one. We conclude that the extreme hydrophobicity of 5α‐androst‐16‐en‐3‐one is probably responsible for the high degree of non‐specific binding noted and for variability in results. This is discussed in relation to other known odorous ligand/receptors in olfactory tissue, particularly that of 5α‐androstan‐3‐one [10].
Comparative effects of cholera and Bordetella pertussis toxins on cyclic AMP and GTP levels and on lipolysis in rat adipocytes incubated in vitro
Authors: Lambert B. , Aublin J.L. , Champion S. , Haye B. , Jacquemin C.
Published: July 2002

The respective effects of cholera and Bordetella pertussis toxins were studied in time and concentration dependent experiments, following glycerol and fatty acid release, GTP and cAMP levels. Cholera toxin, after a lag time of 30 min, stimulated linearly GTP and cAMP accumulation and lipolysis (maximal effect: 2‐fold increase at 5). Pertussis toxin presented a biphasic effect both in time and concentration dependent studies. Up to a maximum reached after 2 h with 1.4 units the stimulation affected GTP (3 fold) and cAMP (7 fold) levels, glycerol and fatty acid release (15 fold). Beyond this, an inhibition occurred, yielding a decrease towards basal values of GTP and cAMP content whereas the glycerol and fatty acid release was stopped. These results, which are the first reporting the fluctuation of the GTP content of intact cells challenged with bacterial toxins, show a close relationship between GTP and cyclic AMP levels and lipolytic activity.
On a sequence similarity between ribosomal protein S5 and DNA binding protein II
Authors: Wilson Keith S. , Kimura Makoto , Dijk Jan
Published: July 2002

Sequence similarity between ribosomal protein S5 and DNA binding protein II from eubacteria is reported and its significance discussed.
Action of influenza virus neuraminidase on gangliosides
Authors: Slepushkin V.A. , Bukrinskaya A.G. , Prokazova N.V. , Zhigis L.S. , Reshetov P.D. , Shaposhnikova J.I. , Bergelson L.D.
Published: July 2002

The action of partly purified neuraminidase (NA) of influenza A virus, a mixture of detergent solubilized NA and haemagglutinin (HA) and of intact virions on gangliosides GTlb, gdla, GDlb, gml was studied. The viral NA transformed gtlb mainly into GDlbWith formation of only minor amounts of GMl. HA was found to inhibit the hydrolysis activity of viral NA. At the same time viral NA transformed GDla quantitatively into gml which was not hydrolyzed by the enzyme. These results suggest that the function of NA is to transfer the ‘primary’ receptor (such as GTlb) into the proper carbohydrate sequence (GDlb‐like) which is proposed to serve as the minimal structure required for influenza virus reception.