bluuebunny/crossref_metadata_2025 huggingface dataset:
Differential photoaffinity labelling of serotonin‐S2 receptors and histamine‐H1 receptors using 7‐azidoketanserin
Authors: Wouters Walter , Van Dun Jacky , Leysen Josée E. , Laduron Pierre M.
Published: July 2002

7‐Azidoketanserin, a potent photoaffinity probe for serotonin‐S2 receptors was shown to irreversibly photoinactivate histamine‐H1receptors as well. The photolabelling of H1‐receptors could be prevented by several selective histamine‐H1 antagonists. In guinea pig cerebellum, a brain area that is highly enriched in H1‐receptors, photolabelling could be directed exclusively to these receptors by adding a high concentration of pipamperone, which selectively blocks serotonin‐S2 receptors. In rat pre‐frontal corex, a region that is enriched in serotonin‐S2 receptors, pyrilamine was used to block H1‐receptors, thereby directing the photolabelling exclusively to S2‐receptors.
Formation of an intramolecular disulfide bond in the mitochondrial adenine nucleotide translocase
Authors: Torok Katalin , Joshi Saroj
Published: July 2002

Adenine nucleotide translocase in electron transport particles or in H+‐ATPase preparation from bovine heart mitochondria is capable of forming both inter‐ and intramolecular disulfide bridges upon reaction with copper‐o‐phenanthroline. We have examined the localisation of the intramolecular disulfide bridge in the protein chain by peptide fragmentation methods. The most likely position of the disulfide bridge is between cysteine 159 and 256, but the possibility of the presence of a second disulfide bridge formed between 129 and 256 cannot be ruled out. Our experimental results support the theoretical model proposed [(1982) FEBS Lett. 144, 250‐254] for the topography of the translocase and provide a more accurate description of the arrangement of some of the hydrophilic segments in the molecule.
Sequence‐specific chemical modification of a 365‐nucleotide‐long DNA fragment with an alkylating oligonucleotide derivative
Authors: Vlassov V.V. , Gaidamakov S.A. , Gorn V.V. , Grachev S.A.
Published: July 2002

An aromatic 2‐chloroethylamino group was attached to the 5'‐terminal phosphate of the oligodeoxyribonucleotide pCCCTCTTTCTT. The oligonucleotide derivative prepared was used for modification of the 365‐nucleotide‐long DNA fragment. It was found that modification of the fragment proceeds in a sequence‐specific way at 3 guanosine residues within the sequence complementary to the oligonucleotide reagent.
Degradation mediated OH radical generation from synthetic cyclic peroxides: ESR studies
Authors: Matsugo Seiichi , Kayamori Nobuko , Hatano Yoshihiko , Ohta Tatsuo , Konishi Tetsuya
Published: July 2002

Hydroxy radical generation on thermal decomposition of synthetic cyclic peroxides was determined by ESR using DMPO as a spin‐trapping reagent. Both the 6‐ and 7‐membered cyclic peroxides, 4‐alkoxy‐2,3‐benzodioxan‐1‐ols and 4‐alkoxy‐2,3‐benzodioxepin‐1‐ols, respectively, which were synthesized according to our idea of a radical‐releasing drug, gave rise to DMPO‐OH signals on heat treatment, i.e. under their degradation conditions. The signals were completely abolished with higher concentrations of OH scavenger.
Probing the adenosine receptor with adenosine and xanthine biotin conjugates
Authors: Jacobson Kenneth A. , Kirk Kenneth L. , Padgett William , Daly John W.
Published: July 2002

Biotin‐containing analogs of a potent agonist (N6
‐phenyladenosine) and a potent antagonist (1,3‐dipropyl‐8‐phenylxanthine) of adenosine receptor activity have been synthesized. A spacer chain to the biotin moiety is attached in both cases to the para‐position of the phenyl ring. Two biotin conjugates of N6
‐phenyladenosine differing only in the length of the spacer chain bind to the adenosine receptor and to avidin simultaneously. The shorter‐chain derivative was more potent in inhibiting binding of N6
‐[3H]lcyclohexyladenosine to rat cerebral cortical membranes (K
i of 11 nM in the absence of avidin, 36 nM for the avidin complex). Three biotin conjugates of 1,3‐dipropyl‐8‐phenylxanthine bound competitively to the adenosine receptor, but only in the absence of avidin. The results are interpreted in terms of the possible orientation of the ligands at the receptor binding site.
Two separate tyrosine protein kinases in human platelets
Authors: Nakamura Shun-ichi , Takeuchi Fumito , Tomizawa Tamami , Takasaki Noriko , Kondo Hiroki , Yamamura Hirohei
Published: July 2002

Tyrosine protein kinase activities were detected in the cytosolic fraction (PC‐TPK) and the particulate fraction (PM‐TPK) in human platelets using the synthetic peptide, E11G1, (Glu‐Asp‐Ala‐Glu‐Tyr‐Ala‐Ala‐Arg‐Arg‐Arg‐Gly) as a substrate. PC‐TPK and PM‐TPK were different in substrate specificities, divalent cation requirements and apparent M
r values. These results strongly suggest that in platelets there exist at least two separate tyrosine protein kinases; one is present in cytosol and the other might be associated with membranes.
Identification and quantitation of O‐phosphoserine in human plasma fibronectin
Authors: Etheredge R.E. , Han S. , Fossel E. , Tanzer M.L. , Glimcher M.J.
Published: July 2002

O‐Phosphoserine was positively identified as the phosphorylated moiety in human plasma fibronectin by 31P‐NMR of intact peptides. These data correlated completely with chemical analyses which demonstrated the presence of O‐phosphoserine at a concentration of 2 residues/molecule. Neither O‐phosphothreonine nor O‐phosphotyrosine was detected in partial acid and partial alkaline hydrolysates, respectively.
Photoaffmity cross‐linking of F1ATPase from the thermophilic bacterium PS 3 by 3'‐arylazido‐β‐alanyl‐8‐azido ATP
Authors: Schäfer Hans-Jochen , Rathgeber Gabriele , Dose Klaus , Masafumi Y. , Kagawa Y.
Published: July 2002

To study the localization of the nucleotide binding sites of coupling factor 1 (TF1) from the thermophilic bacterium PS3 we used the bifunctional (cross‐linking) 3'‐arylazido‐β‐alanyl‐8‐azido ATP (DiN3ATP) for photoaffinity labeling. DiN3ATP is hydrolyzed by TF1 in the absence of ultraviolet light. Irradiation (UV light) of TF1 in the presence of DiN3ATP results in a nucleotide‐specific reduction of ATPase activity and in a nucleotide‐specific formation of different cross‐linked proteins (dimers, trimers, oligomers) formed by the major subunits α and/or β. The results suggest that nucleotide binding sites (one, two, possibly all) are located at the interfaces between these subunits.
Investigation of phospholipase‐lipid interactions by optical detection of triplet state magnetic resonance spectroscopy
Authors: Mao Su-Yau , Maki August H. , de Haas Gerard H.
Published: July 2002

We have investigated the binding of porcine pancreatic phospholipase A2 (PA2) to n‐hexadecylphosphocholine (C16PN) micelles using optical detection of triplet state magnetic resonance (ODMR) spectroscopy. The zero field splittings (zfs) of the single Trp3 residue undergo significant changes upon binding of PA2 to C16PN micelles. Zfs titrations of PA2 vs C16PN indicate that the binding stoichiometry is C16PN:PA2 ~ 25. A reduction of the ¦E¦parameter from 1.227 to 1.135 GHz is postulated to result from Stark effects caused by the binding of a polar group (possibly phosphocholine) near Trp3 in the PA2‐C16PN micelle complex.
Affinity labelling of the GM2‐activator protein
Authors: Neuenhofer Stephan , Sandhoff Konrad
Published: July 2002

In contrast to ganglioside GM2 (GM2), its fatty acid free derivative, lyso‐GM2, and N‐acetyllyso‐GM2 are hydrolyzed by purified human hexosaminidase A (Hex A). The addition of GM2‐activator stimulates the Hex A‐catalyzed hydrolysis of GM2 and that of N‐acetyllyso‐GM2 much more than that of lyso‐GM2. Based on this observation N‐brornoacetyllyso‐GM2 was synthesized as a potential affinity ligand of the GM2‐activator protein. N‐Bromo[3H]acetyllyso‐GM2 inactivated the GM2‐activator selectively and irreversibly by covalent modification.
Sequence similarity between epidermal growth factor precursor and atrial natriuretic factor precursor
Authors: Hayashida Hidenori , Miyata Takashi
Published: July 2002

Computer‐assisted analysis for homology of the EOF precursor revealed the presence of two large duplication units, each comprising 5 non‐EGF‐like homologous segments each of about 40 residues length and 3 or 4 EGF‐like segments. The amino acid sequences of the non‐EGF‐like repeats were subjected to search for homology with 2600 known protein sequences compiled in our database. An unexpected but statistically significant homology has been found, when compared with the atrial natriuretic factor precursor. The functional and evolutionary implications of the homology observed between the two different precursors are also discussed.
Biosynthesis of androgens and pheromonal steroids in neonatal porcine testicular preparations
Authors: Kwan T.K. , Orengo C. , Gower D.B.
Published: July 2002

The biosynthesis of testosterone and 4‐androstene‐3,17‐dione and some 16‐androstenes has been studied in homogenates or subcellular fractions of testes from 3‐week‐old Landrace piglets. Pregnenolone was converted into 5,16‐androstadien‐3β‐ol, 4,16‐androstadien‐3‐one, 5α‐androst‐16‐en‐3‐one and 5α‐androst‐16‐en‐3α‐ and 3β‐ols, but the quantities were some 50 times less than those formed in the mature boar testis. Androgens were also formed in the microsomal fractions but the quantities of 4‐androstene‐3,17‐dione (from side‐chain cleavage of 17‐hydroxyprogesterone) and of testosterone (from reduction of 4‐andro stene‐3,17‐dione) were 50–70 times lower than in the adult animal. The kinetic parameters and cofactor preference of the 3α‐ and 3β‐hydroxysteroid dehydrogenases were determined in the cytosolic, microsomal and mitochondrial fractions of neonatal porcine testes.
Regulation of DNA ligation by the recB protein in Escherichia coli cells
Authors: Syväoja Juhani , Pyhtilä Mauno J.
Published: July 2002

The covalent closing of hydrogen‐bonded λDNA circles in unpurified or slightly purified extracts of Escherichia coli, which was previously shown to require ATP and recB gene products, was found not to require recC gene product. The closing activity in extracts of the λ lysogen that overproduces DNA ligase about 500‐fold over the normal strain was still strictly dependent on ATP, but not on NAD. This suggests that the substrate was not freely available for the action of DNA ligase in recB− extract. ATP could be replaced in the system by a mixture of low concentrations of the 4 dNTPs, but by none of them alone.
Alkaline phosphatase of the calf intestine hydrolyzes phospholipids
Authors: Ehle Heidrun , Müller Elke , Horn Anton
Published: July 2002

Pure alkaline phosphatase of the calf intestine is able to hydrolyze phosphatidylinositol 4,5‐diphosphate (TPI) to phosphatidylinositol and Pi and to dephosphorylate phosphatidic acid. This phosphomonoesterase activity shows a considerably high specifie activity when an incubation medium at neutral pH containing 3 mM deoxycholate is used. The activity is inhibited by low concentrations of Ca2+. The enzyme has no detectable phosphodiesterase activity under the conditions tested.
Comparison of genomes of closely related phages ø29, ø15 and PZA using a rapid method of parallel physical mapping
Authors: Hostomský Zdeněk , Pačes Václav , Zadražil Stanislav
Published: July 2002

The DNAs of phages ø29, ø15 and PZA of Bacillus subtilis were analysed with restriction enzymes EcoRI, HpaI and HindIII. A method was used which permits parallel physical mapping of all three phages, from both ends of their linear genomes. The method is based on transfer of partially digested DNA to DBM paper and sequential hybridization with labelled terminal fragments. It follows from the comparison of the physical maps that phages ø29, ø15 and PZA are closely related and that they probably have arisen from a common ancestor by accumulation of point mutations.
Clostridium pasteurianum glutamine synthetase mechanism
Authors: Krishnan Indira S. , Dua Ramji D.
Published: July 2002

Preliminary chemical modification studies indicated the presence of tyrosine, carboxyl, arginine, histidine and the absence of serine and sulfhydryl residues at or near the active site of Clostridium pasteurianum glutamine synthetase. The conditions for tyrosine modification with tetranitromethane were optimized. The inactivation kinetics follow pseudo‐first‐order kinetics with respect to enzyme and second order with respect to modifier per active site. There was no inactivation at pH 6.5 suggesting the absence of thiol oxidation. The synthetase and transferase reactions followed the same pattern of inactivation on enzyme modification and both were equally protected by glutamate plus ATP. Thus tyrosine residues are present at the active site of the enzyme and are essential for both tranferase and synthetase activities.
Adenosine 5'‐triphosphate stimulates the release of polypeptides from mitochondria
Authors: Felipo Vicente , Grisolía Santiago
Published: July 2002

Adenosine 5'‐triphosphate
Mitochondria
Protein turnover
Bundling of actin filaments by aorta caldesmon is not related to its regulatory function
Authors: Moody Catherine J. , Marston Steven B. , Smith Christopher W.J.
Published: July 2002

Ca2+‐sensitive thin filaments from vascular smooth muscle were disassembled into their constituent proteins, actin, tropomyosin and caldesmon. Caldesmon bound to both actin and to actin‐tropomyosin and inhibited actin‐tropomyosin activation of skeletal muscle myosin MgATPase. It also promoted the aggregation of actin or actin‐tropomyosin into parallel aligned bundles. Quantitative electron microscopy measurements showed that with 1.1 μM actin‐tropomyosin, 1.6 ± 0.5% (n = 3) of the filaments were in bundles. At 0.073 μM, caldesmon inhibited MgATPase activity by 50%, whereas bundling was 3.0 ± 1.3% (n = 4). At 0.37 μM caldesmon, MgATPase inhibition was 83% while 28.1 ± 6.9% (n = 4) of filaments were in bundles. Experiments at 4.4 μM in which MgATPase and bundling were measured in the same samples gave similar results. Small bundles of 2–3 filaments showed the most frequent occurrence at 1.1 μM actin. At 4.4 μM actin the most common bundle size was 3‐5 filaments, with the occasional occurrence of large bundles consisting of up to 120 filaments. The incidence of bundling was the same in the presence and absence of tropomyosin. Thus caldesmon can induce the formation of actin bundles but this property bears no relationship to its inhibition of MgATPase activity.
A new approach to the crystallization of proteins
Authors: Gulewicz K. , Adamiak D. , Sprinzl M.
Published: July 2002

A new method for the crystallization of proteins was developed. Saturated solutions of a protein for crystallization are prepared by chromatography of adsorbed proteins from an insoluble polysaccharide matrix using gradients with decreasing ionic strength, decreasing concentration of organic solvents or an appropriate shift in pH. Insulin, a‐chymotrypsin and lysozyme for which the X‐ray crystal structures are known, were crystallized by this method. X‐ray diffraction photographs were taken to demonstrate the quality of the crystals. In addition to this analytical application, the method may be useful for the crystallization and the simultaneous purification of larger amounts of proteins.
The generation of active fragments of complement receptor type 2 by trypsin digestion
Authors: Micklem Kingsley , Sim Edith , Sim Robert B.
Published: July 2002

B lymphocytes and Raji cells express the complement receptor type 2 (CR2) of 145 kDa which recognises the C3d fragment of C3. When intact cells are treated with trypsin, CR2 is degraded. There is a parallel loss in C3d‐mediated rosetting and in proteins which bind to C3d‐Sepharose. Initially 97 and then 83 kDa fragments of CR2 are produced which retain C3d binding activity. These fragments are associated with the cell surface and mediate rosetting. Purified 125I‐labelled CR2, solubilised in detergent, produces fragments of apparently identical size on treatment with trypsin. The 83 kDa fragment produced by trypsin treatment closely resembles the major C3d binding protein spontaneously released into Raji cell culture medium.
Redox‐dependent activation of 5'‐nucleotidase in rat liver plasma membranes
Authors: Tarakhovsky Alexander M. , Umansky Victor J. , Shlyakhovenko Vladimir A. , Balitsky Konstantin P.
Published: July 2002

Addition of NADH, but not NAD+ or NADPH, to rat liver plasma membranes resulted in the increase of their 5'‐nucleotidase activity. NADH‐dependent activation of 5'‐nucleotidase was significantly suppressed by atebrine, an inhibitor Of NADH dehydrogenase of plasma membranes, and completely abolished by 2,4‐dinitrophenol (2 × 10−4 M) and Triton X‐100 (2%). Inhibitors of electron transfer in the mitochondrial respiratory chain, rotenone and potassium cyanide, failed to affect 5'‐nucleotidase activity in both the presence and absence of NADH. The data obtained give reasons to suggest a redox‐dependent mechanism of 5'‐nucleotidase activation in rat liver plasma membranes.
Vasoactive intestinal peptide receptor activity in human fetal enterocytes
Authors: Gespach Christian , Chastre Eric , Emami Shahin , Mulliez Nicole
Published: July 2002

Functional and specific receptors for vasoactive intestinal peptide (VIP) (determined by their capacity to bind 125I‐VIP and activate adenylate cyclase) and cyclic AMP‐dependent phosphodiesterase activities were characterized in enterocytes of human fetal small intestine between 18 and 23 weeks of gestation. Half‐maximal stimulation of the cyclase and inhibition of 125I‐VIP binding in membrane preparations were respectively observed at 1.4 and 5 × 10−10 M VIP. The peptides structurally related to VIP activated the cyclic AMP generating system at pharmacological doses (10−7M and above) in the following order of potency: VIP > PHI > GRF > secretin. Other peptides or test substances, including GIP, pancreatic glucagon, somatostatin‐14, gastrin, CCK, neurotensin, pancreatic polypeptide, PYY, substance P, histamine and isoproterenol are inactive in this system, while the ubiquitous adenylate cyclase activators NaF, forskolin and prostaglandins were effective. These results, combined with the appearance of intestinal VIP in nerve fibers at 8 weeks and with the morphological and enzymatic maturation at 9–12 weeks of the intestinal mucosa, indicate that this neuropeptide may regulate either the differentiation or function of enterocytes during the early development of human intestinal mucosa.
Relationship of phosphorylation to the oligomerization of SV40 T antigen and its association with p53
Authors: Stürzbecher Horst-Werner , Mörike Martin , Montenarh Mathias , Henning Roland
Published: July 2002

The potential significance of the phosphorylation of SV40 large T antigen for oligomers and complexes with the cellular protein p53 was investigated. We observed that T antigen oligomers remain stable after enzymatic dephosphorylation by alkaline phosphatase up to 80%. Separate analysis of free and p53‐bound T antigen revealed a considerably lower phosphorylation of the p53‐bound subclass. Therefore, a simple correlation between the overall phosphorylation of T antigen and the formation of oligomers and T‐p53 complexes is highly unlikely.
Antiviral activity of phorbol myristate acetate and possible relationships with interferon action
Authors: Premecz György , Markovits Andrea , Földes István
Published: July 2002

Signal‐induced turnover of membrane phospholipids represents a fundamental transducing mechanism that induces a signal cascade resulting in mobilization of calcium, activation of protein kinase C by diacylglycerol, release of arachidonic acid and stimulation of cyclic GMP production. In this pathway tumor‐promoting phorbol esters such as phorbol myristate acetate (PMA) may substitute for diacylglycerol. The interferonlike antiviral effect of PMA described here suggests that the inositol phospholipid‐diacylglycerol‐protein kinase C signal‐transducing mechanism may be involved in interferon action.
Involvement of plastoquinone and lipids in electron transport reactions mediated by the cytochrome b6‐f complex isolated from spinach
Authors: Chain Richard K.
Published: July 2002

The isolation of a cytochrome b
6‐f complex from spinach, which is depleted of plastoquinone (and lipid), is reported. The depleted complex no longer functions as a plastoquinol‐plastocyanin oxidoreductase but can be reconstituted with plastoquinone and exogenous lipids. The lipid classes digalactosyldiacylglycerol, phosphatidylglycerol and phosphatidylcholine were active in reconstitution while monogalactosyldiacylglycerol and sulfoquinovosyldiacylglycerol were not. Neither plastoquinone nor lipid alone fully reconstitutes electron transport in the depleted complex. Saturation of plastoquinol‐plastocyanin oxidoreductase activity in the depleted complex occurs at 1 plastoquinone per cytochrome f.
Sequence analysis of peptide fragments from the intrinsic membrane protein of calf lens fibers MP26 and its natural maturation product MP22
Authors: Ngoc Lien Do , Paroutaud Pierre , Dunia Irene , Benedetti E.Lucio , Hoebeke Johan
Published: July 2002

Calf lens fiber plasma membranes, containing only the intrinsic membrane protein MP26 and its maturation product MP22 were treated with proteolytic enzymes such as trypsin, protease V8 from S. aureus or with chemical agents as CNBr in formic acid. The cleavage products, purified by electrophoresis, were analysed for their amino acid composition and N‐terminal sequences. Proteolysis gave rise to peptides which were mainly shortened at the C‐terminal end of the molecules. While the V8 protease produced a fragment with a similar N‐terminal sequence as the maturation product MP22, trypsin yielded another cleavage product. Chemical hydrolysis yielded large fragments (11–15 kDa) with hydrophobic N‐terminal sequences. Our results suggest that MP26 is characterised by an N‐terminal signal sequence and possesses other hydrophobic domains which could function as untranslocated insertion sequences.
Rat plasma α1‐inhibitor3: a member of the α‐macroglobulin family
Authors: Esnard F. , Gutman N. , El Moujahed A. , Gauthier F.
Published: July 2002

α1 Inhibitor3

α.‐Macroglobulin
Rat plasma
Thiol ester
Heat fragmentation
Early transmembrane events in tumour cell responses observed by stopped‐flow fluorometry
Authors: Utsunomiya Naoko , Tsuboi Masamichi , Nakanishi Mamoru
Published: July 2002

Early transmembrane events of tumour cells (mouse myeloma X5563 and lymphoma RDM4) after binding of a monoclonal antibody against mouse MHC antigen and a mitogenic lectin, Con A, were examined by stopped‐flow fluorometry with 3 different fluorescent probes. The results showed that membrane fluidities of the cells increased first after binding of anti H‐2Kk monoclonal antibody (11‐4.1), then calcium was released from intracellular stores into the cytoplasma, and lastly calcium influx occurred from the external medium into the cytoplasma. While Con A only induced calcium influx from the external medium into the cytoplasma.
The oxygen reaction of the cytochrome d‐terminated respiratory chain of Escherichia coli at sub‐zero temperatures
Authors: Kumar C. , Pooler R.K. , Salmon I. , Chance B.
Published: July 2002

Cytochrome oxidase Escherichia coli Cytochrome b‐d complex
Bacterial electron transport
Low‐temperature technique
Equilibrium between active and inactive forms of rat liver ornithine decarboxylase mediated by L‐ornithine and salts
Authors: Solano F. , Peñafiel R. , Solano M.E. , Lozano J.A.
Published: July 2002

Ornithine decarboxylase
Polyamine
Association equilibrium
Rat liver
Ionic strength
Different kinetic patterns in the α‐chymotrypsin‐catalysed hydrolysis of synthetic ester substrates
Authors: Garrell Joan , M. Cuchillo Claudi
Published: July 2002

Negative cooperativity
Nonhyperbolic kinetics
α‐Chymotrypsin
Hyperbolic kinetics
Hill coefficient
Slow transition
A novel member of the serpin superfamily is encoded on a circular plasmid‐like DNA species isolated from rabbit cells
Authors: Upton C. , Carrell R.W. , McFadden G.
Published: July 2002

A novel member of the serpin family of serine protease inhibitors is presented. A plasmid‐like DNA was isolated from rabbit cells by its homology to the genome of Shope fibroma virus (SFV), a tumorigenic poxvirus of rabbits, and was shown elsewhere to encode a serpin‐like protein [(1986) Mol. Cell. Biol. 6, 265‐276]. Although significant DNA homology exists between the rabbit plasmid serpin open reading frame and the SFV terminal inverted repeat DNA there is no intact serpin counterpart encoded by this region of the SFV genome. The alignment of the novel plasmid‐borne polypeptide with the serpin family of proteins confirms its status within this group.
Kinetics of 125I‐ubiquitin conjugation with and liberation from rabbit reticulocyte stroma
Authors: Dubiel W. , Müller M. , Rapoport S.
Published: July 2002

The breakdown of mitochondria‐containing stroma of rabbit reticulocytes is an ATP‐ and ubiquitin‐dependent process and there is no evidence for an ATP‐dependent but ubiquitin‐independent proteolysis in these cells. The ubiquitin conjugate formation with heat‐denatured stroma proteins is about one‐fifth of that with native stroma. In reticulocytes there exist two mechanisms of ubiquitin liberation from its conjugates with stroma proteins: (i) an ATP‐dependent and hemin‐resistant release of ubiquitin, which is assumed to be the first step in the degradation of ubiquitin conjugates by the protease system, and (ii) a release of ubiquitin catalyzed by an isopeptidase activity.
Microtubule formation and the initial association of tubulin dimers
Authors: Foster Kay E. , Rosemeyer Michael A.
Published: July 2002

Microtubule protein could be prepared in high yield, and could form copious microtubules, in solutions containing glutamate but not in solutions containing only phosphate ions. Correspondingly, tubulin after isolation showed an association equilibrium in the presence of glutamate (or other zwitterions), but not in phosphate buffers. The correlation suggests that this association to tetramers is probably the initial step in the mechanism of microtubule formation.
Internally transposed signal sequence of carp preproinsulin retains its functions with the signal recognition particle
Authors: Wiedmann Martin , Huth Antje , Rapoport Tom A.
Published: July 2002

It is shown that the signal sequence of carp preproinsulin is functional with the dog pancreatic signal recognition particle (SRP) both when present at its normal location at the amino‐terminus of the protein or when engineered to an internal location. Inhibition of translation by SRP in the absence of microsomal membranes, reconstitution by SRP of the translocation competence of high‐salt inactivated microsomes and signal peptide cleavage all occur with the signal sequence being preceded by a highly charged peptide segment of 39 amino acid residues (the distance from the amino‐terminus to the cleavage site of the signal peptidase is increased to 56 residues).
Evidence for a mobile semiquinone in the redox cycle of the mammalian cytochrome bc1 complex
Authors: Rich Peter R. , Wikström Mårten
Published: July 2002

Experimental evidence is presented to demonstrate that cytochromes b of the mammalian cytochrome bc
1 complex may be rapidly oxidised by a pulse of oxidising equivalents which react with cytochrome c
1 even when all cytochrome b is fully reduced before the pulse. The oxidation is sensitive both to antimycin and to myxothiazol. Such behaviour is inconsistent with models in which only the fully oxidised ubiquinone may move beween the centres ‘o’ and ‘i’ of the complex. It is proposed that the charged semiquinone (Q−) may move between these centres, which may constitute separate reaction domains of a single ubiquinone‐binding site. The bearing of this on the mechanism of electron, proton and charge transfer in the complex is discussed.
ANF stimulation of detergent‐dispersed particulate guanylate cyclase from bovine adrenal cortex
Authors: Tremblay Johanne , Gerzer Rupert , Pang Stephen C. , Cantin Marc , Genest Jacques , Hamet Pavel
Published: July 2002

Particulate guanylate cyclase from bovine adrenal cortex can be stimulated by ANF. A 2‐fold stimulation of the enzyme was obtained with 100 nM ANF and a half‐maximal stimulation, with a 5 nM dose. The stimulation by ANF persisted for at least 30 min. Various detergents, such as Triton X‐100, Lubrol PX, cholate, CHAPS, digitonin and zwittergent, stimulated several‐fold the activity of particulate guanylate cyclase. However, only Triton X‐100 dispersed particulate guanylate cyclase without affecting its response to ANF. The dose‐response curve of ANF stimulation of the particulate and the Triton X‐100 dispersed enzyme was similar. The dispersion of a fully responsive guanylate cyclase to ANF will help us to uncover the type of interactions between guanylate cyclase and ANF. It will also be used as a first step for the purification of an ANF‐sensitive particulate guanylate cyclase.
Variants of wheat histone H1 with N‐ and C‐terminal extensions
Authors: Brandt Wolf F. , von Holt Claus
Published: July 2002

Two histone H1 variants have been isolated from wheat germ with M
r in the region 23 000–27 000. Partial sequences from their globular regions are reported.
Nonenzymatic radiolabeling of protein by 32P‐containing nucleotides
Authors: Schmidt Martin C. , Hanna Michelle M.
Published: July 2002

We report a nonenzymatic reaction which results in the radiolabeling of proteins by 32P‐containing nucleoside triphosphates. The labeling reaction does not require any cofactors, but is greatly enhanced by the presence of alcohols. Even under optimal conditions, less than 1% of the protein molecules undergo modification. This nonspecific labeling represents a serious artifact which may become significant in systems involving low levels of specific labeling, such as photoaffinity labeling. Since the reaction is not limited to specific proteins, this may, however, provide a simple and rapid procedure for the preparation of labeled proteins.
The binding of the B‐chain of ricin to Burkitt lymphoma cells
Authors: Manevich Efim M. , Tonevitsky Alexander G. , Bergelson Lev D.
Published: July 2002

It is shown that conformational changes of receptor proteins brought about by binding of a ligand induce changes in the lipid environment of the receptor that can be monitored by fluorescent lipid probes. On this basis a new approach to studies of ligand‐receptor binding is proposed. Using the interaction of the ricin B‐chain with Burkitt lymphoma cells as an example and fluorescent labelled sphingomyelin as a probe, the ligand‐induced changes of fluorescence anisotropy were shown to be concentration‐dependent and to permit determination of the binding constant and the number of receptor‐binding sites. The method was found to be specific and highly sensitive, allowing detection of the action of one RB molecule per cell. Scatchard analysis of the binding of 125I‐RB demonstrated the presence on the cell surface of two binding sites with K
d ~ 10−10 and ~ 10−8 M, respectively. Only the high‐affinity sites were detected by the fluorescence technique. Saturation of these sites resulted in maximum inhibition of protein synthesis.
Bleomycin‐detectable iron in plasma from Plasmodium vinckei vinckei‐infecied mice
Authors: Buffinton G.D. , Cowden W.B. , Hunt N.H. , Clark I.A.
Published: July 2002

(1) Plasma from mice heavily parasitized by Plasmodium vinckei vinckei was found to contain micromolar levels of iron as detected by the ‘bleomycin assay’ (slightly modified) of Gutteridge et al. [(1981) Biochem. J. 199, 263‐265]. Uninfected mouse plasma contained little or no bleomycin‐detectable iron. (2) Plasma ultrafiltrate from infected mice contained no bleomycin‐detectable iron, indicating that such iron was associated with the protein/macromolecule fraction. (3) We speculate that this iron could catalyse reduction of peroxides in vivo and thus play a role in malaria pathology.
GACcodonGGC: a very favorable context for translation errors?
Authors: Laughrea Michael
Published: July 2002

A survey of the literature on the effect of codon context on translation fidelity suggests that contexts such as GACcodonGGC are strong, but not unique, promotors of several types of translation errors.
Genomic organization of rDNA in Pseudomonas aeruginosa
Authors: Hartmann Roland K. , Toschka Holger Y. , Ulbrich Norbert , Erdmann Volker A.
Published: July 2002

We have examined the number and organization of rRNA genes in Pseudomonas aeruginosa by hybridization of restriction nuclease digests of genomic DNA to 3'‐32P‐labelled 23 S, 16 S and 5 S rRNAs and corresponding labelled DNA from the rrnB operon of Escherichia coli. The immediate conclusion from these hybridization data is that there are 4 transcriptional units coding for rDNA in P. aeruginosa. We report here a putative model of the genomic organization of all 4 rDNA operons.
Direct evidence for a coupling between synthesis and export of PhoS in E. coli
Authors: Anba Jamila , Lazdunski Claude , Pages Jean-Marie
Published: July 2002

The accumulation of pre‐PhoS under conditions of PhoS overproduction has been previously described. It is now demonstrated that during the induction of PhoS, a delay in the completion of polypeptide chain elongation can be detected. This delay is related to the extent of jamming of export sites by pre‐PhoS or by other exported proteins. These results suggest that a component required for completion of pre‐PhoS polypeptide becomes limiting, being titrated by the excess of nascent chains bearing signal peptides. This component thus probably acts at an early step in the export pathway.
Express analysis of protein amino acid sequences
Authors: Shlyapnikov S.V. , Bezborodova S.I. , Kulikov V.A. , Yakovlev G.I.
Published: July 2002

The complete amino acid sequence of Penicillium chrysogenum 152A guanyl‐specific RNase has been established using automated Edman degradation of two non‐fractionated peptide mixtures produced by tryptic and staphylococcal protease digests of the protein. The RNase contains 102 amino acid residues: His2, Arg3, Asp7, Asn8, Thr5, Ser11, Glu4, Gln2, Pro4, Gly11, Ala13, Cys4, Val8, Ile3, Leu3, Tyr9, Phe5 (M
r 10 747).
Interaction amongst calcineurin subunits
Authors: Gupta Ramesh C. , Khandelwal Ramji L. , Sulakhe Prakash V.
Published: July 2002

Calcineurin was dissociated into subunits A and B by 6 M urea in the presence (method A) and absence (method B) of MnCI2 and dissociated subunits were isolated by gel filtration in urea in the absence (method B) or presence (method A) of MnCI2. Phosphatase activity was associated with the A subunit isolated by either method. The phosphatase activity () of subunit A isolated by method A was greater (2‐5‐fold) than by method B. Mn2+ increased subunit A phosphatase and calmodulin further increased the enzyme activity. Subunit B isolated by method A or B increased Mn2+ + calmodulin stimulated subunit A phosphatase prepared by method B but interestingly and unexpectedly inhibited such stimulated activity of the subunit A prepared by method A. These results imply the tightly bound cation (in our case, most likely Mn2+) with subunit A dramatically and differentially influences the effects of two Ca2+‐binding proteins, calmodulin and subunit B, on the subunit A phosphatase.
Evidence for a diprotomeric structure of Na,K‐ATPase
Authors: Chetverin Alexander B.
Published: July 2002

Three methods were used to assess protein concentration in membrane‐bound Na,K‐ATPase preparations: standard Lowry assay, Kjeldahl nitrogen determination and amino acid analysis. While the first two methods showed excellent agreement, the third one always gave a lower value which varied drastically depending on the condition of sample treatment before amino acid analysis. This result reinforces the Lowry method in assessing the true concentration of Na,K‐ATPase protein and suggests 250 kDa to be a true estimate of the molecular mass of the smallest ligand‐binding unit of the enzyme. The cyanate method reveals two NH2‐terminal residues of the β‐subunit (NH2‐Ala) and one such residue of the α‐subunit (NH2‐Gly) per ligand‐binding unit. From the data on equimolarity of the α‐ and β‐subunits in Na,K‐ATPase this suggests that the enzyme molecule is composed of two αβ‐protomers, one possessing a modified (presumably an N‐blocked) α‐subunit.
Phytochrome‐mediated accumulation of chloroplast DNA in pea leaves
Authors: Sasaki Yukiko , Nakamura Yosuke , Matsuno Ryuichi
Published: July 2002

Pea seedlings grown for 5 days in the dark were treated with red light for 5 min and grown for 2 more days in the dark. Effects of the red light on chloroplast DNA levels in the pea leaves were examined using probe DNA of the chloroplast‐coded large subunit and nuclear‐coded small subunit of ribulosebisphosphate carboxylase/oxygenase. The gene dosage of the large subunit, but not of the small subunit, was increased by red light. The increase was inhibited by subsequent far‐red light treatment. These results indicate that accumulation of chloroplast DNA in the cell is mediated by phytochrome. Probably the replication of chloroplast DNA is mediated by phytochrome.
Plasminogen‐binding site of the thermostable region of fibrinogen fragment D
Authors: Lezhen T.I. , Kudinov S.A. , Medved' L.V.
Published: July 2002

Affinity chromatography of plasminogen and its proteolytic fragments on immobilized fibrinogen TSD fragment has shown that the latter contains a plasminogen‐binding site which is complementary to the lysine‐binding site(s) of plasminogen molecule 1–3 kringle structures.
Antiviral activity of a chemically stabilized 2‐5A analog upon microinjection into HeLa cells
Authors: Defilippi Paola , Huez Georges , Verhaegen-Lewalle Martine , De Clercq Erik , Imai Jiro , Torrence Paul , Content Jean
Published: July 2002

2‐5A[ppp(A2'p)
n
5'A] has been implicated as a mediator in the antiviral action of interferon. Its direct evaluation as an indicator of virus replication is hampered by two limitations: its inability to penetrate intact cells, and its rapid intracellular degradation by (2'–5')phosphodiesterase. These problems could be overcome by using a microinjection technique whereby a phosphodiesterase‐resistant analog of 2‐A, in which the 2'‐terminals adenosine residue is replaced by 2‐(9‐adenyl)‐6‐hydroxy‐methyl‐4‐hexylmorpholine, was injected into individual HeLa cells before infection with mengovirus or vesicular stomatitis virus (VSV). This comparative assay with two representatives of different virus classes in a single experimental system pointed to the high sensitivity of VSV to inhibition by 2‐5A oligonucleotides, in contrast with the low sensitivity of mengovirus. Microinjection of the hexylmorpholine 2‐5A analog led to a much greater reduction in mengovirus yield than did microinjection of 2‐5A itself.
Effects of gibberellic acid and abscisic acid on levels of translatable mRNA (1→3,1→4)‐β‐D‐glucanase in barley aleurone
Authors: Mundy John , Fincher Geoffrey B.
Published: July 2002

Polyadenylated mRNA has been isolated from barley aleurone layers incubated in the presence and absence of exogenous gibberellic acid and abscisic acid. Immunoprecipitation of in vitro translation products with specific antibodies shows that low levels of translatable mRNA encoding (1 →3,1 →4)‐β‐D‐glucanase 4‐glucanohydrolase (EC 3.2.1.73) can be detected in the absence of the phytohormones. Gibberellic acid causes a 10‐fold increase in levels of translatable mRNA for the enzyme, while abscisic acid treatment suppresses the relative abundance of translatable (l →3,1 →4)‐β‐D‐glucanase mRNA.
ADP‐ribosylation of nuclear proteins is increased by phenobarbital
Authors: Bràz Judite , Celeste Lechner Maria
Published: July 2002

Changes in the ADP‐ribosylation of total proteins and purified histones of rat liver nuclei after phenobarbital treatment (80 , 24 h) have been studied. The [32P]NAD incorporation into total trichloroacetic acid precipitated proteins, in histone H1 and in core histones was evaluated, the specific radioactivities increasing 150, 40 and 8%, respectively. Histones H1 and H2B were the best ADP‐ribose acceptors. Histone H4 did not show any 32P incorporation, as revealed by autoradiography after SDS‐PAGE of the purified histones, in either the control or phenobarbital treated rats. Possible involvement of ADP‐ribosylation of nuclear proteins in the adaptative response of liver to phenobarbital is discussed.
Transition kinetics of the conversion of blue to purple bacteriorhodopsin upon magnesium binding
Authors: Zubov Boris , Tsuji Kinko , Hess Benno
Published: July 2002

Magnesium binding to cation#x2010;depleted blue bacteriorhodopsin (b‐bR) was studied spectrophotometrically as well as by following stopped‐flow kinetics. There exist three kinetically different steps in the binding process, yielding purple bacteriorhodopsin (p‐bR). Since only the firtst step is dependent on the concentration of the reactants, the reaction scheme can be proposed as the simplest model, with MgbR being the first intermediate and ΣI denoting a set of successive intermediates. According to this model k
1, k
−1 and k
2 are calculated to be 2.8 × 104 M−1 · s−1, 5.0 × 10 s−1 and 1 × 10−2 s−1, respectively.
Configuration of the active Mg‐ATP complex in protein kinase C reaction
Authors: Kondo Hiroki , Kinoshita Junko , Matsuba Takao , Sunamoto Junzo
Published: July 2002

To probe the active site structure of protein kinase C stereochemical studies were carried out by using ATPβs. The enzyme utilizes either one of the diastereomers (Sp
and Rp
) of ATPβs almost equally well as a substrate. This result contrasts with that for cyclic AMP‐dependent protein kinase, suggesting that the topography of the nucleotide‐binding site is significantly different between the two kinases.
Prokaryotic initiation factor 2 acts at the level of the 30 S ribosomal subunit
Authors: Gualerzi Claudio O. , Wintermeyer Wolfgang
Published: July 2002

The mechanism of action of initiation factor 2 (IF2) from Escherichia coli during initiation of translation was investigated by kinetic analysis of the binding of N‐AcPhe‐tRNAphe or Phe‐tRNAphe to poly(U)‐programmed 30 S ribosomal subunits. The reaction was studied by using the stopped‐flow technique, monitoring the fluorescence signal of a proflavine inserted next to the anticodon (position 37) of yeast tRNAphe. Both the rate and extent of N‐AcPhe‐tRNAphe binding to 30 S subunits are strongly enhanced by IF2. The effect of IF2 was studied at different stoichiometric ratios between factor, ribosomes, and N‐AcPhe‐tRNAphe, in both the presence and absence of the other two factors. In all cases, the IF2 effect titrates with the 30 S ribosomes. This is also the case in the presence of an equimolar amount of 50 S ribosomal subunits. Furthermore, IF2 was found to stimulate strongly the binding of Phe‐tRNAphe, in spite of the inability of the latter to form a detectable binary complex with IF2. The results are interpreted to mean that IF2 acts in a stoichiometric rather than a catalytic fashion, at the level of the 30 S ribosomal subunit. They do not support a model in which IF2 acts as a carrier for the initiator tRNA.
Phosphorylation of phosphatidylinositol by transverse tubule vesicles and its possible role in excitation‐contraction coupling
Authors: Hidalgo Cecilia , Carrasco M.Angélica , Magendzo Karin , Jaimovich Enrique
Published: July 2002

Phosphorylation of phosphatidylinositol to phosphatidylinositol 4‐monophosphate and to phosphatidyl‐inositol 4,5‐bisphosphate was demonstrated in transverse‐tubule membranes isolated from frog skeletal muscle using [γ‐32P]ATP as substrate. At millimolar concentrations of Mg2+ both phosphorylation reactions were completed within 15 s at 25°C. Isolated sarcoplasmic reticulum vesicles phosphorylated phosphatidyl‐inositol to phosphatidylinositol 4‐phosphate with a lower specific activity than the transverse tubules, and lacked the ability to produce phosphatidylinositol 4,5‐bisphosphate. These findings show, for the first time, that isolated transverse‐tubule membranes carry out one of the steps required to sustain a role for inositol trisphosphate as the physiological messenger in excitation‐contraction coupling in skeletal muscle. The finding that 0.5 mM tetracaine apparently inhibits the phosphorylation of phosphatidylinositol 4‐phosphate to phosphatidylinositol 4,5‐bisphosphate also supports a role for these intermediates in excitation‐contraction coupling.
Fractionation by high‐performance liquid chromatography and characterization of the highly related protamines from the sperm of the marine worm Platynereis dumerilii
Authors: Sellos Daniel
Published: July 2002

The protamines from the emitted sperm nuclei of the marine annelid Platynereis dumerilii have been purified from the whole basic protein by gel filtration and ion‐exchange chromatography and further fractionated by reverse‐phase high‐performance liquid chromatography. Four main fractions were obtained and characterized by their electrophoretic mobilities and amino acid compositions. All these protamines are characterized by high arginine and serine contents and by the presence of equivalent amounts of asparagine, threonine and valine. Each of these fractions is heterogeneous. After re‐fractionation the amino acid composition and the N‐terminal sequence of the samples indicate that each protein corresponds to a variant of the same family of highly related protamines.
L‐(+)‐Tartrate dehydratase from Pseudomonas putida is an iron‐sulphur enzyme
Authors: Kelly Janice M. , Scopes Robert K.
Published: July 2002

The enzyme L‐(+)‐tartrate dehydratase has been isolated from extracts of Pseudomonas putida by a one‐step procedure involving dye‐ligand chromatography. The enzyme loses activity rapidly in the absence of Fe2+; concentrated solutions have a brown colour typical of iron‐sulphur proteins. Analysis of iron and acid‐labile sulphide indicated 3–5 atoms of each per molecule of 100 kDa. The enzyme's structure consists of four subunits, two each of 23 and 27 kDa.
The effects of 1‐aminooxy‐3‐aminopropane and S‐(5'‐deoxy‐5'‐adenosyl)methylthioethylhydroxylamine on ornithine decarboxylase and S‐adenosyl‐L‐methionine decarboxylase from Escherichia coli
Authors: Paulin Lars
Published: July 2002

1‐Aminooxy‐3‐aminopropane (APA) was shown to be a potent competitive inhibitor (K
i = 1.0 nM) of partially purified Escherichia coli ornithine decarboxylase. APA did not inhibit S‐adenosyl‐L‐methionine decarboxylase and spermidine synthase from E. coli. S‐(5'‐Deoxy‐5'‐adenosyl)methylthioethylhydroxylamine (AMA), which is a structural analogue of decarboxylated S‐adenosyl‐L‐methionine, was for the first time shown to be an irreversible inhibitor of bacterial S‐adenosyl‐L‐methionine decarboxylase and a competitive inhibitor (K
i = 47 μM) of bacterial ornithine decarboxylase. AMA had no effect on spermidine synthase.
Complete cDNA sequence for rabbit muscle glycogen phosphorylase
Authors: Nakano Kenichi , Hwang Peter K. , Fletterick Robert J.
Published: July 2002

The cDNA for the nearly full‐length rabbit muscle glycogen phosphorylase mRNA has been isolated and sequenced. The cDNA is rich in G and C nucleotides. This feature is especially striking at the 3rd position of codons, where 86% of the 843 amino acid codons terminate with G or C. Methionine, presumably the initiation residue, is found at position—1, suggesting that the removal of only a single methionine residue precedes the amino‐terminal acetylation at serine. Eight differences between the deduced amino acid sequence and the previously determined protein sequence are discussed.
A simple procedure to determine Ca2+ in oxygen‐evolving preparations from Synechococcus sp
Authors: Kashino Yasuhiro , Satoh Kazuhiko , Katoh Sakae
Published: July 2002

A simple procedure to determine Ca2+ bound to low‐ and high‐affinity sites of Synechococcus oxygen‐evolving particles was developed. The method consists of determination of Ca2+ in the particle suspensions with and without treatment with a chelating resin, Chelex 100, to remove the metal cations contaminating the suspension medium as well as those weakly bound to the particles. It was found that the particles contain one tightly bound Ca2+ per PS II reaction center which cannot be extracted with Chelex 100 and a larger amount of weakly associated and resin‐extractable Ca2+.