bluuebunny/crossref_metadata_2025 huggingface dataset:
Dual effects of the ricin A chain on protein synthesis in rabbit reticulocyte lysate
Authors: OSBORN Rupert W. , HARTLEY Martin R.
Published: March 2005

Ricin A chain caused inhibition of protein synthesis by reticulocyte lysate with concomitant depurination of 28S rRNA. The partial reaction(s) of protein synthesis inhibited was investigated by following the appearance of [35S]methionine from initiator [35S]Met‐tRNA into 40S ribosomal subunits, 80S monosomes and polysomes. Ricin A chain caused an accumulation of [35S]Met in monosomes which did not enter polysomes. In these respects the effects of the ricin A chain resembled those of diphtheria toxin, an inhibitor of elongation‐factor‐2‐catalyzed translocation. This is consistent with the previously proposed site of action of ricin as an inhibitor of elongation. However, the inhibitory effects of the ricin A chain and diphtheria toxin are not equivalent because we observed that the rate of formation of the 80S initiation complex was reduced approximately sixfold with the ricin A chain relative to diphtheria toxin. Analysis of methionine‐containing peptides bound to 80S monosomes in ricin‐A‐chain‐inhibited and diphtheria‐toxin‐inhibited lysates, programmed with globin mRNA, revealed a predominance of Met‐Val, suggesting that the elongation cycle is inhibited at the translocation step. Translocation was also implicated as the step blocked in both the ricin‐A‐chain‐inhibited and diphtheria‐toxin‐inhibited lysates, by the finding that nascent peptide chains were unreactive towards puromycin. It is concluded that ricin‐A‐chain‐modified ribosomes are deficient in two protein synthesis partial reactions: the formation of the 80S initiation complex during initiation and the translocation step of the elongation cycle.
Binding of a photoaffinity analogue of pyridoxal to pyridoxal kinase
Authors: SCHOLZ Glen , KWOK Francis , CHURCHICH Jorge E.
Published: March 2005

The binding of pyridoxal analogues to the structural domains of pyridoxal kinase was studied by fluorescence spectroscopy and chromatographic techniques. Two fragments of 24 and 16 kDa, arising from limited proteolysis of the native enzyme, were separated by ion‐exchange chromatography and used for binding studies with pyridoxal oxime. Fluorometric titrations yielded dissociation constants of 6 and 12.4 μM for pyridoxal oxime bound to the native enzyme and 24‐kDa fragment, respectively. 4‐(4‐Azido‐2‐nitrophenyl)‐pyridoxamine, a new photolabeling reagent, binds irreversibly to the kinase with concomitant loss of catalytic activity. The modified kinase (2.1 mol label/mol dimer) yields two fragments upon limited proteolysis with chymotrypsin. The two fragments were separated by reverse‐phase HPLC and SDS/polyacrylamide gel electrophoresis. Radiolabeled ligand was detected only in the 24‐kDa fragment. It is postulated that the pyridoxal binding site is located in the 24‐kDa structural domain.
M‐phase‐specific cdc2 protein kinase phosphorylates the β subunit of casein kinase II and increases casein kinase II activity
Authors: MULNER‐LORILLON Odile , CORMIER Patrick , LABBÉ Jean‐Claude , DORÉE Marcel , POULHE Robert , OSBORNE Howard , BELLÉ Robert
Published: March 2005

The M‐phase‐specific cdc2 (cell division control) protein kinase (a component of the M‐phase‐promoting factor) was found to activate casein kinase II in vitro. The increase in casein kinase II activity ranged over 1.5–5‐fold. Increase in activity was prevented if ATP was replaced during the activation reaction by a non‐hydrolysable analogue. Alkaline phosphatase treatment of the activated enzyme decreased the activity to the basal level. The β subunit of casein kinase II was phosphorylated by cdc2 protein kinase at site(s) different from the autophosphorylation sites of the enzyme. Phosphoamino acid analysis showed that the β subunit was phosphorylated by cdc2 protein kinase at threonine residues while autophosphorylation involved serine residues. Casein kinase II may be part of the cascade which leads to increased phosphorylation of many proteins at M‐phase and therefore be involved in the pleiotropic effects of M‐phase‐promoting factor.
Vasopressin, insulin and peroxide(s) of vanadate (pervanadate) influence Na+ transport mediated by (Na+, K+)ATPase or Na+/H+ exchanger of rat liver plasma membrane vesicles
Authors: JAKUBOWSKI Josef , JAKOB Andrea
Published: March 2005

Uptake of 22Na+ by liver plasma membrane vesicles, reflecting Na+ transport by (Na+, K+)ATPase or Na+/ H+ exchange was studied. Membrane vesicles were isolated from rat liver homogenates or from freshly prepared rat hepatocytes incubated in the presence of [Arg8]vasopressin or pervanadate and insulin. The ATP dependence of (Na+, K+)ATPase‐mediated transport was determined from initial velocities of vanadate‐sensitive uptake of 22Na+, the Na+‐dependence of Na+/H+ exchange from initial velocities of amiloride‐sensitive uptake.

By studying vanadate‐sensitive Na+ transport, high‐affinity binding sites for ATP with an apparent Km(ATP) of 15 ± 1 μM were observed at low concentrations of Na+ (1 mM) and K+ (1 mM). At 90 mM Na+ and 60 mM K+ the apparent Km(ATP) was 103 ± 25 μM. Vesiculation of membranes and loading of the vesicles prepared from liver homogenates in the presence of vasopressin increased the maximal velocities of vanadate‐sensitive transport by 3.8‐fold and 1.9‐fold in the presence of low and high concentrations of Na+ and K+, respectively. The apparent Km(ATP) was shifted to 62 ± 7 μM and 76 ± 10 μM by vasopressin at low and high ion concentrations, respectively, indicating that the hormone reduced the influence of Na+ and K+ on ATP binding. In vesicles isolated from hepatocytes preincubated with 10 nM vasopression the hormone effect was conserved. Initial velocities of Na+ uptake (at high ion concentrations and 1 mM ATP) were increased 1.6 – 1.7‐fold above control, after incubation of the cells with vasopressin or by affinity labelling of the cells with a photoreactive analogue of the hormone.

The velocity of amiloride‐sensitive Na+ transport was enhanced by incubating hepatocytes in the presence of 10 nM insulin (1.6‐fold) or 0.3 mM pervanadate generated by mixing vanadate plus H2O2 (13‐fold). The apparent Km(Na+) of Na+/H+ exchange was increased by pervanadate from 5.9 mM to 17.2mM. Vesiculation and incubation of isolated membranes in the presence of pervanadate had no effect on the velocity of amiloride‐sensitive Na+ transport.

The results show that hormone receptor‐mediated effects on (Na+,K+)ATPase and Na+/H+ exchange are conserved during the isolation of liver plasma membrane vesicles. Stable modifications of the transport systems or their membrane environment rather than ionic or metabolic responses requiring cell integrity appear to be involved in this regulation.
Vinculin is one of the major endogenous substrates for intrinsic tyrosine kinases in neuronal growth cones isolated from fetal rat brain
Authors: IGARASHI Michihiro , SAITO Shigeru , KOMIYA Yoshiaki
Published: March 2005

Neuronal growth cones, the motile tips of growing neuronal processes, are responsible for the exact guidance of extending neurites. To elucidate the mechanisms of their biochemical signal transduction in growth cones, the growth‐cone‐enriched fraction was isolated biochemically from fetal rat brain and the endogenous protein phosphorylation in the fraction was analyzed under the conditions where tyrosine residues were preferentially phosphorylated. One of the major phosphoproteins was a 130‐kDa slightly acidic protein which reacted with anti‐phosphotyrosine antibody. Its phosphoryl residues were alkali‐stable. Thus, the 130‐kDa protein was concluded to be susceptible to tyrosine phosphorylation. This protein was a component of cytoskeletal proteins thought to be associated indirectly with membranes. All the behavior of the 130‐kDa protein was compatible with the properties of vinculin, a component of focal contacts which are responsible for the stable or motile adhesion between cells or between a cell and the substratum. Immunochemical analyses showed that the 130‐kDa protein was specifically recognized by anti‐vinculin antibody. Therefore, the 130‐kDa protein was concluded to be vinculin. Tyrosine phosphorylation of the protein appeared to be relatively more pronounced in the growth‐cone‐enriched fraction than in adult synaptosomes. The results suggest that tyrosine phosphorylation of vinculin may be regulated developmentally and it may be involved in the functions of growth cones.
Structural and functional characterization of human aromatase P‐450 gene
Authors: TODA Katsumi , TERASHIMA Masako , KAWAMOTO Takeshi , SUMIMOTO Hidetoshi , YOKOYAMA Yuichi , KURIBAYASHI Isao , MITSUUCHI Yasuhiro , MAEDA Tomoho , YAMAMOTO Yasutake , SAGARA Yusuke , IKEDA Hisao , SHIZUTA Yutaka
Published: March 2005

The gene encoding aromatase P‐450 (CYP XIX) has been isolated from two types of human genomic DNA libraries. It spans at least 70 kb and consists of 10 exons. The translational initiation site and the termination site are located in exon 2 and exon 10, respectively. The promoter region of the gene contains a TATA box, a CAAT box and two putative AP‐1 binding sites beginning at –28, –83, –55 and –68 bp, respectively, from the transcriptional initiation site. In addition, a palindromic nucleotide sequence is observed between –209 and –196 and two types of repetitious hexanucleotide (consensus: AATGAA and CCATAAGG) are also present within the regions between –485 and –433 and between –358 and –331. Transient expression studies of chloramphenicol acetyltransferase constructs bearing various lengths of 5′‐flanking region of the gene show that the region between –500 and –243 contains negative cis‐acting element(s), whereas the region between –242 and –183 is required for efficient transcriptional activity. Northern blot analysis demonstrates that the expression of aromatose P‐450 gene is remarkably stimulated by treatment of cells with 12‐O‐tetradecanoyl‐phorbol 13‐acetate. By chloramphenicol acetyltransferase assay the region up to nucleotide position –242 relative to the transcriptional initiation site is shown to participate in the transcriptional responsiveness to this phorbol ester.
The role of an invariant tryptophan residue in α‐bungarotoxin and cobrotoxin
Authors: CHANG Chun‐Chang , KAWATA Yasushi , SAKIYAMA Fumio , HAYASHI Kyozo
Published: March 2005

Ozone oxidation converted the single, invariant, tryptophan residue to N2‐formylkynurenine in α‐bungarotoxin and cobrotoxin. Upon this modification, the lethal toxicity was significantly reduced in cobrotoxin but mostly retained in α‐bungarotoxin. Each neurotoxin containing kynurenine instead of tryptophan retained the same antigenicity as the native toxin. Fluorescence and CD spectroscopy revealed that, although the environment and state of the kynurenine residue were similar, [Kyn29]cobrotoxin was much more sensitive to pH change than α‐[Kyn28]bungarotoxin. In terms of lethal toxicity and conformational stability, the invariant tryptophan residue appears to play a more important role in cobrotoxin, imparting a higher lethal toxicity than that in α‐bungarotoxin, which has a disulfide bond at Cys29–Cys33.
Yeast carbamoyl‐phosphate‐synthetase – aspartate‐transcarbamylase multidomain protein is phosphorylated in vitro by cAMP‐dependent protein kinase
Authors: DENIS‐DUPHIL Michèle , LECAER Jean‐Pierre , HARDIE D. Grahame , CARREY Elizabeth A.
Published: March 2005

The first two steps of de novo pyrimidine synthesis in Saccharomyces cerevisiae are catalyzed by a multifunctional protein, coded by the URA2 gene and which has the carbamoyl‐phosphate (CPSase) synthetase and aspartate transcarbamylase (ATCase) activities. The native enzyme purified from protease‐B‐deficient URA2‐transformed cells, was phosphorylated in vitro using catalytic subunits of pure cAMP‐dependent protein kinase. After electrophoresis under denaturing conditions, a single 240‐kDa species was found to be phosphorylated. Trypsin digestion of this species gave a single, very acidic phosphopeptide upon isoelectric focussing. Purification by HPLC followed by amino acid sequencing of this peptide, showed a phosphoserine at the expected consensus sequence Arg‐Arg‐Phe‐Ser. Knowledge of the URA2 gene sequence allowed the site to be located in the peptide link between dihydroorotase‐like and ATCase domains. Such a location may explain why phosphorylation of the URA2 protein changed neither CPSase and ATCase activities nor their sensitivity to UTP, their common specific inhibitor.
Myosin heavy chain isoform composition in striated muscle after denervation and self‐reinnervation
Authors: JAKUBIEC‐PUKA Anna , KORDOWSKA Jolanta , CATANI Claudia , CARRARO Ugo
Published: March 2005

The total content of myosin heavy chains (MHC) and their isoform pattern were studied by biochemical methods in the slow‐twitch (soleus) and fast‐twitch (extensor digitorum longus) muscles of adult rat during atrophy after denervation and recovery after self‐reinnervation. The pattern of fibre types, in terms of ultrastructure, was studied in parallel.

After denervation, total MHC content decreased sooner in the slow‐twitch muscle than in the fast‐twitch. The ratio of MHC‐1 and the MHC‐2B isoforms to the MHC‐2A isoform decreased in the slow and the fast denervated muscles, respectively. After reinnervation of the slow muscle, the normal pattern of MHC recovered within 10 days and the type 1 isoform increased above the normal. In the reinnervated fast muscle, the 2B/2A isoform ratio continued to decrease. Traces of the embryonic MHC isoform, identified by immunochemistry, were found in both denervated and reinnervated slow and fast muscles. A shift in fibre types was similar to that found in the MHC isoforms. Within 2 months of recovery a tendency to normalization was observed.

The results show that (a) MHC‐2B isoform and the morphological characteristics of the 2B‐type muscle fibres are susceptible to lack of innervation, similar to those of type 1, (b) during muscle recovery induced by reinnervation the MHC isoforms and muscle fibres shift transiently to type 1 in the soleus and to type 2A in the extensor digitorum longus muscles, and (c) the embryonic isoform of MHC may appear in the adult skeletal muscles if innervation is disturbed.
Characterization of a novel calcium‐binding 90‐kDa glycoprotein (BM‐90) shared by basement membranes and serum
Authors: KLUGE Matthias , MANN Karlheinz , DZIADEK Marie , TIMPL Rupert
Published: March 2005

The protein BM‐90 was solubilized from the mouse Engelbreth‐Holm‐Swarm tumor with neutral buffers in molar yields lower (15–30%) than found for other basement membrane proteins (e.g. laminin, BM‐40). The purified protein was shown to be rich in cysteine (5 mol%) and to change in SDS electrophoresis from an 84‐kDa position to a 95‐kDa one upon reduction. BM‐90 was also shown to be a calcium‐binding protein. The N‐terminal sequence of BM‐90, as well as those of several internal peptides, showed no identity with any known protein sequences, indicating that it is a new protein. Specific radioimmunoassays showed no or only minor cross‐reactions with other known basement membrane proteins.

Immunological assays demonstrated BM‐90 to be present in neutral salt extracts from mouse heart and kidney, in serum (20–40 μ/ml) and in the medium of various cultured cells (0.1–1 μ/ml). The protein in these samples was identical in size to BM‐90 purified from the tumor, indicating that negligible degradation occurs during purification. An extracellular matrix localization of BM‐90 was shown by immunofluorescence for Reichert's membrane, lens capsules and other basement membranes. Thus, BM‐90 appears to be a novel basement membrane protein whose functions remain to be studied.
Binding sites of a novel neuropeptide pituitary‐adenylate‐cyclase‐activating polypeptide in the rat brain and lung
Authors: LAM Hing‐Chung , TAKAHASHI Kazuhiro , GHATEI Mohammed A. , KANSE Sandip M. , POLAK Julia M. , BLOOM Stephen R.
Published: March 2005

Pituitary‐adenylate‐cyclase‐activating polypeptide (PACAP) is a novel 38‐amino‐acid neuropeptide isolated from ovine hypothalamic tissues based on its activity of stimulating adenylate cyclase of rat pituitary cells. Binding sites for PACAP were studied in rat tissue membranes using a 27‐amino‐acid N‐terminal derivative of PACAP [PACAP(1–27)] labelled with 125I. Particularly high specific binding sites of 125I‐PACAP(1–27) were noted in the hypothalamus, brain stem, cerebellum and lung. Specific binding sites are also present in the pituitary gland, but at a lower concentration, and mainly in the anterior lobe. Very low concentration of 125I‐PACAP(1–27)‐binding sites were found in the colon, aorta and kidney membranes and no binding sites were detected in the pancreas and testis. Maximal binding of 125I‐PACAP(1–27) was observed at pH 7.4. Interaction of 125I‐PACAP(1–27) with its binding site was rapid, specific and saturable as well as time, pH and temperature dependent. PACAP(1–27) is more potent than PACAP in displacing the binding of 125I‐PACAP(1–27) with brain membranes [concentration that inhibits 50% of the binding (IC50) = 7.45 ± 1.52 nM and 11.45 ± 3.65 nM, respectively); mean ± SEM. n= 4] and lung membranes (IC50= 4.41 ± 0.87 nM and 10.68 ± 3.09 nM, respectively). Vasoactive intestinal peptide displaced the binding of 125I‐PACAP(1–27) in lung membrane (IC50= 16.88 ± 5.14 nM) but not in brain membranes. The equilibrium binding of 125I‐PACAP(1–27) at 4°C was characterized by a single class of binding site for the brain membrane with a dissciation constant (Kd) of 2.46 ± 0.53 nM and a maximal binding capacity (Bmax) of 8.44 ± 3.13 pmol/mg protein, but there were two classes of binding site for lung menbranes with Kd of 1.02 ± 0.51 nM and 5.19 ± 0.99 nM, and Bmax of 2.84 ± 0.72 pmol/mg protein and 9.13 ± 1.89 pmol/mg protein, respectively. These findings suggest that subtypes of PACAP‐binding sites exist and PACAP may have a physiological role in the hypothalamus/pituitary axis as well as in other regions of the brain and lung.
Purification and characterization of acyl carrier protein from two cyanobacteria species
Authors: FROEHLICH John E. , POORMAN Roger , REARDON Eileen , BARNUM Susan R. , JAWORSKI Jan G.
Published: March 2005

The acyl carrier protein (ACP), an essential protein cofactor for fatty acid synthesis, has been isolated from two cyanobacteria: the filamentous, heterocystous, Anabaena variabilis (ATCC 29211) and the unicellular Synechocystis 6803 (ATCC 27184). Both ACPs have been purified to homogeneity utilizing a three‐column procedure. Synechocystis 6803 ACP was purified 1800‐fold with 67% yield, while A. variabilis ACP was purified 1040‐fold with 50% yield. Yields of 13.0 μg ACP/g Synechocystis 6803 and 9.0 μg ACP/g A. variabilis were achieved. Amino acid analysis indicated that these ACPs were highly charged acidic proteins similar to other known ACPs. Sequence analysis revealed that both cyanobacterial ACPs were highly conserved with both spinach and Escherichia coli ACP at the phosphopantetheine prosthetic group region. Examining the probability of α‐helix and ß‐turn regions in various ACPs, showed that cyanobacterial ACPs were more closely related to E. coli ACP than spinach ACP I. Immunoblot analysis and a competitive binding assay for ACP illustrated that both ACPs bound poorly to spinach ACP I antibody. SDS/PAGE and native PAGE of Synechocystis 6803 ACP and A. variabilis ACP showed that cyanobacteria ACPs co‐migrated with E. coli ACP and had relative molecular masses of 18 100 and 17900 respectively. Both native and urea gel analysis of acyl‐ACP products from fatty acid synthase reactions demonstrated that bacterial ACPs and plant ACP gave essentially the same metabolic products when assayed using either bacterial or plant fatty acid synthase. A. variabilis and Synechocystis 6803 ACP could be acylated using E. coli acyl ACP synthetase.
Nuclear magnetic resonance studies of sodium/calcium exchange in frog perfused, beating hearts
Authors: POWELL David , BURSTEIN Deborah , FOSSEL Eric T.
Published: March 2005

This study explores the effect of extracellular Ca2+ concentration ([Ca2+]0), on the intracellular Na+ concentration ([Na+]i, in frog intact hearts using nuclear magnetic resonance spectroscopy, which allows for the measurement of [Na+]i in perfused, beating hearts. Decreases in [Ca2+]0 yielded marked increases in [Na+]i. A similar effect was seen during inhibition of the Na+/K+ pump and was fully reversible. This sensitivity of [Na+]i to [Ca2+]0, previously observed using microelectrodes, supports a crucial physiological role for Na+/Ca2+ exchange in frog intact, beating hearts.
The inhibitory glycine receptor: A ligand‐gated chloride channel of the central nervous system
Authors: LANGOSCH Dieter , BECKER Cord‐Michael , BETZ Heinrich
Published: March 2005

The postsynaptic glycine receptor (GlyR) is a major inhibitory chloride channel protein in the central nervous system. The affinity‐purified receptor contains polypeptides of 48 kDa, 58 kDa, and 93 kDa. The 48‐kDa (α) and 58 kDa (β) subunits span the postsynaptic membrane in a pentameric arrangement to form the anion channel of the receptor. The 93‐kDa polypeptide is cytoplasmically localized and may have an anchoring function. Molecular cloning revealed that different structural characteristics are shared by the membrane‐spanning subunits of the GlyR and those of other ligand‐gated ion channel proteins. Developmental regulation of the GlyR is characterized by alterations in antagonist binding, heterogeneity of α subunits, and increased levels of the 93‐kDa polypeptide. Glycine receptor function can be reconstituted by expression of cloned α subunits in heterologous cell systems. Positive charges found at the presumed mouths of the GlyR channel appear to be important determinants of ion selectivity. These data establish the anion‐conducting GlyR as a homolog of other ligand‐gated ion channel proteins and suggest that the diversity of these channels originates from divergent evolution of a primordial channel protein early in phylogeny.
Effect of isolated C‐terminal fragment of θ‐toxin (perfringolysin O) on toxin assembly and membrane lysis
Authors: IWAMOTO Machiko , OHNO‐IWASHITA Yoshiko , ANDO Susumu
Published: March 2005

θ‐toxin, a thiol‐activated cytolysin, binds cholesterol and assembles on plasma membrane during the lytic process. In order to understand the process at the molecular level, two fragments (T1 and T2) were isolated from a nicked toxin obtained by limited proteolysis with trypsin. Although neither the T1 nor T2 fragment has hemolytic activity, T2 has almost the same potential as native θ‐toxin in its binding affinity for erythrocytes and in its binding specificity for cholesterol. T2, derived from the C‐terminus of the toxin, loses binding activity upon 5,5′‐dithiobis (2‐nitrobenzoic acid) modification of the thiol group. The T2 fragment was found to abolish the hemolytic activity of θ‐toxin completely without any inhibition of θ‐toxin binding to erythrocytes. θ‐toxin normally appears in polymeric form on membranes, while it remains in monomer form in the presence of the T2 fragment, as judged by sedimentation patterns in sucrose density‐gradient centrifugation. These results indicate that without inhibiting binding, the T2 fragment inhibits hemolysis by preventing θ‐toxin from aggregating on membranes, a step that might be essential for the lytic process.
Early events in the import/assembly pathway of an integral thylakoid protein
Authors: REED Janet E. , CLINE Kenneth , STEPHENS Lanie C. , BACOT Karen O. , VIITANEN Paul V.
Published: March 2005

The light‐harvesting chlorophyll a/b protein (LHCP) is nuclear‐encoded and must traverse the chloroplast envelope before becoming integrally assembled into thylakoid membranes. Previous studies implicated a soluble stromal form of LHCP in the assembly pathway, but relied upon assays in which the thylakoid insertion step was intentionally impaired [Cline, K., Fulsom, D. R. and Viitanen, P. V. (1989) J. Biol. Chem. 264, 14225–14232]. Here we have developed a rapid‐stopping procedure, based upon the use of HgCl2, to analyze early events of the uninhibited assembly process. With this approach, we have found that proper assembly of LHCP into thylakoids lags considerably behind trans‐envelope translocation. During the first few minutes of import, two distinct populations of mature‐size LHCP accumulate within the chloroplast. One is the aforementioned soluble stromal intermediate, while the other is a partially (or improperly) assembled thylakoid species. Consistent with precursor/product relationships, both species reach peak levels at a time when virtually none of the imported molecules are correctly assembled. These results confirm and extend our previous interpretation, that upon import, preLHCP is rapidly processed to its mature form, giving rise to a soluble stromal intermediate. They further suggest that the stromal intermediate initially inserts into the thylakoid bilayer in a partially assembled form, which eventually becomes properly assembled into the light‐harvesting complex.
Structural transition of α1‐antitrypsin by a peptide sequentially similar to β‐strand s4A
Authors: SCHULZE Andreas J. , BAUMANN Ulrich , KNOF Siegward , JAEGER Ernst , HUBER Robert , LAURELL Carl‐Bertil
Published: March 2005

Crystal structure studies have shown that cleaved and intact serpins differ essentially in the topology of β‐sheet A. This is five‐stranded in the intact molecules and six‐stranded after cleavage by insertion of strand s4A whose C‐terminus has become free [Löbermann, H., Tokuoka, R., Deisenhofer, J. & Huber, R. (1984) J. Mol. Biol. 177, 531–556; Wright, T. H., Qian, H. X. & Huber, R. (1990) J. Mol. Biol. 213, 513–528]. The structural transition is accømpanied by changes in spectral properties and an increase in thermal stability. We show here that an Nα‐acetyl‐tetradecapeptide with the amino acid sequence of strand s4A, residues 345–358 of human α1‐antitrypsin, associates with intact α1‐antitrypsin and forms a stoichiometric complex with properties very similar to cleaved α1‐antitrypsin. Complex generation has the characteristics of a folding process.
Amino acid sequence of the monomer subunit of the giant extracellular hemoglobin of the aquatic oligochaete, Tubifex tubifex
Authors: STERN Mary S. , VINOGRADOV Serge N. , SHARMA Pawan K. , EREIFEJ Khalil , WALZ Daniel A.
Published: March 2005

The extracellular hemoglobin of the aquatic oligochaete Tubifex tubifex consists of four subunits: a monomer of 16.5 kDa, a disulfide‐bonded trimer of about 50 kDa and at least two subunits of about 30 kDa. The complete amino acid sequence of the monomeric subunit was determined: it consists of 141 amino acid residues and has a molecular mass of 16286 Da including a heme group. 39 residues (28%) were found to be identical with those in the corresponding positions in the monomeric globin chains from Lumbricus terrestris, Pheretima sieboldi, and Tylorrhynchus heterochaetus. Tubifex and Lumbricus are most similar, with 75 amino acid identities (53%). There are eight invariant residues amongst these monomeric globins and the intracellular monomeric globin of Glycera and the human β‐globin. The monomeric globin from Tubifex aligns best with those of group A, globins which have a Cys in their second position and an invariant Lys‐Val‐Lys at positions 9–11 [Gotoh et al. (1987) Biochem. J. 241, 441–445]. The two cysteine residues, at positions 2 and 131, appear to be disulfide‐bonded.
Mutagenesis of the pseudosubstrate site of protein kinase C leads to activation
Authors: PEARS Catherine J. , KOUR Gurdip , HOUSE Colin , KEMP Bruce E. , PARKER Peter J.
Published: March 2005

Protein kinase C is maintained in an inactive state by the action of an inhibitory region within the effector binding domain of the kinase. It has been suggested that a short stretch of amino acids (pseudosubstrate site) mediates this inhibition by binding to the active site and preventing substrate interaction [House, C. and Kemp, B. E. (1987) Science 238, 1726–1728]. A mutated version of protein kinase C‐α containing a glutamic acid for alanine substitution in this region has been analysed for biochemical properties and biological function. Consistent with the importance of this pseudosubstrate site in regulating kinase activity, this altered protein has a significantly increased effector‐independent kinase activity relative to wild‐type protein kinase C‐α and shows increased sensitivity to activation by proteolysis. The increased activity of this protein in the intact cell was confirmed by its ability to stimulate expression from a phorbol‐ester‐inducible reporter construct in a transient transfection system. Expression of a mutant kinase with the pseudosubstrate sequence deleted causes greater induction in this transient expression system, consistent with this kinase being independent of effectors and thus constitutively active.
Independent folding of individual components in hybrid proteins
Authors: SLILATY Steve N. , OUELLET Mariette , FUNG Margaret , SHEN Shi‐Hsiang
Published: March 2005

Inactivation of the Escherichia coli repressor protein, LexA, takes place through a cleavage reaction which hydrolyzes the Ala84‐Gly85 peptide bond near the center of the molecule. The mechanism of cleavage has previously been shown to be an intramolecular reaction stimulated in vitro by elevated pH or by the addition of activated RecA protein. The entire self‐cleavage activity of LexA has been found to lie within a 135‐residue tryptic fragment extending from Leu68 to the end of the protein at Leu202. Since the activity of self‐cleavage is dependent on the proper three‐dimensional structure of the protein, we have used it as a probe to investigate the extend of folding autonomy and functional independence of this 135‐residue carboxy‐terminal domain of LexA by applying a protein fusion approach. A series of twelve different hybrid proteins, containing LexA sequences in a variety of predefined primary structural arrangements, were constructed and evaluated for whether or not self‐cleavage activity has been retained. The results revealed that retention or loss of activity is independent of the nature or size of the foreign protein used. Loss of self‐cleavage was found to be a function of amino‐ or carboxy‐terminal deletions in the self‐cleaving LexA component of the fusion proteins. The present findings, together with the observations of other artificial fusions proteins and the naturally occurring bifunctional and multifunctional proteins, along with the data on helix packing, provide further support for the notion of modular architecture of proteins and suggest that when these autonomous units are fused, they retain their tendency to fold independently of the remainder of the polypeptide to generate physically linked active domains, rather than to fold dependently and yield scrambled structures.
Involvement of pertussis‐toxin‐sensitive G protein in muscarinic‐receptor‐mediated inhibition of K+‐activated 4‐nitrophenylphosphatase activity of cardiac sarcolemma
Authors: DANILENKO Mikhail P. , PANCHENKO Mikhail P. , YESIREV Oleg V. , TKACHUK Vsevolod A.
Published: March 2005

The effects of the cholinergic agonist carbachol on ouabain‐sensitive K+‐activated 4‐nitrophenylphosphatase (K+‐O2NPhPase) activity of rabbit and pig ventricular sarcolemma were examined. Carbachol (0.01–1000 μM) alone had no effect on K+‐O2NPhPase. However, in the presence of GTP (100 μM) or its analog guanosine 5′‐[γ‐thio]triphosphate (GTP[S], 1 μM) the agonist reduced this enzymatic activity (IC50= 0.3 μM) by about 45% in a concentration‐dependent manner. The GTP[S]‐dependent effect of carbachol was blocked by 10 μM atropine, an antagonist of muscarinic acetylcholine receptor (mAcChoR). In the presence of micromolar concentrations of ATP or the GDP analog guanosine 5′‐[β‐thio]diphosphate, carbachol did not change sarcolemmal K+‐O2NPhPase activity. GTP[S] alone reduced this activity (IC50= 2 μM) by about 40% in a concentration‐dependent manner with a lag period of about 3 min. This lag disappeared in the presence of carbachol. Treatment of sarcolemmal membranes with 20 μg/ml pertussis toxin, which catalyzed ADP‐ribosylation of the 40–41‐kDa α‐subunits of inhibitory GTP‐binding protein (Gi), abolished the GTP[S]‐promoted inhibitory effect of carbachol. Immunochemically, these α‐subunits were identified as αi2‐ and αi3‐subunits. It is suggested that the carbachol‐induced inhibition of ouabain‐sensitive K+‐O2NPhPase activity of mammalian myocardial sarcolemma is a result of a negative coupling between mAcChoR and Na+/K+‐ATPase via Gi protein.
Delineation of the minimal portion of the Bacillus sphaericus 1593M toxin required for the expression of larvicidal activity
Authors: ŠEBO Peter , BENNARDO Thérèse , de la TORRE Françoise , SZULMAJSTER Jekisiel
Published: March 2005

The two genes of Bacillus sphaericus 1953M coding for the 51.4‐kDa and 41.9‐kDa proteins are both required for the expression of the active larvicidal toxin in Escherichia coli. The minimal size of the active peptide of the 41.9‐kDa toxin was defined by in vitro deletion analysis of the gene and found to consist of 338 amino acids (38.3 kDa). N‐terminal deletions past the Ile18 residue and C‐terminal deletions past the His352 residue result in the loss of toxic activity and rapid degradation of such modified toxins by host proteases. The minimal active 38.3‐kDa peptide produced in E. coli seems to mimick the stable processed form of the toxin found in larval midguts. However, it still requires the action of the synergistic 51.4‐kDa protein for the larvicidal activity.
A two‐dimensional 1H‐NMR study on Megasphaera elsdenii flavodoxin in the oxidized state and some comparisons with the two‐electron‐reduced state
Authors: van MIERLO Carlo P. M. , van der SANDEN Boudewijn P. J. , van WOENSEL Peter , MÜLLER Franz , VERVOORT Jacques
Published: March 2005

Assignments for the 137 amino acid residues of oxidized Megasphaera elsdenii flavodoxin have been made using the sequential resonance assignment procedure. Great benefit was experienced from assignments of the fully reduced protein. The secondary and tertiary structures of the typical α/β protein remain virtually identical on going from the oxidized to the two‐electron‐reduced state as judged from two‐dimensional NOE spectroscopy. However, functionally important conformation changes in the flavin‐binding region do occur on reduction. Considerable reduction‐state‐dependent chemical shift variations of protons in the immediate vicinity of the isoalloxazine moiety take place. From analysis of these shifts, it can be concluded that ring current effects of the pyrazine part of the flavin diminish on two‐electron reduction.
Basic polycations activate the insulin receptor kinase and a tightly associated serine kinase
Authors: BIENER Yael , ZICK Yehiel
Published: March 2005

The effects of cationic polyamino acids on phosphorylation of the insulin and insulin‐like growth factor 1 receptor kinases were studied and the following observations were made. (a) Polylysine stimulated both tyrosine and serine phosphorylation of the insulin receptor and of additional proteins present in lectin‐purified membrane preparations from rat liver. (b) Polylysine synergized with insulin to enhance phosphorylation of the insulin receptor and of additional proteins (pp40 and pp110). (c) Polylysine effects were more pronounced upon increasing the polylysine chain length. (d) The effect of polylysine was biphasic with an optimum at 100 μg/ml. (e) Polylysine was found ineffective in stimulating the phosphorylation of immobilized insulin receptors. Taken together, these findings support the notion that the action of polylysine involves conformational changes and presumably aggregation of soluble receptors. The same effects of polylysine were obtained with highly purified insulin receptor preparations. Under these conditions polylysine enhanced both serine and tyrosine phosphorylation of the insulin receptor, suggesting that polylysine stimulates the activity of the insulin receptor kinase, and of a serine kinase that is tightly associated with the insulin receptor.
Characterization of two tyrosine‐specific protein kinases from pig spleen
Authors: BATZER Andreas G. , KIRSCH Sabine , HOFER Hans Werner
Published: March 2005

Spontaneously active tyrosine‐specific protein kinases I and II (designated TyrK I and TyrK II) have been purified to electrophoretic homogeneity from a particulate fraction of porcine spleen based on an assay that used poly(4Tyr, Glu) as a substrate. SDS/polyacrylamide gels revealed a doublet of bands of about Mr 51000 for TyrK I and two protein bands of Mr 55000 and 54000 for TyrK II. After incubation in the presence of [γ‐32P]ATP, the bands corresponding to both protein kinases contained phoshotyrosine. The two tyrosine protein kinases showed high activities with poly(Tyr, 4Glu) and poly(Tyr, 3Ala, 6Glu) as substrates and lower activity with angiotensin II. Neither histone, phosvitin, casein nor bovine serum albumin were phosphorylated. Both protein tyrosine kinases were activated by millimolar concentrations of Mg2+ whereas Mn2+ was less effective. The effects of various polyanionic and polycationic substances depended on the nature of the peptide substrate. With poly(Tyr, 4Glu) as a substrate, the substances either inhibited the activities of TyrK I and TyrK II or had no effect. However, activation was observed with angiotensin II as substrate in the presence of polylysine, polyornithine, protamine sulfate, and heparin as effectors. When angiotensin II was used as substrate, activation also occurred by autophosphorylation, in parallel to the phosphate incorporation into the protein kinases. Activation by autophosphorylation was not observed with the synthetic peptide substrates, poly(Tyr, 4Glu) and poly(Tyr, 3Ala, 6Glu).
The identification of cytochromes involved in the transfer of electrons to the periplasmic NO−3 reductase of Rhodobacter capsulatus and resolution of a soluble NO−3 ‐reductase − cytochrome‐c552 redox complex
Authors: RICHARDSON David J. , McEWAN Alastair G. , PAGE M. Dudley , JACKSON J. Baz , FERGUSON Stuart J.
Published: March 2005

The involvement of cytochromes in the electron‐transport pathway to the periplasmic NO−3 reductase of Rhodobacter capsulatus was studied in cells grown photoheterotrophically in the presence of nitrate with butyrate as carbon source. The specific rate of NO−3 reduction by such cells was five times higher than when malate was carbon source. Reduced minus NO−3‐oxidized spectra of cells had peaks in the α‐band region for cytochromes at 552 nm and 559 nm, indicating the involvement of c‐ and b‐type cytochromes in the electron‐transport pathway to NO−3. The total ferricyanide‐oxidizable cytochrome that was also oxidized in the steady state by NO−3‐was greater in cells grown with butyrate rather than malate. Low concentrations of cyanide inhibited NO−3 reduction. Neither CN−, nor a previously characterized inhibitor of NO−3 reduction, 2‐n‐heptyl‐4‐hydroxyquinoline N‐oxide, prevented the oxidation of the cytochromes by NO−3. This suggested a site of action for these inhibitors on the reducing side of the b‐ and c‐type cytochromes involved in electron transport to the NO−3 reductase. The predominant cytochrome in a periplasmic fraction prepared from cells of R. capsulatus grown on butyrate medium was cytochrome c2 but a c‐type cytochrome with an χ‐band reduced absorbance maximum at 552 nm could also be identified. The reduced form of this latter cytochrome, but not that of cytochrome c2, was oxidized upon addition of NO−3 to a periplasmic fraction. The NO−3‐oxidizable cytochrome co‐purified with the periplasmic NO−3 reductase through fractionation procedures that included ammonium sulphate precipitation, gel filtration at low and high salt concentrations, and ion‐exchange chromatography. A NO−3‐reductase ‐ cytochrome‐c552 redox complex that comprised two types of polypeptide, a nitrate reductase subunit and a c‐type cytochrome subunit, was purified. The polypeptides were separated when the complex was chromatographed on a phenyl‐Sepharose hydrophobic chromatography column.
The functional and structural roles of residues Gln114 and Glu117 in elongation factor Tu
Authors: HARMARK Kim , COOL Robbert H. , CLARK Brian F. C. , PARMEGGIANI Andrea
Published: March 2005

The effects of substituting residues Gln114 by Glu and Glu117 by Gln, both situated in the vicinity of the guanine‐nucleotide‐binding pocket, were investigated in the isolated N‐terminal domain (G domain) of elongation factor Tu with respect to the binding of the substrate GDP/GTP, GTPase activity and stability. The major change in the interaction with the guanine nucleotides is a lower affinity for GTP and a reduced GTPase activity when Gln114 is substituted by Glu. This mutation also abolishes most of the selective effects on the GTPase activity induced by the different monovalent cations. Substitution of Glu117 by Gln does not affect the interaction with the guanine nucleotides or the GTPase activity of the G domain in an essential way, but it reduces the stability towards denaturation of the G‐domain · GDP complex. Our results therefore suggest, that Gln114 is involved in keeping a functional conformation of the guanine‐nucleotide‐binding pocket, whereas Glu117 participates in the regulation of the overall conformation of the G domain. Neither of these two residues appears to play a role in the actual GTPase mechanism.
Drosophila contains three genes that encode distinct isoforms of protein phosphatase 1
Authors: DOMBRÁDI Viktor , AXTON J. Myles , BREWIS Neil D. , DA CRUZ E SILVA Edgar F. , ALPHEY Luke , COHEN Patricia T. W.
Published: March 2005

The sequences of two Drosophila and one rabbit protein phosphatase (PP) 1 catalytic subunits were determined from their cDNA. The sequence of Drosophila PP1α1 was deduced from a 2.2‐kb cDNA purified from an embryonic cDNA library, while that for Drosophila PP1β was obtained from overlapping clones isolated from both a head cDNA library and an eye imaginal disc cDNA library. The gene for Drosophila PP1α1 is at 96A2–5 on chrõmosome 3 and encodes a protein of 327 amino acids with a calculated molecular mass of 37.3 kDa. The gene for Drosophila PP1β is localized at 9C1–2 on the X chromosome and encodes a protein of 330 amino acids with a predicted molecular mass of 37.8 kDa. PP1α1 shows 96% amino acid sequence identity to PP1α2 (302 amino acids), an isoform whose gene is located in the 87B6–12 region of chromsome 3 [Dombrádi, V., Axton, J. M., Glover, D. M. Cohen, P. T. W. (1989) Eur. J. Biochem. 183, 603–610]. PP1β shows 85% identity to PP1α1 and PP1α2 over the 302 homologous amino acids. These results demonstrate that at least three genes are present in Drosophila that encode different isoforms of PP1.

Drosophila PP1α1 and PP1β show 89% amino acid sequence identity to rabbit PP1α (330 amino acids) [Cohen, P. T. W. (1988) FEBS Lett. 232, 17–23] and PP1β (327 amino acids), respectively, demonstrating that the structures of both isoforms are among the most conserved proteins known throughout the evolution of the animal kingdom. The presence of characteristic structural differences between PP1α and PP1β, which have been preserved from insects to mammals, implies that the α and β isoforms may have distinct biological functions.
Enzymatic activity of prostate‐specific antigen and its reactions with extracellular serine proteinase inhibitors
Authors: CHRISTENSSON Anders , LAURELL Carl‐Bertil , LILJA Hans
Published: March 2005

Prostate‐specific antigen (PSA) is one of the three most abundant prostatic‐secreted proteins in human semen. It is a serine proteinase that, in its primary structure, manifests extensive similarities with that of the Arg‐restricted glandular kallikrein‐like proteinases. When isolated from semen by the addition of chromatography on aprotinin‐Sepharose to a previously described procedure, PSA displayed chymotrypsin‐like activity and cleaved semenogelin and the semenogelin‐related proteins in a rapid and characteristic pattern, but had no trypsin‐like activity. About one third of the purified protein was found to be enzymatically inactive, due to cleavage carboxy‐terminal of Lys145. Active PSA formed SDS‐stable complexes with α1‐antichymotrypsin, α2‐macroglobulin, and the α2‐macroglobulin‐analogue pregnancy zone protein. PSA formed inhibitory complexes with α1‐antichymotrypsin at a molar ratio of 1:1, a reaction in which PSA cleaved the inhibitor in a position identical to that reported from the reaction between chymotrypsin and α1‐antichymotrypsin. The formation of stable complexes between PSA and α1‐antichymotrypsin occurred at a much slower rate than that between chymotrypsin and α1‐antichymotrypsin, and at a similar or slightly slower rate than that between PSA and α2‐macroglobulin. When added to normal blood plasma in vitro, active PSA formed stable complexes both with α2‐macroglobulin and α1‐antichymotrypsin. This complex formation may be a crucial determinant of the turnover of active PSA in intercellular fluid or blood plasma in vivo.
Molecular and biochemical characterization of the Dio‐9‐resistant pma1‐1 mutation of the H+‐ATPase from Saccharomyces cerevisiae
Authors: VAN DYCK Luc , PETRETSKI Jorge Hudson , WOLOSKER Herman , RODRIGUES Gilberto , SCHLESSER Alain , GHISLAIN Michel , GOFFEAU André
Published: March 2005

The plasma‐membrane H+‐ATPase gene PMA1 was sequenced in four Dio‐9‐resistant strains of Saccharomyces cerevisiae, isolated independently. The same amino acid substitution Ala608→Thr was found in the four mutated strains. The mutant ATPase activity was decreased while the Km value for MgATP was increased. The ATPase efficiency (V/Km) of the mutant was reduced by a factor of 25 under acid conditions (pH 5.5), and by a factor of 10 at physiological pH (pH 6.6). The mutation also strongly reduces the inhibition by vanadate of ATPase activity, suggesting that the altered amino acid is involved in phosphate binding and/or in the E1–E2 transition.
Effector‐dependent conformational changes in protein kinase Cγ through epitope mapping with inhibitory monoclonal antibodies
Authors: CAZAUBON Sylvie , WEBSTER Catherine , CAMOIN Luc , STROSBERG A. Donny , PARKER Peter
Published: March 2005

Three monoclonal antibodies (mAb) directed against the regulatory domain of the protein kinase Cγ (PKCγ); 15G4, 5A2 and 36G9, were shown to display distinct properties with respect to PKCγ kinase activity [Cazaubon, S., Marais, R., Parker, P. & Strosberg, A.D. (1989) Eur. J. Biochem. 182, 401–406]. The mAb 5A2 and 36G9, which act as potent inhibitors of the cofactor‐dependent kinase activity, can no longer bind PKCγ in the presence of phosphatidylserine and phosphatidylserine/phorbol ester, respectively; 15G4 binding is not influenced by effectors. Due to this functional relationship between the inhibitory mAb‐ and cofactor‐binding sites, we sought to localize the mAb epitopes with respect to the functional sites of PKCγ. For this purpose, several deletions were introduced at the 5′ end of the PKCγ cDNA and the mutant proteins were expressed in Escherichia coli. The determination of the immunoreactivity of the deleted PKCγ proteins shows that the amino acid residues essential to the binding of 5A2 and 36G9 are directly adjacent to the second cysteine‐rich motif: these are contained in the sequences at positions 151–163 and 164–197, respectively. In addition, various deletions around the C1 region of the regulatory domain allowed the identification of the second cysteine‐rich motif as a functional binding site for phorbol dibutyrate. These deletion studies thus demonstrate that the epitopes recognized by the inhibitory mAbs 5A2 and 36G9 are distinct from the cofactor‐binding sites. This suggests that the binding of phosphatidylserine and phorbol ester induce conformational changes in the regulatory domain of PKC, which are thus responsible for the loss of the 5A2 and 36G9 immunoreactivity of the native protein. In this conformational state, PKCγ conserves its ability to interact with the non‐inhibitory mAb 15G4. By using synthetic peptides, the 15G4 epitope was localized to the sequence 297–310 in the V3 variable region. This indicates that the flexibility of the V3 region, which delimits the C‐terminus of the regulatory domain, may not be necessary for the allosteric activation of PKC.

In view of these results, we propose that PKC activation by its cofactors results in intramolecular changes which allow the enzyme to bind exogenous substrates.