bluuebunny/crossref_metadata_2025 huggingface dataset:
Slow exchanging protons in the Z‐form of G‐C and A‐C alternating polymers by using a rapid dialysis method
Authors: Hartmann B. , Ramstein J. , Leng M.
Published: July 2002

Using a dialysis method we have measured the hydrogen exchange (HX) kinetics in poly(dG‐dC)·poly(dG‐dC), poly(dG‐m5dC)·poly(dG‐m5dC), poly(dG‐br5dC)·poly(dG‐br5dC) and platinated poly(dA‐br5dC)·poly(dG‐dT) under experimental conditions in which these polymers adopt the Z‐conformation. The latter polymer has one slow exchanging proton with a half‐time of about 2 h, whereas the other G‐C alternating polymers display a slow class of two protons with exchange half‐time of about 6 h. These exchange half‐times are independent of ionic strength and of the nature of the salt for all these polymers in the Z‐form. The slow proton exchange appears to be strongly correlated to the Z‐conformation but rather independent of the Z‐DNA sequence. The comparison of the proton exchange rates with the corresponding B⇋Z transition rates is not in favour of the same rate limiting step for both processes.
Murine interleukin‐1 receptor: Differences in binding properties between fibroblastic and thymoma cells and evidence for a two‐chain receptor model
Authors: Bird T.A. , Gearing A.J.H. , Saklatvala J.
Published: July 2002

The concentration of porcine interleukin‐1β (pIL1β) required to elicit half‐maximal IL2 production from NOB‐1, a subline of murine thymoma EL4, was 100‐fold greater than for p1Lα. In contrast, similar doses of each type of IL1 stimulated increased lactate production by Balb/C 3T3 fibroblasts. Receptor‐bound 125I‐IL1α was displaced with equal efficiency by both unlabelled forms from 3T3 cells, but a 20‐fold lower affinity for p1L1β was observed using NOB‐1. Crosslinking experiments suggested that the IL1 receptors on each line consisted of two polypeptides of 80 and 100 kDa. The results provide the first evidence for a multiple‐component IL1 receptor within which IL1α and IL1β may bind at different loci, and suggest the receptors may have evolved differently in the two lines.
Ribonucleotide reductase in cell extracts of Methanobacterium thermoautotrophicum
Authors: Hogenkamp H.P.C. , Follmann H. , Thauer R.K.
Published: July 2002

Cell free extracts of Methanobacterium thermoautrophicum were found to catalyze the reduction of CDP and GDP provided that the cells were harvested, disrupted and the extracts assayed under strictly anaerobic conditions. Ribonucleotide reduction is not affected by adenosylcobalamin and cell extracts do not catalyze the tritium exchange reaction between [5‐3H2]adenosylcobalamin and water. The reduction of CDP requires ATP and that of GDP requires dTTP as positive allosteric effectors. In contrast, dATP at low concentration stimulates CDP reduction while at higher concentrations it inhibits the reductase reaction.
Anomalous behavior of human leukocyte interferon subtypes on polyacrylamide gel electrophoresis in the presence of dodecyl sulfate
Authors: Ohara Osamu , Teraoka Hiroshi
Published: July 2002

35S‐labeled human leukocyte interferon (IFN) subtypes produced in a cell‐free system derived from Escherichia coli were analyzed by polyacrylamide gel electrophoresis in the presence of SDS (SDS‐PAGE). Some IFN subtypes anomalously showed lower electrophoretic mobilities than those expected from their formula molecular masses. The results with hybrid IFNs and esterification suggest that this anomaly of IFN subtypes on SDS‐PAGE is due to the introduction of one or two negative charges in the middle of the molecule.
Carbachol and sodium fluoride, but not TSH, stimulate the generation of inositol phosphates in the dog thyroid
Authors: Graff I. , Mockel J. , Laurent E. , Erneux C. , Dumont J.E.
Published: July 2002

In dog thyroid slices prelabeled with myo‐[2‐3H]inositol, carbachol (10−710−4 M) and NaF (10‐20 mM) stimulated IP1, IP2 and IP3 generation. These effects did not require the presence of extracellular calcium. Atropine and PDBu inhibited the action of the cholinergic agonist. No effect of TSH (1–100 ) could be detected on PIP2 hydrolysis and IP production. These results suggest that (i) IP3 could play a role in the metabolic actions of carbachol in the thyroid; (ii) a G‐protein coupling the hormone‐receptor binding to phospholipase C activation exists in the thyroid membrane; (iii) the well known TSH‐induced increased PI turnover does not result in IP3 accumulation.
Evidence for bile acid glucosides as normal constituents in human urine
Authors: Marschall H.-U. , Egestad B. , Matern H. , Matern S. , Sjövall J.
Published: July 2002

A glucosyltransferase catalysing formation of bile acid glucosides was recently isolated from human liver microsomes. In order to investigate the potential occurrence of such bile acid derivatives in vivo, a method was devised for their isolation and purification from urine. Conditions were established with the aid of glucosides of radiolabelled, unconjugated glycine and taurine conjugated bile acids prepared enzymatically using human liver microsomes. Analysis by gas chromatography and mass spectrometry of methyl ester trimethylsilyl ether derivatives indicated the excretion of glucosides of nonamidated hyodeoxycholic, chenodeoxycholic, deoxycholic, ursodeoxycholic and cholic acids and of glycine and taurine conjugated chenodeoxycholic and cholic acids. Additional compounds were present giving mass spectral fragmentation patterns typical ofdi‐ and trihydroxy bile acid glycosides. Semiquantitative estimates indicated a total daily excretion of about 1 μmol.
Two rat homologues of human cystatin C
Authors: Esnard Annick , Esnard Frédéric , Faucher Didier , Gauthier Francis
Published: July 2002

Two immunochemically related forms of cystatin C‐like inhibitors which differ in their M
r app and isoelectric point have been found both in urine and seminal vesicles of rats. Amino‐terminal sequences of these two cystatins are identical within the same fluid and exhibit a high degree of homology with that of human cystatin C. However, cystatins C purified from urine lack eight residues at their amino‐terminal end when compared to those of seminal vesicles. The occurrence of two cystatin C‐like components in rat fluids has been found to be due to the presence of a glycosylated form reported here as cystatin Cg which specifically binds concanavalin A and is susceptible to endo‐β‐N‐acetylglucosaminidase treatment.
Contractile activity enhances the synthesis of hexokinase II in rat skeletal muscle
Authors: Weber F.E. , Pette D.
Published: July 2002

An 11‐fold increase in hexokinase activity and the hexokinase II isoform was found in rat tibialis anterior muscle after 7 days of chronic, low‐frequency stimulation. In vivo labeling studies showed that this increase in enzyme protein content was related to an approx. 30‐fold increase in [35S]methionine incorporation.
Ion‐conducting channels in a Gram‐positive bacterium
Authors: Zoratti Mario , Petronilli Valeria
Published: July 2002

The patch‐clamp technique was used to obtain information on the existence and properties of ion channels in giant protoplasts obtained from the Gram‐positive bacterium Streptococcus faecalis. The membrane proved to contain a pore with numerous conductance states, ranging from 10 pS to several nanosiemens. Application of a slight pressure differential across the membrane resulted in the activation of the channel. The pressure sensitivity points to a relationship between this channel and one recently discovered in E.coli spheroplasts [(1987) Proc. Natl. Acad. Sci. USA 84, 2297–2301] suggesting that pores of this type might be widespread among prokaryotes.
Subunit III of ruminant procarboxypeptidase A‐S6 complexes and pancreatic proteases E a new family of pancreatic serine endopeptidases?
Authors: Cambillau Christian , Kerfelec Brigitte , Sciaky Martine , Chapus Catherine
Published: July 2002

Subunit III (BSIII) of the bovine ternary complex of procarboxypeptidase A‐S6 (PCPA‐S6), a defective serine endopeptidase‐like protein, actively synthesized by the pancreas of some ruminant species, is highly homologous to human protease E (HPE). Both proteins possess the same atypical disulfide bridge in position 98–99b. They are structurally related to porcine elastase 1 and human elastase 2 (about 56% identity). However, in contrast to those two enzymes which have an overall positive net charge, BSIII and HPE are negatively charged. Three‐dimensional models of BSIII and HPE have been constructed from the crystallographic structure of porcine pancreatic elastase 1. The inhibitor‐binding site for TFAI in these three proteins seems to be very similar; the atypical disulfide bridge does not seem to be involved in this binding site. The specific structural features of BSIII and HPE strongly support the assumption that BSIII is a truncated protease E and that both proteins belong to a separate serine endopeptidase family.
Alterations in the cleavage site of the signal sequence for the secretion of human lysozyme by Saccharomyces cerevisiae
Authors: Nagahora Hitoshi , Fujisawa Hirofumi , Jigami Yoshifumi
Published: July 2002

The amino acids corresponding to the cleavage site of a hybrid preprotein containing a chicken lysozyme signal and a mature portion of human lysozyme were altered. The processing of mutant signals of −3Pro and −3Asp/−1Ala decreased remarkably, while that of −2Pro was 75% of that of the native signal. The major cleavage site of −3Pro was the same as that of the native signal, but that of the −2Pro and −3Asp/−1Ala signals was shifted one residue closer to the N‐terminal side than the original site. The cleavage of the −2Pro signal, which was identical to the native processing of pheasant prelysozyme, suggested that the signal peptidases in yeast and bird are similar.
Isolation of human pancreastatin fragment containing the active sequence from a glucagonoma
Authors: Sekiya K. , Ghatei M.A. , Minamino N. , Bretherton-Watt D. , Matsuo H. , Bloom S.R.
Published: July 2002

The primary structure of a human pancreastatin‐like peptide was determined from a pancreatic glucagonoma. The 28‐amino acid peptide was identified using a specific antibody raised against porcine pancreastatin 1–49 and showed a 75% sequence homology with porcine pancreastatin 22–49 and bovine chromogranin A 267–294. Several forms of pancreastatin‐like immunoreactivity were found in human endocrine tumors of which the purified peptide was the smallest and contained the active sequence of pancreastatin.
Isolation and characterization of a (1 → 3)‐β‐glucan endohydrolase from germinating barley (Hordeum vulgare): amino acid sequence similarity with barley (1 → 3, 1 → 4)‐β‐glucanases
Authors: Høj Peter Bordier , Slade Amanda M. , Wettenhall Richard E.H. , Fincher Geoffrey B.
Published: July 2002

A (1 → 3)‐β‐glucan 3‐glucanohydrolase (EC 3.2.1.39) has been purified approx. 190‐fold from extracts of germinating barley. The enzyme has an apparent M
r 32 000, a pI of 8.6, and a pH optimum of 5.6. Analysis of hydrolysis products released from the (1 → 3)‐β‐glucan, laminarin, shows that the enzyme is an endohydrolase. Sequence analysis of the 46 NH2‐terminal amino acids of the (1 → 3)‐β‐glucanase reveals 54% positional identity with barley (1 → 3,1 → 4)‐β‐glucanases (EC 3.2.1.73) and suggests a common evolutionary origin for these two classes of β‐glucan endohydrolases. The barley (1 → 3)‐β‐glucanase also exhibits significant similarity with a (1 → 3)‐β‐glucanase from tobacco.
A novel protein, p19/6.8, specific for cardiac and slow skeletal muscle
Authors: Kluxen Franz-Werner
Published: July 2002

Upon in vitro translation of mRNAs from slow (soleus) muscles of the mouse a hitherto undescribed translation product has been detected that was absent from fast sekeletal muscles and was termed p19/6.8 according to its position in 2‐dimensional gels. mRNA for p19/6.8 was also found in the ventricle of the heart. p19/6.8 was not detectable by Coomassie blue staining but could be characterised by fractionation of in vivo labelled muscle tissue. It was found to sediment with the particulate fraction at 14 000 × g. The expression of p19/6.8 mRNA appears to be down‐regulated in muscles with phasic activity.
Starvation or diabetes decreases the content but not the mRNA of 6‐phosphofructo‐2‐kinase in rat liver
Authors: Crepin Karine M. , Darville Martine I. , Hue Louis , Rousseau Guy G.
Published: July 2002

In rat liver, the activity of 6‐phosphofructo‐2‐kinase (PFK‐2) decreases upon starvation and in diabetes. Cyclic AMP‐dependent phosphorylation of the enzyme is not sufficient to account for this decrease. PFK‐2 content was therefore measured by immunotitration and relative PFK‐2 mRNA levels were determined by hybridization with cDNA probes. The data are compatible with a posttranscriptional mechanism of regulation that involves decreased translational efficiency of PFK‐2 mRNA and(or) increased turnover of the PFK‐2 protein.
Interactions of pure βγ‐subunits of G‐proteins with purified β1‐adrenoceptor
Authors: Im Mie-Jae , Holzhöfer Andreas , Böttinger Heiner , Pfeuffer Thomas , Helmreich Ernst J.M.
Published: July 2002

The role of the βγ‐subunits in the interaction of G‐proteins was examined with β1‐adrenoceptors purified from turkey erythrocytes and pure βγ‐subunits prepared from turkey erythrocytes and bovine brain. On a non‐denaturing polyacrylamide gel, the mobility of βγ‐subunits was increased when incubated with β1‐adrenoceptor and the β1‐adrenergic agonist l‐(−)‐isoproterenol, whereas on incubation with the antagonist l‐alprenolol the mobility was unchanged. Furthermore, the β1‐adrenoceptor was retarded on a Sephadex G‐50 column equilibrated with βγ‐subunits and agonist. No retardation occurred in the presence of antagonist. These data suggest a direct interaction of activated β1‐adrenoceptors with isolated βγ‐subunits of G‐proteins.
Three fast myosin heavy chains in adult rat skeletal muscle
Authors: Bär A. , Pette D.
Published: July 2002

A new fast myosin heavy chain isoform was electrophoretically detected in adult rat skeletal muscles. It was present at high levels in diaphragm and, therefore, designated as MHCIId. Appreciable amounts of MHCIId were detected in tongue musculature, the extraocular muscles, and in the deep red portions of various fast muscles. Its concentration in fast‐twitch muscle was greatly increased by chronic stimulation.
Identification of the phosphoserine residue in histone H1 phosphorylated by protein kinase C
Authors: Jakes Scott , Hastings Teresa G. , Reimann Erwin M. , Schlender Keith K.
Published: July 2002

The site‐specific phosphorylation of bovine histone H1 by protein kinase C was investigated in order to further elucidate the substrate specificity of protein kinase C. Protein kinase C was found to phosphorylate histone H1 to 1 mol per mol. Using N‐bromosuccinimide and thrombin digestions, the phosphorylation site was localized to the globular region of the protein, containing residues 71–122. A tryptic peptide containing the phosphorylation site was purified. Modification of the phosphoserine followed by amino acid sequence analysis demonstrated that protein kinase C phosphorylated histone H1 on serine 103. This sequence, Gly97‐Thr‐Gly‐Ala‐Ser‐Gly‐Ser(PO4)‐Phe‐Lys105, supports the contention that basic amino acid residues C‐terminal to the phosphorylation site are sufficient determinants for phosphorylation by protein kinase C.
Epinephrine and the Ca2+ ionophore A23187 synergistically induce platelet aggregation without protein kinase C activation
Authors: Olbrich Christine , Siess Wolfgang
Published: July 2002

Aspirin‐pretreated, 32P‐prelabeled, washed human platelets resuspended in a buffer containing apyrase and 2% plasma were exposed to epinephrine and the Ca2+ ionophore A23187. Epinephrine potentiated platelet aggregation (not secretion), the production of [32P]phosphatidic acid and myosin light chain phosphorylation induced by A23187. No phosphorylation of the 40 kDa protein, the substrate of protein kinase C, was observed. We conclude that G1‐protein activation evoked by epinephrine and Ca2+ mobilization caused by A23187 represents a novel synergism for platelet aggregation and that protein kinase C activation, under these conditions is not needed for platelet aggregation.
Isoenzyme patterns of protein kinase C and a phospholipid‐dependent but Ca2+‐inhibited enzyme fraction in the crude extracts of different tissues
Authors: Faragó Anna , Farkas Gyöngyi , Mészáros György , Buday László , Antoni Ferenc , Sepr”odi J’anos
Published: July 2002

We compared the protein kinase C isoenzyme patterns of crude extracts of rabbit brain, cerebellum, spleen, thymus and human and pig granulocytes. The isoenzymes were fractionated by hydroxyapatite chromatography and the protein kinase C activity was determined with a synthetic oligopeptide substrate. In the extracts of several tissues we also observed an enzyme fraction which was activated by phosphatidylserine+diacylglycerol but inhibited by Ca2+.
cDNA and derived amino acid sequence of the hypusine containing protein from Dictyostelium discoideum
Authors: Sandholzer Ute , Centea-Intemann Mihaela , Noegel Angelika A. , Lottspeich Friedrich
Published: July 2002

The eukaryotic translation initiation factor eIF‐4D is the only protein known to contain the unusual amino acid hypusine, a posttranslationally modified lysine. For the production of monoclonal antibodies the hypusine‐containing protein (HP) was isolated from Dictyostelium discoideum. Using these monoclonal antibodies, a full‐length cDNA clone was isolated from a λgt11 library. The D. discoideum HP consists of 169 amino acids and has a molecular mass of 18.3 kDa. It is encoded by a single gene. Tryptic and cyanogen bromide peptides were prepared from the purified protein and sequenced. The hypusine residue is located at amino acid position 65 of the HP. The corresponding mRNA of approx. 0.6 kb is present throughout the life cycle of D. discoideum.
Selective cleavage at lysine of the 50 kDa‐20 kDa connector loop segment of skeletal myosin S‐1 by endoproteinase Arg‐C
Authors: Bertrand R. , Derancourt J. , Kassab R.
Published: July 2002

The reaction of endoproteinase Arg‐C on the skeletal myosin head heavy chain was investigated through characterization of peptides and amino acid sequence analysis. The protease splits exclusively the 50 kDa‐20 kDa junction at the lysine cluster spanning residues 639–641 and does not affect any other protease‐sensitive region of the entire myosin heavy chain. The sensitivity of the cleavage to actin and nucleotide binding makes this protease a very specific conformational probe of S‐1. The nicked S‐1 derivative, containing an intact NH2‐terminal 75 kDa fragment, may serve as a tool for gaining further insights into the domain structure and function of the myosin head.
N‐terminal sequencing of photosystem II low‐molecular‐mass proteins 5 and 4.1 kDa components of the O2‐evolving core complex from higher plants
Authors: Ikeuchi Masahiko , Takio Koji , Inoue Yorinao
Published: July 2002

High resolution gel electrophoresis in the low‐molecular‐mass region combined with electroblotting using polyvinylidene difluoride membranes enabled us to sequence the low‐molecular‐mass proteins of photosystem II membrane fragments from spinach and wheat. The determined N‐terminal sequences, all showing considerable homology between the two plants, involved two newly determined sequences for the 4.1 kDa protein and one for the 5 kDa proteins. The sequence of the 4.1 kDa protein did not match any part of the chloroplast DNA sequence from tobacco or liverwort, suggesting that it is encoded by the nuclear genome. In contrast, the sequence of the 5 kDa protein matched ORF38, which is located just downstream of psbE and psbF in the chloroplast DNA and is assumed to be co‐transcribed with them. These two components were associated with the O2‐evolving core complex. Sequences of other low‐molecular‐mass proteins confirmed the previous identification as photosystem II components.
Interaction of iodoacetamidofluorescein‐labelled tropomyosin with deoxyribonuclease I
Authors: Burtnick L.D. , Wong M.K.C.
Published: July 2002

5‐Iodoacetamidofluorescein (IAF) reacted with rabbit cardiac muscle tropomyosin (TM) to yield a highly fluorescent product, IAF‐TM. The extent of labelling reached one fluorescein group per TM molecule in solutions at pH 8.5. While fluorescence polarization values for IAF‐TM solutions were unaffected by the presence or absence of KCl, addition of pancreatic deoxyribonuclease I (DNase I) resulted in a 10% drop, suggestive of a greater freedom of motion of the fluorescein label in the presence of DNase I. Furthermore, a 15% increase in slopes of Stern‐Volmer plots for IAF‐TM in the presence of DNase I demonstrated a greater susceptibility of the fluorescein group to dynamic quenching by iodide. These results suggest that interaction between DNase I and TM produces a localized unfolding of the coiled coil near the IAF reactive site on TM.
Localisation of the binding site for the initiating substrate on the RNA polymerase from Sulfolobus acidocaldarius
Authors: Grachev Mikhail A. , Mustaev Arkadij A. , Zaychikov Evgeny F. , Lindner Anton J. , Hartmann Guido R.
Published: July 2002

RNA polymerase from the archaebacterium Sulfolobus acidocaldarius was chemically modified with AMP o‐formylphenyl ester followed by reduction with borohydride. The modified protein catalyzes the labeling of its own largest subunit when incubated with [α‐33P]UTP in the presence of poly[d(A‐T)]. On cleaving of the labeled protein using cyanogen bromide, hydroxylamine or amino acid‐specific endoproteinases for a very brief period, the pattern and size of the radioactive fragments formed are best explained by attachment of the label between Gly843 and Met895 of the largest subunit. In this region there exists a highly conserved sequence which is also found in other archaebacterial, eukaryotic and prokaryotic RNA polymerases. This suggests that the binding site for the initiating substrate of RNA polymerases has been conserved during evolution.
Mapping of β‐adrenoceptor coupling domains to Gs‐protein by site‐specific synthetic peptides
Authors: Palm Dieter , Münch Gerald , Dees Christian , Hekman Mirko
Published: July 2002

Peptides corresponding to the known sequence of turkey erythrocyte β1‐adrenergic receptor were synthesized and the effects on receptor‐mediated cyclase activation were measured. Peptides corresponding to the first and second intracellular loops (T61‐71 and T138‐159) inhibited at micromolar concentrations the hormone‐dependent cyclase activation in turkey erythrocyte membranes. In contrast, the peptide corresponding to the C‐terminal part of the third intracellular loop (T284‐295) increased the cyclase activity in a hormone‐independent manner. Peptides T338‐353 and T2‐10 and a number of synthetic peptides unrelated to the β‐adrenoceptor had no effect.
Heterogeneity of native rat liver elongation factor 2
Authors: Marzouki Abdelkader , Lavergne Jean-Pierre , Reboud Jean-Paul , Reboud Anne-Marie
Published: July 2002

The high heterogeneity of native rat liver EF‐2 prepared from either 105000 x g supernatant or microsome high‐salt extract was detected by two‐dimensional equilibrium isoelectric focusing‐SDS‐polyacrylamide gel electrophoresis in the presence of 9.5 M urea. Five spots were always detected, all of M
r 95000, which were not artefactual for their amount varied when EF‐2 was specifically ADP‐ribosylated by diphtheria toxin in the presence of NAD+, and/or phosphorylated on a threonine residue by a Ca2+/calmodulin‐dependent protein kinase (most likely Ca2+/calmodulin‐dependent protein kinase III described by others [(1987) J. Biol. Chem. 262, 17299–17303; (1988) Nature 334, 170–173]). Results of ADP‐ribosylation and/or phosphorylation experiments with either unlabeled or labeled reagents ([14C]NAD and [32P]ATP) strongly suggest that our preparation contained native ADP‐ribosylated and native phosphorylated forms which could be estimated at about 20% and 40% of the whole EF‐2. Phosphorylated and ADP‐ribosylated forms of EF‐2 could be ADP‐ribosylated and phosphorylated, respectively, but a native form both ADP‐ribosylated and phosphorylated was not detected. Our results also suggest the existence of a minor native form of EF‐2 and of its phosphorylated and ADP‐ribosylated derivatives.
Elastase released from human granulocytes stimulated with N‐formyl‐chemotactic peptide prevents activation of tumor cell prourokinase (pro‐uPA)
Authors: Schmitt Manfred , Kanayama Naohiro , Henschen Agnes , Hollrieder Angelika , Hafter Reimar , Gulba Dietrich , Jänicke Fritz , Graeff Henner
Published: July 2002

Proteolytic enzymes released from granulocytes upon stimulation with the chemotactic N‐formyl peptide FNLPNTL (in the presence of cytochalasin B) prevented activation of tumor cell single‐chain urokinase‐type plasminogen activator (pro‐uPA) by plasmin. Elastase was identified by the use of eglin C (elastase inhibitor) and a monoclonal antibody to elastase as the functional proteolytic enzyme in granulocyte supernatants. Action of purified granulocyte elastase on pro‐uPA generated enzymatically inactive two‐chain uPA linked by disulfide bridges which was indistinguishable by SDS‐PAGE from plasmin‐generated HMW‐uPA. The major elastase cleavage site in pro‐uPA was located between Ile159 and Ile160; a minor one between Thr165 and Thr166. Elastase cannot substitute for plasmin in the proteolytic activation of pro‐uPA to enzymatically active HMW‐uPA. However, when pro‐uPA was first activated by plasmin to form enzymatically active HMW‐uPA, this enzymatic activity was not impaired by subsequent elastase treatment.
A purified complex from Xenopus oocytes contains a p47 protein, an in vivo substrate of MPF, and a p30 protein respectively homologous to elongation factors EF‐1γ and EF‐1β
Authors: Bellé Robert , Derancourt Jean , Poulhe Robert , Capony Jean-Paul , Ozon René , Mulner-Lorillon Odile
Published: July 2002

A high molecular mass complex isolated from Xenopus laevis oocytes contains three main proteins, respectively p30, p36 and p47. The p47 protein has been reported to be an in vivo substrate of the cell division control protein kinase p34cdc2. From polypeptide sequencing, we now show that the p30 and the p47 correspond to elongation factor EF‐1β and EF‐1γ. Furthermore, the p30 and p36 proteins were phosphorylated in vitro by casein kinase II.
Inhibition by islet‐activating protein, pertussis toxin, of retinoic acid‐induced differentiation of human leukemic (HL‐60) cells
Authors: Tohkin Masahiro , Iiri Taroh , Ui Michio , Katada Toshiaki
Published: July 2002

Human promyelocytic leukemic (HL‐60) cells were induced to differentiate into neutrophil‐ or macrophage‐like cells by incubation of the cells with retinoic acid, dibutyryl cyclic AMP (Bt2cAMP) or phorbol 12‐myristate 13‐acetate (PMA). Differentiation was determined by an increase in the percentage of morphologically mature cells. The retinoic acid‐induced differentiation of HL‐60 cells was, but the Bt2cAMP‐ or PMA‐induced one was not, inhibited by prior exposure of the cells to islet‐activating protein (IAP), pertussis toxin. The IAP‐induced inhibition was correlated with the toxin‐catalyzed ADP‐ribosylation of a membrane GTP‐binding protein with a molecular mass of 40 kDa. Thus, the IAP‐substrate GTP‐binding protein appears to be involved in the retinoic acid‐induced differentiation of HL‐60 cells.
Orientation of the chromophore transition moment in the 4‐leaved shape model for pea phytochrome molecule in the red‐light absorbing form and its rotation induced by the phototransformation to the far‐red‐light absorbing form
Authors: Tokutomi Satoru , Mimuro Mamoru
Published: July 2002

Orientation of the chromophore transition moment in pea phytochrome (subunit molecular mass, 114 kDa) in the red‐light‐absorbing form (Pr) and its rotation induced by the phototransformation to the far‐red‐light‐absorbing (Pfr) form was studied by measuring linear dichroism of oriented phytochrome prepared by a ‘gel squeezing method’. The phytochrome in Pr showed a negative linear dichroism. On the contrary, the Pfr form showed positive linear dichroism. Assuming that the molecular axis for the two‐fold rotational symmetry in the ‘4‐leaved shape model’ proposed recently for the dimeric molecular structure of the phytochrome [(1989) FEBS Lett. 247, 139–142], is oriented in parallel with the direction of the external constraint, the average azimuthal angle between the molecular axis and the transition moment of the chromophore was calculated as 59° or 121° for Pr and 52° or 128° for Pfr. These results explain well the polarotropic responses by phytochrome observed in plant organisms.
Comparison of partial amino acid sequences of two protamine 2 variants from stallion sperm Structural evidence that the variants are products of different genes
Authors: Pirhonen Arja , Linnala-Kankkunen Annika , Mäenpää Pekka H.
Published: July 2002

Protamine 1 and two protamine 2 variants were isolated from stallion sperm and separated by acetic acid‐urea gel electrophoresis. After electroblotting onto polyvinyldifluoride filters, their amino‐terminal amino acid sequences were determined by pulse‐liquid peptide sequencing. The sequences of the two protamine 2 variants are homologous but slightly different in length and amino acid composition and indicate for the first time the existence of two different genes for this protamine species.
The yeast pyruvate kinase gene does not contain a string of non‐ preferred codons: Revised nucleotide sequence
Authors: McNally Teresa , Purvis Ian J. , Fothergill-Gilmore Linda A. , Brown Alistair J.P.
Published: July 2002

The sequence of the gene encoding pyruvate kinase from Saccharomyces cerevisiae was re‐determined because of failures with oligonucleotide‐directed mutagenesis experiments involving a region thought to contain a string of five contiguous non‐preferred codons. This region was found to be difficult to sequence and was shown to have three extra bases when compared with the published sequence [(1983) J. Biol. Chem. 258, 2193–2201]. The revised sequence demonstrates that the yeast pyruvate kinase gene does not have a cluster of non‐preferred codons, and that it therefore is not an example of the class of genes which possibly exhibit translational control by the presence of non‐preferred codons.
Phospholipase D activation by the mitogens platelet‐derived growth factor and 12‐O‐tetradecanoylphorbol 13‐acetate in NIH‐3T3 cells
Authors: Ben-Av Pazit , Liscovitch Mordechai
Published: July 2002

The effect of mitogens on phospholipase D activity was investigated in NIH‐3T3 fibroblasts by measuring the accumulation of phosphatidylpropanol, produced by phospholipase D phosphatidyl transferase activity when 1‐propanol acts as the phosphatidyl group acceptor. Platelet‐derived growth factor (PDGF) and 12‐O‐tetradecanoylphorbol 13‐acetate (TPA) stimulated phosphatidylpropanol production by the cells. The dose‐response relationships for activation of phospholipase D and stimulation of thymidine incorporation by PDGF and TPA were comparable. The possibility that activation of phospholipase D is utilized by mitogens as a trans‐membrane pathway for signalling cell growth is discussed.
Surface enhanced resonance Raman scattering (SERRS) as a probe of the structural differences between the Pr and Pfr forms of phytochrome
Authors: Rospendowski B.N. , Farrens D.L. , Cotton T.M. , Song P.-S.
Published: July 2002

Surface enhanced resonance Raman scattering (SERRS) spectra have been obtained from the active, far‐red light absorbing (Pfr) and biologically inactive (Pr) forms of phytochrome adsorbed on silver colloids. Substantial differences between the SERRS spectra of the two forms in the low and high wavenumber regions are observed using 406.7 nm wavelength excitation. These differences reinforce those seen with 413.1 nm wavelength excitation in the high wavenumber region. Simultaneously, extensive differences are observed in the SERRS obtained from the same form in the low wavenumber region using 406.7 nm, as compared with 413.1 nm wavelength excitation. The relative intensity differences observed for the two forms, and those obtained using two slightly different excitation wavelengths to illuminate the same form, suggest that some type of subtle, proteincontrolled structural variation is responsible for the spectroscopic differences. A Z → E isomerization during the Pr → Pfr phototransformation is consistent with the SERRS data, although the overall chromophore conformations are most likely conserved for the native Pr‐ and Pfr‐phytochrome species. Slight out‐of‐plane ring twisting, accompanying the Pr → Pfr photoisomerization, may be responsible for the large difference in the spectroscopic properties of the native Pr and Pfr chromophores.
Evidence from EPR spectroscopy that phosphorylation of Ser‐40 in bovine adrenal tyrosine hydroxylase facilitates the reduction of high‐spin Fe(III) under turnover conditions
Authors: Andersson Kristoffer K. , Haavik Jan , Martinez Aurora , Flatmark Torgeir , Petersson Leif
Published: July 2002

The tetrahydrobiopterin‐dependent iron‐enzyme tyrosine hydroxylase (TH) catalyses the rate‐limiting step in catecholamine biosynthesis. Electron paramagnetic resonance (EPR) data show that following phosphorylation of Ser‐40 by protein kinase A, the enzyme‐bound Fe(III), coordinated to catecholamines, can be reduced by 6‐methyl‐tetrahydropterin under turnover conditions. The 8‐fold increase in product formation upon phosphorylation can partly be explained by an increase in the fraction of active TH, by dissociation of the endogenous catecholamine inhibitors.
Retinoic acid regulation of the expression of retinoic acid receptors in wild‐type and mutant embryonal carcinoma cells
Authors: Song Soobong , Siu Chi-Hung
Published: July 2002

Retinoic acid; Retinoic acid receptor; Embryonal carcinoma
The primary structure of rat ribosomal protein S24
Authors: Chan Yuen-Ling , Paz Veronica , Olvera Joe , Wool Ira G.
Published: July 2002

The amino acid sequence of rat ribosomal protein S24 was deduced from the sequence of nucleotides in a recombinant cDNA. S24 contains 133 amino acids and has a molecular mass of 15 413. Hybridization of the cDNA to digests of nuclear DNA suggests that there are 12–16 copies of the S24 gene. The mRNA for the protein is about 600 nucleotides in length. Rat S24 is homologous to Xenopus laevis S19 and related to Halobacterium morismortui ribosomal protein S 15.
Measurement of the redox state of the ubiquinone pool in Rhodobacter capsulatus membrane fragments
Authors: Zannoni Davide , Moore Anthony L.
Published: July 2002

The dependence of the respiratory rate on the redox poise of the quinone pool was investigated in wild type and mutant membranes of Rhodobacter capsulatus. A linear relationship has been found between these two parameters only when succinate was oxidized by the bc
1 complex. Conversely, a marked nonlinear relationship was observed between the Q‐pool reduction level and the respiratory rate when O2 uptake occurred via the alternative oxidase. In addition, it was found that this latter pathway was not engaged until Q‐pool reduction level reached approximately 25%. These results are discussed within the framework of a homogeneous pool regulating both photosynthetic and respiratory fluxes.
Characterization of the β‐chain N‐terminus heterogeneity and the α‐chain C‐terminus of human platelet GPIIb
Authors: Calvete J.J. , Schäfer W. , Henschen A. , González-Rodríguez J.
Published: July 2002

Human platelet glycoprotein IIb (GPIIb) and IIIa (GPIIIa) form a Ca2+‐dependent heterodimer, the integrin GPIIb/IIIa, which functions as the fibrinogen receptor at the surface of activated platelets. GPIIB and GPIIIa are synthesized as single polypeptides from single messages and their amino acid sequences were derived from their cDNAs. The GPIIb precursor is proteolytically processed to yield the known disulphide‐bonded two‐chain (GPIIbα and GPIIbβ) covalent structure found in mature GPIIb. Our present protein chemical and mass spectrometric analyses indicate that the GPIIb precursor is proteolytically cleaved at two or three sites, to give rise to an homogeneous α‐chain (GPIIb 1–856) single disulphidebonded to one of the two β‐chains, which are present in a nearly 1:1 ratio: GPIIb β1 (860–1008), with pyroglutamic acid as its blocked N‐terminal residue; and GPIIbβ2 (872–1008), with the already known N‐terminal sequence. These results satisfy the previously observed electrophoretic size‐heterogeneity of the β‐chain, confirmed the potential cleavage sites in the junction region, and indicate a probable dual proteolytic processing of GPIIb, which may be relevant to the rest of the two‐chain α‐subunits of the integrin family.
Granulosa cells as a source and target organ for tumor necrosis factor‐α
Authors: Zolti Mati , Meirom Ruth , Shemesh Mordechai , Wollach David , Mashiach Shlomo , Shore Laurence , Ben Rafael Zion
Published: July 2002

Tumor necrosis factor (TNF‐α), a 17 kDa cytokine, is a product of activated macrophages which was recently shown to be produced by rat and bovine granulosa cells. In the present work, human granulosa cells derived from preovulatory follicles were used. It was demonstrated that human granulosa cells produce TNF‐α (5–10 units/300 000 cells per 15 h). This production was increased by addition of follicle‐stimulating hormone or by a combination of human chorionic gonadotrophin and CSF to the culture media. TNF was also found in bovine follicular fluid and the concentration was higher in the periovulatory than mid‐cycle follicles. TNF‐α was found to increase prostaglandin F‐2α production by human granulosa cells (P < 0.001). We conclude that granulosa cells are both a source and target organ for TNF‐α.
Proline residues undergo structural changes during proton pumping in bacteriorhodopsin
Authors: Gerwert Klaus , Hess Benno , Engelhard Martin
Published: July 2002

The role of proline residues in the proton pump mechanism of bacteriorhodopsin is investigated by Fourier‐transform infrared (FTIR) difference spectroscopy using [15N]‐proline labelled bacteriorhodopsin. Due to the isotopic shifts of absorbance bands, proline vibrations can be assigned to bands at 1616 cm−1 and between 1460 cm−1 and 1400 cm−1. At 1616 cm−1 a decrease of an absorbance band is observed in the intermediates K, L, M and in the dark adapted state (DA). This absorbance change can be correlated to a light induced H‐bond alteration of a X‐Pro amide carbonyl group. Isotopic shifts of difference bands between 1450 cm−1 and 1400 cm−1 are detected in K and M but are not observed in L and DA. From these frequency shifts of proline vibrations it is concluded that two proline residues undergo structural changes of their X‐Pro C‐N peptide bonds during proton pumping. The results support the suggestion that the more flexible X‐Pro C‐N bonds are used as hinges for a functional important structural motion of the protein backbone.
Autolysis of proproteinase E in bovine procarboxypeptidase A ternary complex gives rise to subunit III
Authors: Pascual Roger , Vendrell Josep , Avilés Francesc X. , Bonicel Jacques , Wicker Catherine , Puigserver Antoine
Published: July 2002

Extracts of bovine pancreatic tissue are shown by HPLC to contain two distinct ternary complexes of procarboxypeptidase A (subunit I), chymotrypsinogen C (subunit II) and either proproteinase E or subunit III. It is shown that proproteinase E in the complex generates subunit III by removal of 13 N‐terminal residues when the former is allowed to autolyze in solution or when catalytic amounts of isolated active proteinase E are added to it. Autolysis of proproteinase E was accompanied by the loss of potential activity towards specific synthetic substrates and occurred at a higher rate in pancreatic juice than in pancreatic tissue extracts, even when both were processed in the presence of serine protease inhibitors. We conclude that subunit III (also called truncated protease E) is an autolytic product of proproteinase E and not an ab initio component of the native ternary complex.
Properties and nature of a cysteine proteinase inhibitor located in keratohyalin granules of rat epidermis
Authors: Takahashi Masae , Tezuka Tadashi , Towatari Takae , Katunuma Nobuhiko
Published: July 2002

The pI4.7, 14.5 kDa hematoxylin‐stainable protein (HSP) from rat epidermis inhibited the activities of the cysteine proteinases papain, ficin, cathepsins B, H and L with similar inhibitory characteristics as recombinant cystatin‐α. Proteinases of other classes were not inhibited. The inhibitory activity of HSP was heat stable in the wide pH range of 3.0–10.0. Polyclonal antibodies against HSP cross‐reacted with cystatin‐α and the molecular mass of HSP was similar to that of cystatin‐α, though its isoelectric point was different. The in vivo location of both HSP and cystatin‐α is on keratohyalin granules in epidermis as detected by indirect immunofluorescence technique using individual antibodies. Therefore it is highly probable that HSP is a cystatin‐α derivative or a very similar proteinase inhibitor belonging to a family of cystatins.
Superinduction of ornithine decarboxylase (ODC) by actinomycin D is due to stimulation of ODC mRNA translation
Authors: Wallon U.Margaretha , Persson Lo , Heby Olle
Published: July 2002

Inhibition of transcription by treatment with actinomycin D caused superinduction of the ornithine decarboxylase (ODC) activity in Ehrlich ascites tumor cells. Experiments with cycloheximide ruled out the possibility that this superinduction was due to stabilization of ODC. Instead the ODC activity exhibited a more rapid turnover in the presence of actinomycin D (t

=36 min) than in its absence (t

= 56 min). The superinduction was found to coincide with an increased rate of ODC synthesis, as determined by measuring the incorporation of [35S]methionine into immunoreactive ODC protein. The steady‐state level of ODC mRNA was unchanged, indicating an effect on the translational efficiency.
Identification of the sites in myelin basic protein that are phosphorylated by meiosis‐activated protein kinase p44mpk
Authors: Sanghera Jasbinder S. , Aebersold Ruedi , Morrison Hamish D. , Bures Edward J. , Pelech Steven L.
Published: July 2002

Myelin basic protein serves as a convenient substrate for detection of a 44 kDa protein‐serine/threonine kinase (p44
mpk
) that is activated near the time of germinal vesicle breakdown in maturing echinoderm and amphibian oocytes. In vitro phosphorylation by purified p44
mpk
from sea star oocytes was primarily on threonine residues on a single tryptic peptide of bovine brain myelin basic protein. Amino acid composition analysis of the isolated posphopeptide revealed that it was rich in proline residues. Automated solid‐phase sequencing by Edman degradation identified the major site as Thr‐97 in the sequence NIVTPRTPPPSQGK, which corresponds to residues 91–104 in bovine brain myelin basic protein. Thr‐94 was also phosphorylated by p44
mpk
to a very minor extent.
Isolation and functional expression of pituitary peptidylglycine α‐amidating enzyme mRNA
Authors: Kato Ichiro , Yonekura Hideto , Yamamoto Hiroshi , Okamoto Hiroshi
Published: July 2002

We demonstrate that, in rat pituitary, peptidylglycine α‐amidating enzyme was encoded by at least 5 distinct mRNAs. Southern blot and ribonuclease protection analyses revealed that the mRNAs arose through alternative splicing. A variant lacking the transmembrane domain‐coding sequence was a major mRNA species for the enzyme in the pituitary. When the cDNAs were expressed in COS‐7 cells, the variant was the most efficient in producing a secretory form (37 kDA) of the enzyme.
EF‐hand motifs in inositol phospholipid‐specific phospholipase C
Authors: Bairoch Amos , Cox Jos A.
Published: July 2002

Computer sequence analysis with a linear weight matrix revealed the presence of one canonical EF‐hand motif in the δ isoform of inositol phospholipid‐specific phospholipase C and one ancestral EF‐hand in the γ form.
Electrophoretic separation by an improved method of fast myosin HCIIb‐,HCIId‐, and HCIIa‐based isomyosins with specific alkali light chain combinations
Authors: Termin Angelika , Pette Dirk
Published: July 2002

An improved method of electrophoresis under nondenaturing conditions separated three electrophoretically distinct isomyosin triplets when applied to rat fast‐twitch muscles displaying a predominance of one of the fast myosin heavy chain HCIIb, HCIId or HCIIa. The three isomyosin triplets, named FM1b‐FM3b, FM1d‐FM3d, FM1a‐FM3a, correspond to the three possible alkali light chain (I.C.) combinations (L.C.??? homodimer, LC??? LC3f heterodimer, and LC3f homodimer) with each fast HC isoform. Different proportions of these various isomyosins suggested specific affinities of light chains LC??? and LC3f for the fast heavy chain isoforms.
Footprinting studies on the sequence‐selective binding of pentamidine to DNA
Authors: Fox Keith R. , Sansom Clare E. , Stevens Malcolm F.G.
Published: July 2002

The sequence‐selective binding of pentamidine, an antimicrobial aromatic diamidine, has been investigated by footprinting studies on two different DNA fragments using DNase I, micrococcal nuclease and hydroxyl radical as probes. Each probe reveals drug‐induced protection from cleavage in AT‐rich regions. The best binding sites consist of at least 5 consecutive AT base pairs. Three or less AT pairs do not constitute a pentamidine binding site.
Identification of hematoxylin‐stainable protein in epidermal keratohyalin granules as phosphorylated cystatin α by protein kinase C
Authors: Takahashi Masae , Tezuka Tadashi , Towatari Takae , Katunuma Nobuhiko
Published: July 2002

The hematoxylin‐stainable protein (HSP) in keratohyalin granules of the newborn rat epidermis was found to have the same amino acid composition and the same inhibitory and immunological properties as cystatin α. However, only its pi value (4.7) differed from that of cystatin α (5.3). Alkaline phosphatase treatment of HSP changed its pI value from 4.7 to 5.3. This pI change was inhibited by EDTA, an inhibitor of alkaline phosphatase. Furthermore, 32P from [γ‐32P]ATP was incorporated into recombinant cystatin α by a protein kinase C (PKC) preparation in the presence of phosphatidyl serine and Ca2+ ions as co‐factors. The incorporation increased dose‐dependently with the added cystatin α and was inhibited significantly by H‐7. a specific inhibitor of PKC. SDS‐PAGE autoradiography of the 32P‐labeled proteins showed that 32P was incorporated into the cystatin α. This incorporation was not observed by the action of cAMP‐dependent protein kinase. Therefore, it is highly possible that the HSP is a phosphorylated cystatin α and that the phosphorylation is catalyzed specifically by PKC.
Desensitization is a property of the cholinergic binding region of the nicotinic acetylcholine receptor, not of the receptor‐integral ion channel
Authors: Kuhlmann Jürgen , Okonjo Kehinde O. , Maelicke Alfred
Published: July 2002

The reversible acetylcholine esterase inhibitor (−)‐physostigmine (eserine) is the prototype of a new class of nicotinic acetylcholine receptor (nAChR) activating ligands: it induces cation fluxes into nAChR‐rich membrane vesicles from Torpedo marmorala electric tissue even under conditions of antagonist blocked acetylcholine binding sites (Okonjo, Kuhlmann, Maclicke, Neuron, in press). This suggests that eserine exerts its channel‐activating property via binding sites at the nAChR separate from those of the natural transmitter. We now report that eserine can activate the channel even when the receptor has been preincubated (desensitized) with elevated concentrations of acetylcholine. Thus the conformational state of the receptor corresponding to desensitization is confined to the transmitter binding region, leaving the channel fully activatable — albeit only from other than the transmitter binding site(s).