bluuebunny/crossref_metadata_2025_split huggingface dataset:
Processing of human choriogonadotropin and its receptors by cultured pig Leydig cells
Authors: BERNIER Michel , CLERGET Magali , MOMBRIAL Charlotte F. , SAEZ José M. 
Published: March 2005

We have examined the process by which human choriogonadotropin/luteinizing hormone (hCG/LH) receptors are regulated in cultured porcine Leydig cells. Treatment of Leydig cells with human choriogonadotropin, cholera toxin, forskolin and cyclic 8‐bromoAMP (8‐BrcAMP) produced a loss of surface receptors without modification of the binding affinity. This negative regulation of the number of receptors mediated by maximal concentrations of hCG was higher than that induced by the other agents. The extent of receptor loss in cells treated with increasing concentrations of hCG was highly correlated with their capacity to stimulate cAMP production. However, there was little correlation between down‐regulation and cAMP production of these cells treated by hCG plus forskolin or cholera toxin plus forskolin, where a synergistic cAMP production was obtained. Following exposure of Leydig cells to both hCG and 8‐BrcAMP, the surface receptor disappearance began after an initial lag period of about 6–8 h. Thereafter a 50% loss of surface receptor was observed in the next 8‐h incubation. Monensin with hCG shortens this lag period before initiation of receptor loss. Kinetic studies with 125I‐hCG, in the presence or absence of monensin, showed that the half‐life of the receptor‐bound hormone complexes at the cell surface was 10.5 h and 8 h respectively. Therefore, the steady state of the surface receptor during the lag phase of 8 h is probably related to recycling of internalized receptors and/or translocation of preformed receptors. Cycloheximide and actinomycin D inhibit hCG‐mediated and 8‐BrcAMP‐mediated down‐regulation. Cycloheximide lengthens ligand‐receptor complexes at the surface by slowing down the rate of internalization (half‐life of 20 h), but this mechanism is not enough per se to explain the effect of cycloheximide. Pulses of hCG or 8‐BrcAMP for 4 h and 8 h sufficed to induce nearly maximal down‐regulation. However, it was possible to attenuate this triggering effect by adding cycloheximide after pulse of the cells. Thus, even after removal of the triggering agent (hCG or 8‐BrcAMP), the loss of surface receptor could be triggered by a protein‐sensitive signal.Taken as a whole these results indicate that a coordinated interaction is involved in the cell‐surface hCG/LH receptor regulation. The apparent steady state of the number of receptors during the first hours of stimulation passed through a reuptake of internalized receptors. It is also proposed that the stimulatory agent may deplete the surface pool of receptors by progressively preventing the return of internalized receptors to the surface by a process sensitive to the newly synthesized proteins, rather than by increasing their rate of internalization.
Cytochalasin D alters the rate of synthesis of some HEp‐2 cytoskeletal proteins
Authors: TANNENBAUM Janet 
Published: March 2005

1. The most abundant proteins of HEp‐2 cells were resolved by two‐dimensional gel electrophoresis. The protein spots corresponding to several cytoskeletal proteins (vimentin, α‐tubulin, β‐tubulin, α‐actinin, tropomyosins, and cytokeratins) were identified by comigration with protein markers or by immunological techniques.2. After treatment of HEp‐2 cells with 0.2 μM or 2.0 μM cytochalasin D for 20 h, radioautograms of two‐dimensional gel patterns of lysates from cells pulse‐labeled with [35S]methionine indicated that the drug altered the rate of synthesis of some proteins. The relative rate of synthesis of the identified cytoskeletal proteins was measured. Synthesis of α‐actinin, the higher‐molecular‐mass pair of tropomyosins and actin were similarly increased with cytochalasin D treatment, suggesting coordinate induction. Vimentin and tubulin synthesis was depressed. One cytokeratin exhibited an increase in synthesis comparable to actin, another was increased to a lesser extent and one was decreased.
Translocation of proteins into rat liver mitochondria
Authors: ONO Hideyu , TUBOI Syozo 
Published: March 2005

The precursor polypeptides of a large subunit of succinate dehydrogenase and ornithine aminotransferase (the enzymes which are located in the mitochondrial inner membrane and matrix respectively) were synthesized as a larger molecular mass than their mature subunits, when rat liver RNA was translated in vitro. These precursor polypeptides were also detected in vivo in ascites hepatoma cells (AH‐130 cells). When the 35S‐labeled precursor polypeptides were incubated with isolated rat liver mitochondria at 30°C in the presence of an energy‐generating system, these two precursors were converted to their mature size and the 35S‐labeled mature‐size polypeptides associated with mitochondria. Furthermore, these mature‐size polypeptides were recovered from their own locations, the inner mitochondrial membrane and the matrix.The precursor of ornithine aminotransferase incubated with rat liver mitochondria at 0°C was specifically and tightly bound to the surface of the mitochondria even in the presence of an uncoupler of oxidative phosphorylation. This precursor, bound to the mitochondria, was imported into the matrix when the mitochondria were reisolated and incubated at 30°C in the presence of an energy‐generating system, suggesting that a specific receptor may be involved in the binding of the precursor.The processing enzyme for both precursor polypeptides seemed to be located in the mitochondrial matrix and was partially purified from the mitochondria. A metal‐chelating agent strongly inhibited the processing enzyme and the inhibition was recovered by the addition of Mn2+ or Co2+.
Target size analysis of opioid receptors
Authors: OTT Susanne , COSTA Tommaso , WÜSTER Michael , HIETEL Bernhard , HERZ Albert 
Published: March 2005

Target size analysis of opioid receptor is complicated by the presence of multi‐exponential inactivation curves. Irradiation of intact frozen tissue proved essential to eliminate such artifacts, due to indirect irradiation effects. Upon irradiation condition, opioid binding activity was inactivated in a single mono‐exponential manner.Identical inactivation curves were obtained for μ, δ and κ binding activities in brain membranes from rat, guinea‐pig and frog and in NG 108‐15 cells: the molecular mass obtained was 98 ± 2 kDa. However, when opioid binding was assayed in the presence of Na+, Mg2+ and GTP, the molecular mass was found to be only 56 ± 4.4 kDa.We suggest that the opoid recognition site comprises a unit of 56 kDa and that in the absence of Na+, Mg2+ and GTP an additional membrane component of 40–44 kDa is necessary for high‐affinity opioid binding.
Structure of initiation factor eIF‐3 from rat liver
Authors: BEHLKE Joachim , BOMMER Ulrich‐Axel , LUTSCH Gudrun , HENSKE Annemarie , BIELKA Heinz 
Published: March 2005

On the basis of hydrodynamic, electron microscopic and biochemical investigations a new model of the structure of initiation factor eIF‐3 is proposed.From sedimentation and diffusion coefficients of 16.35 S and 2.13 × 10−7 cm2/s, respectively, as well as from sedimentation equilibrium measurements, a molecular mass of about 650 kDa was determined for isolated eIF‐3. This is in agreement with molecular mass estimations by sodium dodecyl sulphate gel electrophoresis. A partial specific volume of 0.723 cm3/g was determined by means of the amino acid composition and the specific volume increments of the amino acids. From this value and from the molecular mass, a volume of 780 nm3 was calculated for eIF‐3.In electron micrographs of isolated eIF‐3, images with triangular profiles and side lengths of 14 nm, 16 nm, and 17 nm have been observed. Taking into account the calculated volume and considering the triangular image as one face of the particle, it is suggested that eIF‐3 has the shape of a flat triangular prism with a height of about 7 nm and the above‐mentioned side‐lengths.This model is in agreement with results of electron microscopic investigations of eIF‐3 in native small ribosomal subunits [Lutsch, G., Benndorf, R., Westermann, P., Bommer, U.‐A. & Bielka, H. (1986) Eur. J. Cell Biol. 40/2, in press]. The high frictional ratio of about 1.7 also supports eIF‐3 to be rather of a flat than of a globular shape.
Purification and characterization of a neutral processing mannosidase from calf liver acting on (Man)9(GlcNAc)2 oligosaccharides
Authors: SCHWEDEN Jürgen , LEGLER Günter , BAUSE Ernst 
Published: March 2005

A processing mannosidase acting on (Man)9(GlcNAc)2 oligosaccharides, Man9 mannosidase, has been purified 2190‐fold from calf liver crude microsomes by a four‐step procedure involving (a) differential salt/detergent extraction, (b) affinity chromatography on AH‐Sepharose 4B with N‐5‐carboxypentyl‐1‐deoxy‐mannojirimycin as ligand, (c) ConA‐Sepharose and (d) DEAE‐Sephacel chromatography. (Man)9 mannosidase has a subunit molecular mass of 56 kDa and does not bind to ConA‐Sepharose, indicating the absence of high‐mannose oligosaccharides. The enzyme has a pH optimum close to pH 6.0 and requires divalent cations for activity, Ca2+ being most effective. It is inhibited by 1‐deoxymannojirimycin (dMM), N‐methyl‐dMM and N‐5‐carboxypentyl‐dMM with Ki= 7 μM, 75 μM, and 140 μM, respectively.Man9 mannosidase cleaves three of the four α1,2‐linked mannose residues from the (Man)9(GlcNAc)2 oligosaccharide, does not hydrolyse the remaining (Man)6(GlcNAc)2 structure and is not active against aryl α‐mannosides. This pronounced substrate specificity points to the participation of Man9 mannosidase in the N‐linked processing pathway and, in addition, clearly distinguishes this enzyme from the mannosidases reported previously. As Man9 mannosidase appears to act in the processing sequence immediately after the three glucose residues have been removed from the (Glc)3(Man)9(GlcNAc)2 intermediate, we assume that the enzyme is located in the endoplasmic reticulum.
Activation of phosphorylase kinase through autophosphorylation by membrane component phospholipids
Authors: NEGAMI Akira I. , SASAKI Hiromasa , YAMAMURA Hirohei 
Published: March 2005

Phosphatidic acid (PtdOH) has been shown not only to stimulate autophosphorylation and autoactivation of phosphorylase kinase of rabbit skeletal muscle but also to decrease the apparent Ka for Ca2+ on autophosphorylation sharply [Negami et al. (1985) Biochem. Biophys. Res. Commun. 131, 712–719]. In this study we investigated the interaction between PtdOH and other phospholipids on autophosphorylation and autoactivation of this enzyme.
Acidic phospholipids, such as phosphatidylserine (PtdSer), phosphatidylinositol (PtdIns) and PtdOH, stimulated this reaction about 2–4‐fold, and the approximate Ka values of this reaction were 10 μg/ml, 6.3 μg/ml and 30 μg/ml respectively.
The molar ratio of PtdIns and PtdSer with maximal effect on autophosphorylation was about 1:1. Under these conditions PtdOH stimulated the initial velocity of autophosphorylation about 5.2‐fold.
When fully autophosphorylated, about 12–13 mol phosphate per tetramer (αβγδ) were incorporated in the presence of mixed acidic phospholipids (PtdOH:PtdIns:PtdSer = 2:1:1), which was about twice as much as values observed without effectors.
In the presence of mixed acidic phospholipids there was a concomitant enhancement of kinase activity, about 30–40‐fold at pH 6.8 and 2.5–3‐fold at pH 8.2.
Mixed acidic phospholipids sharply decreased an apparent Ka for Ca2+ from 4 × 10−5 M to 8 × 10−7 M.
With mixed acidic phospholipids as effectors this autophosphorylation occurred through an intramolecular mechanism. Based on these results, autophosphorylation and autoactivation of phosphorylase kinase in the presence of acidic phospholipids may account for an important regulatory mechanism of glycogenolysis in muscle contraction.
Binding of gossypol to purified tubulin and inhibition of its assembly into microtubules
Authors: MEDRANO Francisco Javier , ANDREU Jose Manuel 
Published: March 2005

Gossypol is a polyphenolic pigment, which is employed as a male antifertility drug. It inhibits, among other reported effects, the growth of cultured mammalian cells, spermiogenesis, flagellar motility in Trypanosoma and sperm, dynein ATPase and the lactate dehydrogenase X (LDH‐X) isozyme.
We have characterized the non‐covalent binding of gossypol to purified calf brain tubulin in 10 mM phosphate buffer, 0.1 mM GTP pH 7.0 at 25°C. Equilibrium measurements were performed by difference spectroscopy. A peak at 435 nm was produced by the perturbation of gossypol light absorption upon binding to tubulin. The experimental isotherm was fitted by 1.96 ± 0.06 gossypol binding sites per tubulin molecule, with identical apparent equilibrium binding constants of (7.5 ± 1.1) × 104 M−1. The complex formed could be separated from free gossypol by gel chromatography.
Binding of gossypol was independent of the presence of 0.1 mM GTP in the buffer. Gossypol did not affect the binding of ligands to the colchicine site. Gossypol interacted with vinblastine but apparently did not bind to the vinblastine sites of tubulin. Gossypol did not displace anilinonaphthalene sulphonate (ANS) bound to tubulin, but caused a strong (fivefold) quenching of its fluorescence. This indicated that gossypol probably binds in the vicinity of the ANS site of tubulin.
Gossypol inhibited in vitro microtubule assembly at the same concentration range employed in the binding studies. An increase in the critical protein concentration required for polymerisation was observed, most simply interpreted by a stoichiometric mechanism. Gossypol did not induce any noticeable distortion of the microtubules observed under the electron microscope. This compound constitutes a new tubulin ligand and an inhibitor of microtubule assembly in vitro.
Characterization of functional domains in human tissue‐type plasminogen activator with the use of monoclonal antibodies
Authors: HOLVOET Paul , LIJNEN Henri Roger , COLLEN Désiré 
Published: March 2005

Two murine monoclonal antibodies (MA‐2G6 and MA‐1C8), secreted by hybridomas obtained by fusion of myeloma cells with spleen cells from mice immunized with human tissue‐type plasminogen activator (t‐PA), inhibited the activity of t‐PA on fibrin plates. MA‐2G6 inhibited the amidolytic activity of t‐PA and did not react with t‐PA in which the active‐site serine was blocked with diisopropylfluorophosphate nor with t‐PA in which the active‐site histidine was alkylated by reaction with d‐Ile‐Pro‐Arg‐CH2Cl. This indicated that MA‐2G6 is directed against an epitope covering the active site of t‐PA. MA‐1C8 did not inhibit the amidolytic activity of t‐PA, but abolished both the binding of t‐PA to fibrin and the stimulatory effect of fibrin on the activation of plasminogen by t‐PA. Thus MA‐1C8 is directed against an epitope which covers the fibrin‐binding site of t‐PA. The A and B chains of partially reduced two‐chain t‐PA were separated by immunoadsorption on immobilized MA‐1C8 and MA‐2G6. The purified B chain reacted with MA‐2G6 but not with MA‐1C8 and activated plasminogen following Michaelis‐Menten kinetics with kinetic constants similar to those of intact t‐PA (Km= 100 μM and kcat= 0.02 s−1). However, fibrin or CNBr‐digested fibrinogen did not stimulate the activation of plasminogen by the B chain.The purified A chain reacted with MA‐1C8 but not with MA‐2G6. It bound to fibrin with an affinity similar to that of intact t‐PA but did not activate plasminogen.It is concluded that the active center of t‐PA is located in the B chain and the fibrin‐binding site in the A‐chain. Both functional domains are required for the regulation by fibrin of the t‐PA‐mediated activation of plasminogen.
Biosynthesis and intracellular processing of carbonic anhydrase in Chlamydomonas reinhardtii
Authors: TOGURI Toshihiro , MUTO Shoshi , MIYACHI Shigetoh 
Published: March 2005

Carbonic anhydrase (CA) of Chlamydomonas reinhardtii is a glycoprotein of 35 kDa which is localized outside the plasma membrane. The activity of CA was increased when the CO2 concentration during photoautotrophic growth was decreased to air level. After decreasing the CO2 concentration from 4% to 0.04%, several polypeptides including CA were induced continuously or transiently. To investigate the biosynthesis and intracellular processing of CA, the cells of wall‐less mutant CW‐15, which secretes CA into the culture medium, were pulse‐labeled with radioactive arginine, chased, and radioactive proteins were immunoprecipitated with anti‐CA serum.A 42‐kDa polypeptide with isoelectric point (pI) of 7.1–7.3 was first synthesized. Within 5 min the molecular mass of this polypeptide was decreased to 35 kDa and it was then secreted into the culture medium within 30 min. This indicates that the former is the precursor form and the latter the mature form of CA. The primary translation product from poly(A)‐rich RNA in a cell‐free reticulocyte lysate system from a rabbit was a 38‐kDa polypeptide. This was cotranslationally converted into the 42‐kDa precursor in vitro in the presence of dog pancreatic microsomal membranes. As the 42‐kDa precursor had a high affinity to concanavalin A, it was assumed to have a high‐mannose‐type oligosaccharide. The mature enzyme had a pI of 6.1–6.2 and was composed of more than two isoforms, which had a complex‐type oligosaccharide with low affinity to concanavalin A. Chemical deglycosylation of the mature enzyme by trifluoromethanesulfonic acid indicated that the molecular mass of the polypeptide moiety was 32 kDa and the difference between this and the primary translation product suggests that cleavage of the polypeptide occurs during its biosynthesis.
Pectinesterase
Authors: MARKOVIČ Oskar , JÖRNVALL Hans 
Published: March 2005

The first primary structure of a pectinesterase has been determined by analysis of the main form of this plant enzyme from tomatoes. The analysis was untraditional in the sense that few data were obtained by frequently used methods. Thus, digestion with trypsin, Glu‐specific protease and several other enzymes gave limited cleavages and highly insoluble products. Instead, the determination was to a large extent based on peptides derived from chemical cleavages with CNBr at Met, N‐chlorosuccinimide at Trp and hydroxylamine at Asn‐Gly, plus an enzymatic cleavage at modified cysteine residues after chemical derivatizations.The structure shows the protein chain to be 305 residues long, with four half‐cystine residues and five tryptophan residues. The N‐terminus has a free α‐amino group (from isoleucine). Two types of residue, tyrosine and histidine, have been functionally implied in the catalytic activity of the enzyme. Tyrosine is common in the protein (at 18 positions). However, only two histidine residues are found, and both are close to tyrosine residues in the primary structure (His‐128 separated by one intervening residue from Tyr‐126, and His‐269 adjacent to Tyr‐268), defining segment(s) of possible interest in relation to the active site.
Nucleotide sequence of the pntA and pntB genes encoding the pyridine nucleotide transhydrogenase of Escherichia coli
Authors: CLARKE David M. , LOO Tip W. , GILLAM Shirley , BRAGG Philip D. 
Published: March 2005

A 3240‐base‐pair DNA fragment spanning the pyridine nucleotide transhydrogenase (pnt) genes of Escherichia coli has been sequenced. The sequence contains two open‐reading frames, pntA and pntB of 1506 and 1386 base pairs, coding for the transhydrogenase α and β subunits, respectively. The coding sequences are preceded by a promoter‐like structure and are most likely co‐transcribed. Each coding sequence is preceded by a Shine‐Dalgarno sequence.The amino‐terminal amino acid sequences were determined from the purified α and β subunits of the transhydrogenase. These sequences agree with those predicted from the nucleotide sequences of the pntA and pntB genes. The predicted relative molecular masses of 53906 (α) and 48667 (β) are close to the values obtained by analysis of the subunits by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. Several hydrophobic regions large enough to span the cytoplasmic membrane were observed in each subunit. These results indicate that transhydrogenase is an intrinsic membrane protein.
Interaction of vasoactive intestinal peptide (VIP) and N‐terminally modified VIP analogs with rat pancreatic, hepatic and pituitary membranes
Authors: ROBBERECHT Patrick , COY David H. , DE NEEF Philippe , CAMUS Jean‐Claude , CAUVIN Annick , WAELBROECK Magali , CHRISTOPHE Jean 
Published: March 2005

Six vascoactive intestinal peptide (VIP) analogs inhibited [125I]iodo‐VIP and [125I]iodo‐helodermin binding to high‐affinity VIP receptors in rat hepatic membrances. They also stimulated adenylate cyclase activity through these receptors, their decreasing order of potency being VIP > [d‐Ala4]VIP > [d‐Asp3]VIP > [d‐Ser2]VIP > [d‐His1]VIP > [d‐Phe2]VIP > [d‐Arg2]VIP, with the latter two peptides acting as partial agonists only.All VIP analogs tested on rat pancreatic membranes were able to stimulate adenylate cyclase, their order of potency being very similar to that observed on hepatic membranes. [d‐Ser2]VIP, [d‐His1]VIP, [d‐Arg2]VIP and [d‐Phe2]VIP were partial agonists with an intrinsic activity of, respectively, 0.8, 0.7, 0.35 and 0.09 as compared to that of VIP = 1.0. [d‐Phe2]VIP competitively and selectively inhibited VIP‐stimulated adenylate cyclase activity (Ki= 0.1 μM).On male rat anterior pituitary homogenates the order of potency of the peptides was VIP > [d‐Ala4]VIP > [d‐Asp3]VIP > [D‐Ser2]VIP > [D‐His1] and [d‐His1]VIP acted as partial agonists. Besides, [d‐Phe2]VIP and [d‐Arg2]VIP were inactive as well as unable to inhibit VIP‐stimuated adenylate cyclase activity.These results indicated that (a) the efficacy of VIP receptor/effector coupling depended on the tissue tested; (b) the possibility exists to design a VIP antagonist by appropriate modification in the N‐terminal moiety of the molecule.