bluuebunny/crossref_metadata_2025 huggingface dataset:
Developmental regulation of metallothionein mRNA, zinc and copper levels in rainbow trout, Salmo gairdneri
Authors: OLSSON Per‐Eric , ZAFARULLAH Muhammad , FOSTER Randy , HAMOR Thomas , GEDAMU Lashitew
Published: March 2005

The metallothionein (MT) gene expression profile was followed in rainbow trout during early embryo development and in liver and gonads during the period of sexual maturation. The hepatic MT mRNA levels increase at the end of sexual maturation in both male and female rainbow trout. Although both isoforms of MT mRNA accumulate in the liver, there is a preferential increase in MT‐A in the female liver. Concomitantly with this increase in MT there is a redistribution of zinc and copper to MT. In the juvenile female there is an abundance of MT mRNA in the ovaries. This is correlated to high levels of zinc in the MT fraction upon Sephadex G‐75 chromatography. During ovary development the MT mRNA levels and the MT‐bound zinc levels drop, with an increase in zinc being bound to high‐molecular‐mass proteins. At ovulation most of the zinc is found in the membrane portion upon centrifugation. In contrast to the ovaries, there are no apparent changes in either tracemetal distribution or MT mRNA levels during testis development. In the developing embryo there is an increase in MT‐bound copper at gastrulation. This is accompanied by an increase in both isoforms of MT mRNA. At hatch both the copper and zinc levels increase in the MT fraction, with a concomitant increase in mainly MT‐A mRNA. These findings indicate that the variations in MT mRNA levels during development are closely associated with metal regulation.
Isolation and structure determination of the intact sialylated N‐linked carbohydrate chains of recombinant human follitropin expressed in Chinese hamster ovary cells
Authors: HÅRD Karl , MEKKING Albert , DAMM Jan B. L. , KAMERLING Johannis P. , DE BOER Willem , WIJNANDS Robert A. , VLIEGENTHART Johannes F. G.
Published: March 2005

Biologically active recombinant human follitropin has been expressed in Chinese hamster ovary cells. The carbohydrate chains of the recombinant glycoprotein hormone were enzymatically released by peptide‐N4‐(N‐acetyl‐β‐glucosaminyl)asparagine amidase F. The oligosaccharides were separated from the N‐deglycosylated protein by gel‐permeation chromatography on Bio‐Gel P‐100, and fractionated by a combination of FPLC on Mono Q and HPLC on Lichrosorb‐NH2. The structures of the carbohydrate chains were determined by 500‐ or 600‐MHz 1‐NMR spectroscopy. The following types of carbohydrates occur: monosialylated diantennary (10%), disialylated diantennary (43%), disialylated tri‐antennary (5%), trisialylated tri‐antennary (13%), trisialylated tri'‐antennary (8%), and tetrasialylated tetraantennary (12%) N‐acetyllactosamine type of carbohydrate chains, all bearing exclusively α2‐3‐linked N‐acetylneuraminic acid (Neu5Ac). Previously, for pituitary follitropin mono‐, di‐, tri‐, tri′‐, and tetra‐antennary oligosaccharides containing α2‐3‐ as well as α2‐6‐linked Neu5Ac residues were reported. The bisecting GlcNAc residues present in native follitropin were not detected in the recombinant glycoprotein. Of the oligosaccharides 29% have an α1‐6‐linked Fuc residue at the asparagine‐bound GlcNAc, whereas this amount is about 50% in pituitary follitropin. In some of the tri‐, tri′‐ and tetra‐antennary oligosaccharide fractions small amounts (< 5%) of compounds were detected having one or more additional N‐acetyllactosamine units.
Dissimilar effects of the protein kinase C inhibitors, staurosporine and H‐7, on cholecystokinin‐induced enzyme secretion from rabbit pancreatic acini
Authors: EDERVEEN Antoine G. H. , VAN EMST‐DE VRIES Sjenet E. , DE PONT Jan Joep H. H. M. , WILLEMS Peter H. G. M.
Published: March 2005

The effects of two putative inhibitors of protein kinase C activity, staurosporine and H‐7, on partially purified protein kinase C and amylase secretion from isolated rabbit pancreatic acini were investigated. Staurosporine dose‐dependently inhibited amylase release stimulated by an optimal concentration of cholecystokinin C‐terminal octapeptide. At a concentration of 100 nM, the drug inhibited the secretory response to the secretagogue by approximately 50%. At the same concentration, staurosporine inhibited 12‐O‐tetradecanoylphorbol 13‐acetatestimulated enzyme secretion by 90%. Moreover, the potentiating effect of this phorbol ester on cholecystokinin‐induced amylase release was completely abolished in the presence of staurosporine. Interestingly, amylase release was decreased to the level observed with the combination of cholecystokinin and staurosporine. In contrast, H‐7, potentiated rather than inhibited cholecystokinin‐stimulated enzyme secretion, whereas the secretory response to 12‐O‐tetradecanoylphorbol 13‐acetate was not affected by the drug. Both staurosporine and H‐7, however, inhibited protein kinase C purified from exocrine pancreatic tissue. Kinetic analysis revealed that both compounds inhibited protein kinase C competitively with respect to ATP. The Ki value for staurosporine was 0.55 nM and for H‐7 13.5 μM. Our results obtained with staurosporine are in line with a stimulatory role of protein kinase C in cholecystokinin‐induced enzyme secretion from the exocrine pancreas. The results obtained with H‐7 emphasize that care has to be taken in interpreting the biological effects of this drug.
Inhibition by aminoacyl‐chloromethane protease inhibitors of superoxide anion production by phorbol‐ester‐stimulated human neutrophils
Authors: CONSEILLER Emmanuel C. , SCHOTT Dominique , LEDERER Florence
Published: March 2005

In a previous paper, we described the kinetic characteristics of the inhibition exerted by the protease inhibitors tosylphenylalanyl and tosyllysyl chloromethanes on superoxide production by human polymorphonuclear leukocytes when stimulated by phorbol esters [E. C. Conseiller & F. Lederer (1989) Eur. J. Biochem. 183, 107–114]. The results suggested the existence of a specific target which was affinity labeled by the inhibitors. The target appeared to be neither a protease, nor intracellular enzymes which can be inhibited in vitro by the chloromethanes (protein kinase C, hexokinase and enzymes of the hexose monophosphate shunt). In the present work, using the cell‐free reconstitution assay for superoxide production, we substantiate the hypothesis that the chloromethanes, target is on the plasma membrane. We have radiolabeled the membranes of cells inactivated before or after phorbol ester stimulation, using either [3H]KBH4 reduction after reaction with unlabeled inactivator, or tritiated tosylphenylalanyl chloromethane. In all cases, besides a certain background of non‐specific labeling, a radioactive band of Mr 15000 can be observed upon SDS/PAGE of radiolabeled membranes. We suggest that it is the chemical modification of this protein which is responsible for inactivation of superoxide production. Its identity and its role in the oxidative burst remain to be determined.
Interaction of acetyl‐CoA fragments with rat liver acetyl‐CoA carboxylase
Authors: RABINKOV Aaron G. , VELIKODVORSKAYA Vera V. , KOPELEVICH Viacheslav M. , TOLOSA Emma A. , GUNAR Vladimir I.
Published: March 2005

The interaction of acetyl‐CoA fragments with rat liver acetyl‐CoA carboxylase has been studied. Dephosphorylated acetyl‐CoA did not actually differ from acetyl‐CoA in its substrate properties. Non‐nucleotide analogues of the substrate, S‐acetylpantatheine and it's 4′‐phosphate, also possess substrate properties (Vmax= 1.5% and 15% of the maximal rate value of acetyl‐CoA carboxylation, respectively). The nucleotide fragment in the acetyl‐CoA molecule produces a marked effect on the thermodynamics of the substrate‐enzyme interaction, and is apparently involved in activation and appropriate orientation of the acetyl group in the active site. The better substrate properties of S‐acetylpantetheine 4′‐phosphate and the inhibitory properties of pantetheine 4′‐phosphate, compared to the unphosphorylated analogues, evidence an important role of the 5′‐β‐phosphate of 3′‐phosphorylated ADP residue in acetyl‐CoA binding to the enzyme.
Blocking of Na+/K+ transport by the MgPO4 complex analogue Co(NH3)4PO4 leaves the Na+/Na+‐exchange reaction of the sodium pump unaltered and shifts its high‐affinity ATP‐binding site to a Na+‐like form
Authors: BUXBAUM Engelbert , SCHONER Wilhelm
Published: March 2005

Inactivation of Na+/K+‐ATPase activity by the MgPO4 complex analogue Co(NH3)4PO4 leads, in everted red blood cell vesicles, to the parallel inactivation of 22Na+/K+ flux and 86Rb+/Rb+ exchange, but leaves the 22Na+/Na+‐exchange activity and the uncoupled ATP‐supported 22Na+ transport unaffected.

Furthermore, inactivation of purified Na+/K+‐ATPase by Co(NH3)4PO4 leads to a parallel decrease of the capacity of the [3H]ouabain receptor site, when binding was studied by the Mg2+/Pi‐supported pathway (ouabain‐enzyme complex II) but the capacity of the ouabain receptor site was unaltered, when the Na+/Mg2+/ATP‐supported pathway (ouabain‐enzyme complex I) was used. No change in the dissociation constants of either ouabain receptor complex was observed following inactivation of Na+/K+‐ATPase.

When eosin was used as a marker for the high‐affinity ATP‐binding site of the E1 conformation, formation of stable E′2· Co(NH3)4PO4 complex led to a shift in the high‐affinity ATP‐binding site towards the sodium form. This led to an increase in the dissociation constant of the enzyme complex with K+, from 1.4 mM with the unmodified enzyme to 280 mM with the Co(NH3)4PO4‐inactivated enzyme.

It was concluded, that the effects of Co(NH3)4PO4 on the partial activities of the sodium pump are difficult to reconcile with an α,β‐protomeric enzyme working according the Albers‐Post scheme. The data are consistent with an α2,β2 diprotomeric enzyme of interacting catalytic subunits working with a modified version of the Albers‐Post model.
Sequence of subunit c of the sodium ion translocating adenosine triphosphate synthase of Propionigenium modestum
Authors: LUDWIG Wolfgang , KAIM Georg , LAUBINGER Werner , DIMROTH Peter , HOPPE Jürgen , SCHLEIFER Karl. H.
Published: March 2005

The 30 N‐terminal amino acid residues of the purified ATPase c subunit of Propionigenium modestum have been determined. An oligonucleotide mixture was derived from this sequence and used as probe for cloning the corresponding gene in Escherichia coli. The nucleotide sequence of the gene has been determined and compared with those of ATPase c subunits from other bacteria and chloroplasts. Peculiar sequence similarities are found only at the C‐terminus between the c subunits of the ATPases from P. modestum and from Vibrio alginolyticus, another putative Na+‐translocating ATPase.
Secondary structure and dosage of soluble and membrane proteins by attenuated total reflection Fourier‐transform infrared spectroscopy on hydrated films
Authors: GOORMAGHTIGH Erik , CABIAUX Véronique , RUYSSCHAERT Jean‐Marie
Published: March 2005

Attenuated total reflection Fourier‐transform infrared spectroscopy of thin hydrated films of soluble and membrane protein included in a phospholipid bilayer is shown to provide useful information as to the secondary structure of the protein. The analysis of the amide I band of deuterated samples by Fourier self‐deconvolution followed by a curve fitting was performed by a new procedure in which all the input parameters are generated by the computer rather than by the investigator. The results of this analysis provide a correct estimation of the α‐helix and β‐sheet structure content with a standard deviation of 8.6% when X‐ray structures are taken as a reference. We also show that the orientation of the different secondary structures resolved by the Fourier self‐deconvolution/curve‐fitting procedure and of the phospholipid acyl chains can be simultaneously evaluated for membrane proteins reconstituted in a lipid bilayer. Of special interest for reconstitution of membrane proteins, the lipid/protein ratio can be accurately and quickly determined from the infrared spectrum.
myo‐Inositol oxygenase from rat kidneys
Authors: KOLLER Franz , KOLLER Elisabeth
Published: March 2005

myo‐Inositol from rat kidneys, an oligomeric protein with apparent molecular mass of about 270 kDa can be dissociated under mild conditions to structured 16.8‐kDa monomers. This dissociation can be reversed at high protein concentrations at room temperature. The corresponding apparent dimerization constant K2app = 1.38 × 105 M−1, the corresponding rate constant k2= 350 s−1· M−1, and the apparent constant for the association of dimers, K4app = 2.7 × 106 M−1.

Reassociation is significantly enhanced in the presence of the substrate and iron(II) (K2app= 9.8 × 105 M−1; K4app= 3.75 × 106 M−1, k2= 1750 s−1· M−1, at 20 mM myo‐inositol and 0.5 mM FeSO4). Under these conditions almost 100% of the original enzymatic activity was reconstituted.

Monomers, with or without bound ligands, lack catalytic activity, whereas the dimer is likely to be the elementary active enzyme‐building unit. The effects of myo‐inositol on the dimerization lead to the conclusion that this step is both mediated and facilitated by the substrate.
A novel testis‐specific 105‐kDa protein related to the 90‐kDa heat‐shock protein
Authors: ITOH Hideaki , TASHIMA Yohtalou
Published: March 2005

Brain 90‐ and 100‐kDa heat‐shock proteins (HSP90 and HSP100) were purified and antibodies against them prepared. The two antibodies were very specific and did not cross‐react with each other. In rat, immunoblotting with the anti‐HSP90 antibody showed the most abundant presence of HSP90 in testis as well as brain, compared with lung, liver, spleen, kidney, cardiac muscle, ovarium and uterus. The anti‐HSP90 antibody showed the presence of a new 105‐kDa protein in rat testis. This novel 105‐kDa protein was also detected in brain at a very low concentration but not in HeLa cells or other organs including the uterus and ovarium. The testis 105‐kDa protein was purified from rat testis; although it was clearly separable from HSP90 by two‐dimensional gel electrophoresis, Q‐Sepharose and hydroxyapatite column chromatographies, the properties of this protein were very similar to HSP90. The similarity was higher than 60% on peptide mapping with trypsin digestion, the 105‐kDa protein cross‐reacted with anti‐HSP90 antibody, both were bound similarly to heparin‐Sepharose gel and both are located in the cytosol fraction. When the 105‐kDa protein was fractionated by HPLC, a molecular mass of 195 kDa was calculated, indicating that it is composed of two identical subunits, similarly to HSP90. The 105‐kDa protein did not react with the anti‐HSP100 antibody. There was a slight similarity between the 105‐kDa protein and HSP100 on the peptide mapping. HSP100 was present in the microsomal fraction as well as in the cytosol. It is concluded that the 105‐kDa protein is a testis‐specific and HSP90‐related protein.
Subspecies of arrestin from bovine retina
Authors: WEYAND Ingo , KÜHN Hermann
Published: March 2005

Arrestin (also named 48‐kDa protein or S‐antigen) binds to photoexcited and phosphorylated rhodopsin and thereby prevents activation of cGMP phosphodiesterase (EC 3.1.4.35) by transducin in retinal rods. We report here that retinal arrestin consists of several subspecies (isoelectric points between pH 5.5–6.2), which can be separated by FPLC anion‐exchange chromatography and by FPLC chromatofocusing resulting in highly enriched individual subspecies. The entire heterogeneity pattern of arrestin is present in rod outer segments, independently of whether arrestin originated from the outer or mostly from the inner segment of rod cells. The different subspecies show a similar binding behavior to photoexcited rhodopsin phosphorylated to various degrees and they quench the cGMP phosphodiesterase activity equally well. In the presence of rod outer segment membranes, arrestin is phosphorylated light‐dependently by protein kinase C (0.2 mol phosphate/mol arrestin). This implies that the heterogeneity of arrestin is not primarily due to phosphorylation. Arrestin from different individuals exists as four isoelectric focusing patterns which occur with remarkably different frequencies in calf and cattle. The complexity of the IEF pattern does not increase with aging. Distinct subspecies of arrestin may reflect differences in their primary structure, or may result from differentially regulated post‐translational modifications in individuals.
Inhibition of factor X and factor V activation by dermatan sulfate and a pentasaccharide with high affinity for antithrombin III in human plasma
Authors: OFOSU Frederick A. , CHOAY Jean , ANVARI Nooroldin , SMITH Lindsay M. , BLAJCHMAN Morris A.
Published: March 2005

There is evidence that by catalyzing thrombin inhibition, several glycosaminoglycans can inhibit the thrombinmediated amplification reactions of coagulation and thereby delay prothrombin activation. The two amplification reactions can apparently be catalysed by endogenously generated factor Xa and thrombin. This study provides evidence which suggests that on a molar basis, an agent which can only catalyse thrombin inhibition is approximately 10 times more effective than an agent which can only catalyse factor Xa inhibition in their ability to inhibit intrinsic prothrombin activation. We determined the concentrations of each of heparin, dermatan sulfate and a pentasaccharide with high affinity for antithrombin III, to delay intrinsic prothrombin activation for at least 15s. Heparin catalyses both thrombin and factor Xa inhibition; dermatan sulfate catalyses only thrombin inhibition, while the pentasaccharide only catalyses factor Xa inhibition. Efficient prothrombin activation, which coincided with both factor X activation and factor V proteolysis, was first observed 45s after CaC12 was added to contactactivated plasma. Heparin (∼ 0.1 μM) prolonged by at least 30 s the time required for the activation of the three clotting factors to begin. The minimum concentrations of the pentasaccharide and dermatan sulfate to delay the activation of prothrombin, factors X and V were ∼ 50 μM and ∼ 5 μM, respectively. Thus, each anticoagulant could inhibit intrinsic prothrombin activation only when it inhibited activation of both factors X and V. A combination of ∼ 5 μM pentasaccharide and ∼ 0.05 μM dermatan sulfate similarly delayed the activation of all three clotting factors. Thus, while catalysis of thrombin inhibition is a more effective pathway than catalysis of factor Xa inhibition for delaying prothrombin activation, the simultaneous catalysis of thrombin and factor Xa inhibition can synergistically improve the ability of a sulfated polysaccharide to delay prothrombin activation.
Degradation of insulin in isolated liver endosomes is functionally linked to ATP‐dependent endosomal acidification
Authors: DESBUQUOIS Bernard , JANICOT Michel , DUPUIS Aline
Published: March 2005

The degradation of insulin in isolated liver endosomes and the relationships of this process with ATP‐dependent endosomal acidification have been studied. Incubation of endosomal fractions containing 125I‐insulin in isotonic KCl at 30°C resulted in a rapid loss of insulin integrity as judged from trichloroacetic acid precipitability, Sephadex G‐50 chromatography, immunoreactivity and receptor binding ability, with a maximum at pH 5–6 (t1/2: 10, 10, 6 and 6 min, respectively). On a log/log plot, the amount of acid‐soluble products generated was linearly related to the amount of insulin associated with endosomes (slope, 0.80). Upon incubation, virtually all acid‐soluble products diffused out of endosomes as judged from their solubility in aqueous poly(ethyleneglycol). In permeabilized endosomes, intact insulin was also released in part extraluminally, but only when degradation was inhibited did this release increase with lowering pH. ATP shifted the pH for maximal insulin degradation to about 7.5–8.5 and caused endosomal acidification as judged from the uptake of acridine orange and the fluorescence of internalized fluorescein‐labeled dextran and galactosylated bovine serum albumin (φ about 0.8–0.9). GTP, ITP and UTP exerted comparable effects but with lower potencies. The ability of ATP to alter the pH dependence of insulin degradation was maximal in the presence of Cl−, other anions being less effective (Br− < gluconate = SO42– < NO3−= sucrose = mannitol) and/or inhibitory (NO3−). Na+, K+ and Li+ supported more effectively ATP‐dependent insulin degradation than did choline. Divalent cations were required for the ATP effect (Mg2+= Mn2+ < Co2+ < Ni2+= Zn2 < Ca2+). Little or no effects of ATP occurred in the presence of proton ionophores such as monensin and carbonyl cyanide chlorophenylhydrazone and inhibitors of the proton ATPase such as N‐ethylmaleimide. The abilities of nucleotides, ions and inhibitors to support or inhibit ATP‐dependent insulin degradation were well correlated with their abilities to affect ATP‐dependent acidification. The acidotropic agents chloroquine and quinacrine caused a leftward shift in the pH dependence of insulin degradation and a decrease in maximal degradation; in the presence of ATP, chloroquine almost completely inhibited degradation at pH 5–9. It is concluded that ATP‐dependent acidification, in part by enhancing the dissociation of the insulinreceptor complex, is required for optimum degradation of insulin within liver endosomes.
Inositol phospholipid metabolism in human platelets stimulated by ADP
Authors: VICKERS John D. , KINLOUGH‐RATHBONE Raelene L. , PACKHAM Marian A. , MUSTARD J. Fraser
Published: March 2005

ADP‐induced changes in inositol phospholipids, phosphatidic acid and inositol phosphates of human platelets have been studied in detail, using not only 32P labelling, but also by examining changes in amounts of the phospholipids, their labelling with [3H]glycerol and their specific radioactivities; changes in the labelling of inositol phosphates in platelets prelabelled with [3H]inositol were also measured. During the early (10 s) stage of reversible ADP‐induced primary aggregation in a medium containing fibrinogen and with a concentration of Ca2+ in the physiological range (2 mM), the amounts of phosphatidylinositol 4,5‐bisphosphate (PtdInsP2) and phosphatidylinositol 4‐phosphate (PtdInsP) decreased (by 11.2 ± 4.9% and 11.3 ± 5.3%, respectively) while the labelling, but not the amount, of phosphatidic acid increased. The decreases do not appear to be attributable to the action of phospholipase C because the specific radioactivity of phosphatidic acid labelling with [3H]glycerol was not significantly increased at 10 s (although the initial specific radioactivities of the inositol phospholipids and PtdCho were more than double that of phosphatidic acid), and no increases in the labelling of inositol trisphosphate (InsP3), inositol bisphosphate (InsP2) or inositol phosphate (InsP) were detectable at 10 s. Shifts in the interconversions between PtdInsP2 and PtdInsP, and PtdInsP and PtdIns may occur. By 30 to 60 s, when deaggregation was beginning, the amounts of PtdInsP2, PtdInsP and phosphatidic acid were not different from those in unstimulated platelets, but large increases in the 32P‐labelling and [3H]glycerol labelling of phosphatidic acid were observed. Formation of [3H]inositol‐labelled InsP3 was not detectable at any time in association with ADP‐induced primary aggregation, indicating that degradation of PtdInsP2 by phospholipase C is not appreciably stimulated by ADP. These findings were compared with those obtained when platelets were aggregated by ADP in a medium without added of Ca2+ in which secondary aggregation associated with thromboxane A2 (TXA2) formation and release of granule contents occurs. At 10 s (during primary aggregation) the changes were similar in the two media. At 30 s and 60 s (during secondary aggregation in the low‐Ca2+ medium), the increases in PtdInsP2, PtdInsP and phosphatidic acid in platelets suspended in the absence of added Ca2+ were larger than those in platelets suspended in the presence of 2 mM Ca2+. In the absence of added Ca2+. ADP‐induced increases in the labelling of InsP3. InsP2 and InsP which were probably due to the effects of TXA2 since they were abolished by aspirin. Thus, ADP‐induced primary aggregation differs from that induced by agonists such as thombin and TXA2 and from the secondary phase of ADP‐induced aggregation that occurs in a medium containing an unphysiological, low concentration of Ca2+, in that aggregation occurs without evidence of degradation of PtdInsP2 by phospholipase C.
Microtubule‐associated‐protein (MAP) kinase activated by nerve growth factor and epidermal growth factor in PC12 cells
Authors: GOTOH Yukiko , NISHIDA Eisuke , YAMASHITA Takashi , HOSHI Minako , KAWAKAMI Minoru , SAKAI Hikoichi
Published: March 2005

Treatment of PC12 cells with either nerve growth factor (NGF), a differentiating factor, or epidermal growth factor (EGF), a mitogen, resulted in 7–15‐fold activation of a protein kinase activity in cell extracts that phosphorylated microtubule‐associated protein (MAP) 2 on serine and threonine residues in vitro. Both the NGF‐activated kinase and the EGF‐activated kinase could be partially purified by sequential chromatography on DEAE‐cellulose, phenyl‐Sepharose and hydroxylapatite, and were identical with each other in their chromatographic behavior, apparent molecular mass (∼40 kDa) on gel filtration, substrate specificity, and phosphopeptide‐mapping pattern of MAP2 phosphorylated by each kinase. Moreover, both kinases were found to be indistinguishable from a mitogen‐activated MAP kinase previously described in growth‐factor‐stimulated or phorbol‐ester‐stimulated fibroblastic cells, based on the same criteria. Kinase assays in gels after SDS/polyacrylamide gel electrophoresis revealed further that the NGF‐ or EGF‐activated MAP kinase in PC12 cells, as well as the EGF‐activated MAP kinase in fibroblastic 3Y1 cells resided in two closely spaced polypeptides with an apparent molecular mass of ∼40 kDa. In addition, these MAP kinases were inactivated by either acid phosphatase treatment or protein phosphatase 2A treatment. These results indicate that MAP kinase may be activated through phosphorylation by a differentiating factor as well as by a mitogen. MAP kinase activation by EGF was protein kinase C independent; it reached an almost maximal level 1 min after EGF treatment and subsided rapidly within 30–60 min. On the other hand, NGF‐induced activation of MAP kinase was partly protein kinase C dependent and continued for at least 2–3 h.
The C‐terminal part of the CDC25 gene product plays a key role in signal transduction in the glucose‐induced modulation of cAMP level in Saccharomyces cerevisiae
Authors: VAN AELST Linda , BOY‐MARCOTTE Emmanuelle , CAMONIS Jacques H. , THEVELEIN Johan M. , JACQUET Michel
Published: March 2005

In the yeast Saccharomyces cerevisiae, addition of glucose to cells grown under glucose‐derepressed conditions induces a transient rise in the intracellular level of cAMP. This modulation requires functional elements of the cAMP‐producing pathway, adenylate cyclase, ras proteins and the product of the CDC25 gene. To determine whether or not the CDC25 gene product is a transducing element in the signal‐transmission pathway leading from glucose to ras adenylate cyclase we have made use of the mutated allele RAS2Ile152 whose gene product uncouples the product of CDC25 from adenylate cyclase, but does not promotes other secondary phenotypes. The transient increase in cAMP is lost in cells lacking a functional CDC25 gene product, although they produce a normal amount of cAMP with the RAS2Ile152 gene. This result demonstrates the requirement of CDC25 for mediation of glucose signal transmission. The fact that cells grow normally on glucose in the absence of glucose‐induced cAMP signaling confirms that this signaling pathway is not essential for growth on glucose. To further analyze the role of the CDC25 gene product we have made use of truncated versions of the gene. The results show that the C‐terminal part of the gene alone is able to mediate glucose‐induced activation of the RAS adenylate cyclase pathway.
Binding of ellipticine to β‐lactoglobulin
Authors: DODIN Guy , ANDRIEUX Marc , KABBANI Hanan Al
Published: March 2005

The unprotonated form of the anti‐tumor alkaloid ellipticine binds to β‐lactoglobulins A and B from bovine milk with an affinity constant of 7 ± 3 × 105 M−1. There is one binding site/dimeric protein molecule (the stable form at medium pH). The attachment site is not the β‐barrel nor the hydrophobic site identified as the retinol site in β‐lactoglobulin but a domain located at the interface of the two monomeric units where the ligand lies close to Trp61 of both polypeptide chains. The positive binding enthalpy observed in temperature‐jump relaxation experiments is overcome by a strong entropy increase, tentatively thought to result from water release at the binding domain. Accordingly, desolvation is assumed to be the rate‐determining step in the process of ellipticine binding.
Dicyclohexylcarbodiimide‐binding proteins related to the short circuit of the proton‐pumping activity of photosystem II
Authors: JAHNS Peter , JUNGE Wolfgang
Published: March 2005

In photosynthesis of higher plants, photosystem II drives electron transfer from the water‐oxidizing manganese centre at the lumental side to bound plastoquinone at the stromal side of the thylakoid membrane. Proton release into the lumen and proton uptake from the stroma, i.e. net proton pumping, follows as consequence of vectoral electron transport. The proton pumping activity can be short circuited by covalent modification with N,N′‐dicyclohexylcarbodiimide (cHxN)2C of certain proteins in the 20–28‐kDa range. After modification, protons from water oxidation are no longer released into the thylakoid lumen, but instead transferred through the photosystem complex to protonate the photoreduced bound quinone at the other side of the membrane [Jahns, P., Polle, A. & Junge, W. (1988) EMBO J. 7, 589–594]. Here we identify the pertinent (cHxN)2C‐binding proteins by amino acid sequence analysis and localize (cHxN)2C‐binding sites within their primary structure. The proteins that are associated with the proton short circuit are light‐harvesting chlorophyll‐a/b‐binding proteins. Our results imply that in addition to acting as antennae they may serve another function: the funneling into the thylakoid lumen of protons, which are liberated in the water‐oxidizing Mn centre.
Tissue distribution and quantitation of the mRNAs for three rat tachykinin receptors
Authors: TSUCHIDA Kunihiro , SHIGEMOTO Ryuichi , YOKOTA Yoshifumi , NAKANISHI Shigetada
Published: March 2005

The family of mammalian tachykinin receptors consists of substance P receptor (SPR), neuromedin K receptor (NKR) and substance K receptor (SKR). In this investigation, tissue and regional distributions of the mRNAs for the three rat tachykinin receptors were investigated by blot‐hybrdization and RNase‐protection analyses using the previously cloned receptor cDNAs. SPR mRNA is widely distributed in both the nervous system and peripheral tissues and is expressed abundantly in the hypothalamus and olfactory bulb, as well as in the urinary bladder, salivary glands and small and large intestines. In contrast, NKR mRNA is predominantly expressed in the nervous system, particularly in the cortex, hypothalamus, and cerebellum, whereas SKR mRNA expression is restricted to the peripheral tissues, being abundant in the urinary bladder, large intestine, stomach and adrenal gland. Thus, the mRNAs for the three tachykinin receptors show distinct patterns of expression between the nervous system and peripheral tissues. Blot‐hybridization analysis in combination with S1 nuclease protection and primer‐extension analyses revealed that there are two large forms of SKR mRNA expressed commonly in the peripheral tissues, and two additional small forms of the mRNA expressed specifically in the adrenal gland and eye. These analyses also showed that the multiple forms of SKR mRNA differ in the lengths of the 5′ mRNA portions, and that the two small forms of the mRNA, if translated, encode a truncated SKR polypeptide lacking the first two transmembrane domains. This investigation thus provides the comprehensive analysis of the distribution and mode of expression of the mRNAs for the multiple peptide receptors and offers a new basis on which to interpret the diverse functions of multiple tachykinin peptides in the CNS and peripheral tissues.
Radiolabel‐transfer cross‐linking demonstrates that protein 4.1 binds to the N‐terminal region of β spectrin and to actin in binary interactions
Authors: BECKER Pamela S. , SCHWARTZ Martin A. , MORROW Jon S. , LUX Samuel E.
Published: March 2005

Erythrocyte protein 4.1 plays a major role in stabilizing the spectrin‐actin junction of the erythrocyte membrane skeleton. The particular sites on spectrin responsible for the binding of actin and protein 4.1 have not been specifically defined, although the general region of the ‘tail’ end, opposite the self‐association site, has been deduced by electron microscopy. Using a photoactivtable, radiolabel‐transfer cross‐linker, 1‐[N‐(2‐hydroxy‐5‐azidobenzoyl)‐2‐aminoethyl]‐4‐(N‐hydroxysuccin‐imidyl)‐succinate, we have determined that the binding site for protein 4.1 on spectrin resides in the N‐terminal region of β spectrin within a sequence homologous to the actin‐binding region of α actinin. Moreover, this technique provided clear evidence for a direct binding interaction between actin filaments and protein 4.1 that was confirmed by rapid‐sedimentation assays. In summary, use of radiolabel‐transfer cross‐linking has enabled assignment of the protein‐4.1‐binding site on erythrocyte spectrin and has identified a previously ill‐defined binary interaction between protein 4.1 and F‐actin.
Glycosylation of the envelope glycoprotein from a polytropic murine retrovirus in two different host cells
Authors: GEYER Hildegard , KEMPF Roland , SCHOTT Hans‐Henning , GEYER Rudolf
Published: March 2005

A polytropic recombinant retrovirus containing the envelope gene of Friend mink cell focus‐inducing virus plus the remainder of the genome of an amphoropic murineleukemia virus was propagated on mouse embryo fibroblasts and mink lung cells. Virus particles, metabolically labeled with [2‐3H]mannose, were harvested from the culture supernatants and lysed with detergents. The viral envelope glycoprotein was isolated from the lysates by immunoaffinity chromatography and purified by preparative SDS/PAGE. Oligosaccharides were liberated by sequential treatment of tryptic glycopeptides with endo‐ß‐N‐acetylglucosaminidase H and peptide‐N4‐(N‐acetyl‐ß‐glucosaminyl)asparagine amidase F and fractionated by high‐performance liquid chromatography. Individual glycans were characterized chromatographically, by methylation analyses and in part, by enzymic microsequencing.

The results demonstrated that viral glycoproteins, synthesized in mouse embryo fibroblasts, carried as major constituents partially fucosylated diantennary, 2,4‐ and 2,6‐branched triantennary and tetraantennary complex type N‐glycans with 0–4 sialic acid residues and only small amounts of high‐mannose type species with 5–9 mannose residues. As a characteristic feature, part of the complex type glycans contained additional Gal(α1–3) substituents. Glycoprotein obtained from virions propagated on mink lung cells, contained partially fucosylated diantennary and 2,4‐branched triantennary oligosaccharides with 1–3 sialic acid residues, in addition to trace amounts of high‐mannose type species with 8 or 9 mannose residues. Thus, the results reveal that predominantly, the complex type N‐glycans of the retroviral envelope glycoprotein display cell‐specific variations including differences in oligosaccharide branching, sialylation and subtitution by additional Gal(α1–3) residues.
Spectroscopic studies on the metal‐ion‐binding sites of Co2+‐substituted D‐xylose isomerase from Streptomyces rubiginosus
Authors: SUDFELDT Christoph , SCHÄFFER Andreas , KÄGI Jeremias H. R. , BOGUMIL Ralf , SCHULZ Hans‐Peter , WULFF Stephanie , WITZEL Herbert
Published: March 2005

The coordination sphere of the two metal‐binding sites/subunit of the homotetrameric D‐xylose isomerase from Streptomyces rubiginosus has been probed by the investigation of the Co2+‐substituted enzyme using electronic absorption, CD and magnetic circular dichroic spectroscopies in the visible region. The spectrum of the high‐affinity site (B site) has an absorption coefficient, ɛ545, of 18 M−1, indicating a distorted octahedral complex geometry. The spectrum of the low‐affinity site (A site) shows two absorption maxima at 505 nm and 586 nm with ɛ values of 170 M−1 cm−1 and 240 M−1 cm−1, respectively, which indicates a distorted tetrahedral or pentacoordinated complex structure as also observed for the enzyme from Streptomyces violaceoruber [Callens et al. (1988) Biochem. J. 250, 285–290] having the same feature but lower ɛ values. The first 4 mol Co2+ added/mol apoenzyme occupy both sites nearly equally. Subsequently the Co2+ located in the A site slowly moves into the B site. After equilibrium is reached, the next 4 mol Co2+/mol again occupy the A site with its typical spectrum, restoring full activity. Addition of 4 mol Cd2+ or Pb2+/mol Co4‐loaded derivative displaces the Co2+ from the B site to form the Pb4/Co4 derivative containing Co2+ in the A site, reducing activity fourfold while the Pb4/Pb4 species is completely inactive. In contrast, Eu3+ displaces Co2+ preferentially from the A site. Thus, the high‐and low‐affinity sites may be different for different for different cations. After addition of the substrates D‐xylose, D‐glucose and D‐fructose and the inhibitor xylitol the intense Co2+ A‐site spectrum of both the active Co4/Co4 derivative and the less active Pb4/Co4 derivative decreases, indicating that these compounds are bound to the A site, changing the distorted tetrahedral or pentacoordinated symmetry there to a distorted octahedral complex geometry.
On the limited ability of superoxide to release iron from ferritin
Authors: BOLANN Bjørn J. , ULVIK Rune J.
Published: March 2005

Reductive release of iron from ferritin may catalyze cytotoxic radical reactions like the Haber‐Weiss reaction. The ability of ·O−2 to mobilize Fe(II) from ferritin was studied by using the xanthine/xanthine oxidase reaction, with and without superoxide dismutase, and with bathophenanthroline sulphonate as the chelator. Not more than one or two Fe(II)/ferritin molecules could be released by an ·O−2‐dependent mechanism, even after repeated exposures of ferritin to bursts of ·O−2.

The amount of releaseable iron depended on the size and the age of the iron core, but not on the iron content of the protein shell of ferritin which was manipulated by chelators and addition of FeCl3. The kinetic characteristics of the ·O−2 mediated iron release indicated the presence of a small pool of readily available iron at the surface of the core. The very limited ·O−2 dependent release of iron from ferritin is compatible with a protective role of ferritin against toxic iron‐catalyzed reactions.
Isolation and characterization of two glycoproteins from hyaline cartilage
Authors: ANAGNOSTIDES Stavros T. , ALETRAS Alexis J. , LYMBERI Peggy , TSIGANOS Constantine P.
Published: March 2005

Two acidic glycoproteins of molecular mass 92 kDa and 56 kDa were purified from 4 M guanidine hydrochloride extracts of chick sternal cartilage, by density gradient centrifugation, ion‐exchange chromatography, gel chromatography and SDS/PAGE. The glycoproteins differed in their amino acid and carbohydrate compositions. They were identified by the immunoblotting technique in extracts of chick articular cartilage from various sites and in extracts of cartilage from other species. The proteins are synthesized by the chondrocytes and show a partial cross‐reactivity between their antisera.
Cloning and nucleotide sequence of cDNA encoding human liver serine – pyruvate aminotransferase
Authors: NISHIYAMA Kozo , BERSTEIN Gabriel , ODA Toshiaki , ICHIYAMA Arata
Published: March 2005

Cloned cDNAs for human liver serine – pyruvate aminotransferase (Ser – PyrAT) were obtained by screening of a human liver cDNA library with a fragment of cDNA for rat mitochondrial Ser – PyrAT as a probe. Two clones were isolated from 50 000 transformants. Both clones contained approximately 1.5 kb cDNA inserts and were shown to almost completely overlap each other on restriction enzyme mapping and DNA sequencing. The nucleotide sequence of the mRNA coding for human liver Ser – PyrAT was determined from those of the cDNA clones. The mRNA comprises at least 1487 nucleotides, and encodes a polypeptide consisting of 392 amino acid residues with a molecular mass of 43039 Da. The amino acid composition determined on acid hydrolysis of the purified enzyme showed good agreement with that deduced from the nucleotide sequence of the cDNA. In vitro translation of the mRNA derived from one of the isolated clones, pHspt12, as well as that of mRNA extracted from human liver, yielded a product of 43 kDa which reacted with rabbit anti‐(rat mitochondrial Ser – PyrAT) serum. Comparison of the deduced amino acid sequences of human Ser – PyrAT and the mature form of rat mitochondrial Ser – PyrAT revealed 79.3% identity. Although human Ser – PyrAT appears to be synthesized as the mature size, the 5′‐noncoding region of human Ser – PyrAT mRNA contains a nucleotide sequence which would encode, if translated, an amino acid sequence similar to that of the N‐terminal extension peptide of the precursor for rat mitochondrial Ser – PyrAT.
Properties of purified squalene‐hopene cyclase from Bacillus acidocaldarius
Authors: OCHS Dietmar , TAPPE Cord H. , GÄRTNER Peter , KELLNER Roland , PORALLA Karl
Published: March 2005

The squalene‐hopene cyclase from Bacillus acidocaldarius cytoplasmic membrane, was purified to homogeneity by solubilization with Triton X‐100, chromatography on DEAE‐cellulose, phenyl Sepharose and two gel‐filtration columns. The enzyme monomer had a molecular mass of 75 kDa. The sequence of the first 23 amino acids was determined by Edman degradation. The enzyme activity was efficiently inhibited by n‐alkyldimethylammonium halides with alkyl chain lengths between 12 and 18 C atoms. Inhibition was also observed with (5‐hydroxycarvacryl)trimethylammonium chloride 1‐piperidine carboxylate, dodecyldimethylamine N‐oxide, azasqualene and farnesol. Competitive inhibition with dodecyltrimethylammonium bromide, (5‐hydroxycarvacryl)trimethylammonium chloride 1‐piperidine carboxylate and dodecyldimethylamine N‐oxide was demonstrated by Lineweaver‐Burk plots.
Cloning and sequence analysis of the genes encoding the dihydrolipoamide acetyltransferase and dihydrolipoamide dehydrogenase components of the pyruvate dehydrogenase multienzyme complex of Bacillus stearothermophilus
Authors: BORGES Adolfo , HAWKINS Christopher F. , PACKMAN Leonard C. , PERHAM Richard N.
Published: March 2005

A 2641‐bp EcoRl fragment of DNA that encodes the C‐terminal part of the dihydrolipoyl acetyltransferase (E2) component and the dihydrolipoamide dehydrogenase (E3) component of the pyruvate dehydrogenase complex of Bacillus stearothermophilus has been cloned in Escherichia coli. Its nucleotide sequence was determined. A 705‐bp truncated open reading frame was located at the 5′ end of the insert which, together with the 588‐bp truncated open reading frame at the 3′ end of another EcoRI fragment of B. stearothermophilus DNA previously cloned and sequenced [Hawkins, C. F., Borges, A. & Perham, R. N. (1990) Eur. J. Biochem. 191, 337–446], was identified as the gene, pdhC, encoding the E2 polypeptide chain. Direct sequence analysis of the purified E2 chain confirmed that the two EcoRl fragments are adjoining in the B. stearothermophilus genome. The E3 gene, pdhD, begins just 4 bp downstream from the stop codon of the pdhC gene. The amino acid sequences deduced from the pdhC and pdhD genes correspond to proteins of 427 amino acids (E2, Mr 46265) and 469 amino acids (E3, Mr 49193), respectively. Both genes are preceded by potential ribosome‐binding sites and the E3 gene is followed by a stem‐loop structure characteristic of rho‐independent transcription terminators. The B. stearothermophilus E2 and E3 chains exhibit substantial sequence similarity with the corresponding subunits of other 2‐oxo‐acid dehydrogenase multienzyme complexes. The cloning and sequence analysis described here complete the description of the gene cluster (pdhA, B, C and D) which encodes the B. stearothermophilus pyruvate dehydrogenase multienzyme complex.
Involvement of the hydrophobic patch of azurin in the electron‐transfer reactions with cytochrome c551 and nitrite reductase
Authors: van de KAMP Mart , SILVESTRINI Maria Chiara , BRUNORI Maurizio , Van BEEUMEN Jozef , HALI Frits C. , CANTERS Gerard W.
Published: March 2005

The electron‐transfer reactions of site‐specific mutants of the blue copper protein azurin from Pseudomonas aeruginosa with its presumed physiological redox partners cytochrome c551 and nitrite reductase were investigated by temperature‐jump and stopped‐flow experiments. In the hydrophobic patch of azurin Met44 was replaced by Lys, and in the His35 patch His35 was replaced by Phe, Leu and Gln. Both patches were previously thought to be involved in electron transfer. 1H‐NMR spectroscopy revealed only minor changes in the three‐dimensional structure of the mutants compared to wild‐type azurin. Observed changes in midpoint potentials could be attributed to electrostatic effects. The slow relaxation phase observed in temperature‐jump experiments carried out on equilibrium mixtures of wild‐type azurin and cytochrome c551 was definitively shown to be due to a conformational relaxation involving His35. Analysis of the kinetic data demonstrated the involvement of the hydrophobic but not the His35 patch of azurin in the electron transfer reactions with both cytochrome c551 and nitrite reductase.
Triiodothyronine increases rat apolipoprotein A‐I synthesis and alters high‐density lipoprotein composition in vivo
Authors: APOSTOLOPOULOS Jim J. , MARSHALL Jeannette F. , HOWLETT Geoffrey J.
Published: March 2005

The effects of altered serum 3,3′,5‐triiodothyronine levels on rat lipoprotein metabolism were examined. Daily injections of the hormone (50 μg/100 g body mass) over a period of six days led to an increase of 6.4‐fold in the hepatic mRNA level for apolipoprotein(apo)A‐I, and a 21% increase in serum apoA‐I levels. 12 h after a single injection of 3,3′,5‐triiodothyronine the rate of [14C]leucine incorporation into apoA‐I increased 2.1 fold. Conversely, in hypothyroid rats there was a decrease in hepatic mRNA levels for apoA‐I and a decreased rate of [14C]leucine incorporation into apoA‐I. The increase in hepatic apoA‐I mRNA levels following 3,3′,5‐triiodothyronine treatment occurred prior to significant changes in serum triacylglycerol levels. High‐density lipoprotein (HDL) particles isolated from the serum of hyperthyroid rats were smaller and enriched in apoA‐I compared to apoA‐IV and apoE. Similar changes in HDL composition were observed following in vitro incubations of normal rat serum with purified rat apoA‐I. The results suggest that during altered thyroid status, changes in serum HDL size and composition occur in association with significant changes in apoA‐I gene expression.