bluuebunny/crossref_metadata_2025 huggingface dataset:
Ribonucleases of diverse specificities in rabbit brain nuclei
Authors: PANTOPOULOS Kostas , GEORGATSOS John G.
Published: March 2005

A salt extract of rabbit brain nuclei contains three endoribonucleases, designated RNases Y, A and R, which produce acid‐soluble products when incubated at near‐neutral pH in the absence of metal ions. RNases Y and A yield products with the monoesterified phosphate at the 3′ position, through 2′,3′‐(cyclic)phosphate intermediates. Oligonucleotides terminating with a 2′,3′‐(cyclic)phosphate are the end‐products of the action of RNase R. Double‐stranded substrates are highly resistant to the action of all enzymes. On the basis of limited hydrolysis of end‐labelled 5S RNA, the three enzymes differ in their preference for the susceptible phosphodiester bond. Thus, RNase Y hydrolyses preferentially the YpN bond, RNase A the ApN bond and RNase R the RpU bond where R is guanosine in most cases. The advantages and disadvantages of using homopolyribonucleotides and dephosphorylated dinucleotides and trinucleotides in determining various aspects of the specificity of RNases are discussed.
The components of merocyanine‐540 absorption spectra in aqueous, micellar and bilayer environments
Authors: KASCHNY Peter , GOÑI Félix M.
Published: March 2005

Spectral‐data‐processing and curve‐fitting techniques have been applied to the decomposition of merocyanine‐540 absorption spectra in aqueous, micellar and bilayer environments. The various resolved component bands have been assigned to dye monomers, dimers, or larger aggregates, either in polar or non‐polar environments. The analysis of spectral parameters (λmax and integrated intensity) of the overall spectra and of each component has revealed that merocyanine 540 is a useful probe in studies of membrane structure and dynamics using visible‐absorption spectroscopy. In particular, the monomer λmax and the integrated intensity, i.e. area, of the dimer population are very useful in this respect. The monomer λmax is especially sensitive to polarity changes and is thus useful, e.g. in the precise determination of critical micellar concentrations. The fractional area of the dimer increases with the packing density of the phospholipid‐hydrocarbon region near the interface and is thus very sensitive to changes in vesicle curvature and to the presence of sterols or intrinsic polypeptides in the bilayer.
Interaction between the carboxyl groups of Asp127 and Asp129 in the active site of Escherichia coli phosphofructokinase
Authors: LAINE Romuald , DEVILLE‐BONNE Dominique , AUZAT Isabelle , GAREL Jean‐Renaud
Published: March 2005

The pH dependence of the enzymic properties of the phosphofructokinase from Escherichia coli was compared to those of two mutants in which one carboxyl group of the active site has been removed from either Asp127 or Asp129. All measurements of activity were made in the presence of allosteric activator ADP or GDP to eliminate any cooperative process. Asp129 is a crucial residue for the activity of phosphofructokinase since its conversion to Ser decreases the catalytic activity by 2—3 orders of magnitude in both the forward and reverse reactions, but the ionization of Asp129 is not directly related the pH dependence of phosphofructokinase activity. This pH dependence is however modified by the Asp129 → Ser mutation, which decreases the pK of another residue, Asp127, by as much as pH of 1.5. The side chain of Asp127 has the catalytic role proposed earlier: its deprotonated form acts as a base in the forward reaction, and its protonated form acts as an acid in the reverse reaction. The protonated form of Asp127 is also required for the binding of fructose 1,6‐bisphosphate. The electrostatic interaction between the carboxyl groups of Asp127 and Asp129 seems different in free phosphofructokinase to that in enzyme/substrate complexes, suggesting that a conformational change occurs upon substrate binding. The pH dependence of phosphofructokinase activity involves one other ionizable group with a pK of approximately 6 which does not belong to the side chains of Asp127 or Asp129.
Differential scanning calorimetry of chicken erythrocyte nuclei
Authors: GIARTOSIO Anna , WANG Changqing , D'ALESSIO Silvana , FERRARO Anna , ALTIERI Fabio , EUFEMI Margherita , TURANO Carlo
Published: March 2005

Investigation of structural features of native chromatin requires the use of intact nuclei, a turbid material which cannot be analyzed by optical methods. Differential scanning calorimetry does not require optically clear samples and has been proved by a number of authors to be a powerful tool in this field of study. By this technique, chicken erythrocyte nuclei were found to undergo at least four thermal transitions, centered at 59, 74, 88 and 98°C. The highest temperature transition is strongly dependent on age and storage conditions of the nuclei. Adequate storage conditions overcame this problem and reproducible scans were obtained over a period of several months. This technical improvement has permitted the reconsideration of the occurrence of the fourth calorimetric transition, previously believed to be displayed only in replicating nuclei. Evidence gathered in the presence of perturbants and possible ligands allows the assignment of the four transitions to a nuclear protein scaffold, histones, nucleosomal DNA and a superstructured form of DNA. Moreover, it suggests that the higher‐order structure is stabilized by fibronectin‐like proteins.
Control of human coagulation by recombinant serine proteases
Authors: CITARELLA Franca , AIUTI Alessandro , LA PORTA Claudia , RUSSO Giulia , PIETROPAOLO Concetta , RINALDI Monica , FANTONI Antonio
Published: March 2005

The availability of engineered serine proteases allows one to study the activation, substrate specificity and regulation of human coagulation and fibrinolytic activities. Human coagulation factor XII is composed of the protease catalytic region at the C‐terminus, a hinge proline‐rich region and regulatory domains at the N‐terminus. From cDNA clones coding for factor XII, two DNA molecules were constructed, one being full length and the other being deleted of exons coding for the regulatory domains. Engineered factor‐XII cDNA species were inserted by a homologous recombination technique into vaccinia viruses, which were used to infect the human hepatoma cell line HepG2. Two recombinant proteins were prepared from the culture media and identified by their antigenic properties and electrophoretic mobilities. The recombinant protein of larger size was identified as the full‐length factor XII of 80 kDa and its specific activities and activation patterns, determined both by the coagulation and the amidolytic assays, are very similar to these of native human factor XII. The recombinant protein of smaller size was identified as a 319‐amino‐acid‐deleted factor‐XII protein of 32 kDa, containing only the entire protease region and part of the proline‐rich hinge. This protein was expected to be the ‘minimal’ portion of factor XII able to sustain protease activity, but unable to recognize substrates and surfaces necessary to activate the contact phase of coagulation. However, this ‘minimal’ factor‐XII protein displays a marked protease activity and, although lacking five regulatory domains of factor XII, is bound and activated by negative charges and promotes coagulation with high efficiency.
Diphenylene iodonium as an inhibitor of the NADPH oxidase complex of bovine neutrophils
Authors: DOUSSIÈRE Jacques , VIGNAIS Pierre V.
Published: March 2005

Diphenylene iodonium (Ph2I), a lipophilic reagent, is an efficient inhibitor of the production of O2‐ by the activated NADPH oxidase of bovine neutrophils. In a cell‐free system of NADPH oxidase activation consisting of neutrophil membranes and cytosol from resting cells, supplemented with guanosine 5′‐[γ‐thio]triphosphate, MgCl2 and arachidonic acid, or in membranes isolated from neutrophils activated by 4β‐phorbol 12‐myristate 13‐acetate, addition of a reducing agent, e.g. NADPH or sodium dithionite, markedly enhanced inhibition of the NADPH oxidase by Ph2I. The membrane fraction was found to contain the Ph2I‐sensitive component(s). In the presence of a concentration of Ph2I sufficient to fully inhibit O2‐ production (around 10 nmol/mg membrane protein), addition of catalytic amounts of the redox mediator dichloroindophenol (Cl2Ind) resulted in a by‐pass of the electron flow to cytochrome c, the rate of which was about half of that determined in non‐inhibited oxidase. A marked increase in the efficiency of this by‐pass was achieved by addition of sodium deoxycholate. The Cl2‐Ind‐mediated cytochrome c reduction was negligible in membranes isolated from resting neutrophils. At a higher concentration of Ph2I (100 nmol/mg membrane protein), the Cl2Ind‐mediated cytochrome c reductase activity was only half inhibited, which indicated that, in the NADPH oxidase complex, there are at least two Ph2I sensitive components differing by their sensitivity to the inhibitor. At low concentrations of Ph2I (≤ 10 nmol/mg protein), the spectrum of reduced cytochrome b558 in isolated neutrophil membranes was modified, suggesting that the component sensitive to low concentrations of Ph2I is the heme binding component of cytochrome b558. Higher concentrations of Ph2I were found to inhibit the isolated NADPH dehydrogenase component of the oxidase complex. A number of membrane and cytosolic proteins were labeled by [125I]Ph2I. However, the radiolabeling of a membrane‐bound 24‐kDa protein, which might be the small subunit of cytochrome b558, responded more specifically to the conditions of activation and reduction which are required for inhibition of O2‐ production by Ph2I. The O2‐‐generating form of xanthine oxidase was also inhibited by Ph2I. Inhibition of xanthine oxidase, a non‐heme iron flavoprotein, by Ph2I had a number of features in common with that of the neutrophil NADPH oxidase, namely the requirement of reducing conditions for inhibition of O2‐ production by Ph2I and the induction of a by‐pass of electron flow to cytochrome c by Cl2Ind in the inhibited enzyme, suggesting some similarity in the molecular organization of the two enzymes.
Purification, phosphorylation and control of the guanine‐nucleotide‐exchange factor from rabbit reticulocyte lysates
Authors: OLDFIELD Susan , PROUD Christopher G.
Published: March 2005

A simple, improved procedure for the isolation of guanine‐nucleotide‐exchange factor (GEF) and for eukaryotic initiation factor 2 (eIF‐2) from rabbit reticulocyte lysates has been developed using ion‐exchange chromatography on S‐Sepharose, Q‐Sepharose, Mono Q and Mono S. The majority of the eIF‐2 is separated from GEF at an early stage in the procedure and the remaining small amount of eIF‐2 · GEF complex is separated from the bulk of the GEF by FPLC on Mono S. The procedure yields approximately 2 mg each of eIF‐2 and GEF, of 90% and greater than 80% purity, respectively, from the blood of ten rabbits. All fractions of purified GEF contain four subunits of molecular masses 84, 66, 54 and 39 kDa, with various amounts of a fifth, 30‐kDa subunit.

The modulation of GEF activity was investigated using the highly purified factor in a guanine‐nucleotide‐exchange assay. The activity of GEF was stimulated by physiological concentrations of the polyamines, spermine and spermidine, but was unaffected by another polycationic compound, polylysine. Activity was also found to be inhibited by 1 mM NADP+ or NAD+, and this inhibition was overcome by the presence of 1 mM NADPH. Stoichiometric amounts of GEF were unable to release GDP from eIF‐2 · GDP complexes in the absence of free guanine nucleotides, suggesting that GEF operates by a ternary‐complex mechanism.

Casein kinase 1 or casein kinase 2 can each phosphorylate the largest subunit (84 kDa) of GEF. These enzymes both phosphorylate serine residues in GEF but they phosphorylate distinct sites, as demonstrated by phosphopeptide mapping following proteolytic or cyanogen bromide digestion. Neither of these kinases phosphorylated any of the other subunits of GEF to any significant extent and several other kinases were inactive against GEF. No effect of phosphorylation on activity could be demonstrated.
Role and environment of the conserved Lys27 of snake curaremimetic toxins as probed by chemical modifications, site‐directed mutagenesis and photolabelling experiments
Authors: HERVÉ Mireille , PILLET Laurence , HUMBERT Paul , TRÉMEAU Odile , DUCANCEL Frédéric , HIRTH Christian , MÉNEZ André
Published: March 2005

The positive charge of Lys27 was supressed by chemical means in two short‐chain curaremimetic toxins, namely erabutoxin a (Ea) from Laticauda semifasciata and toxin α from Naja nigricollis. This modification leads to a decrease in the binding affinity of the toxins for the nicotinic acetylcholine receptor, which range 6–15‐fold, as judged from both the data reported here and those previously described in the literature. A negatively charged glutamate residue has been introduced at position 27 of erabutoxin a by site‐directed mutagenesis. This change provokes a 120‐fold decrease in the affinity, which reflects a major alteration of toxin‐receptor cognate events.

Using toxin‐α derivative harbouring a photoactive group at Lys27, we probed the toxin local environment in a receptor‐bound state by photocoupling experiments. The δ chain was the predominant coupling target, in contrast to previous observations indicating that a photoactive probe on Lys47 predominantly labelled the α chain. The toxin derivative weakly labelled the α and γ chains but not the β chain. The toxin may therefore interact with subunits other than the α chain, at least in the vicinity of Lys27.
Purification of the main somatostatin‐degrading proteases from rat and pig brains, their action on other neuropeptides, and their identification as endopeptidases 24.15 and 24.16
Authors: DAHMS Peter , MENTLEIN Rolf
Published: March 2005

The main somatostatin‐degrading proteases were purified from rat and pig brain homogenates and characterized as thiol‐and metal‐dependent endoproteases. Two types of proteases with apparent native and subunit molecular masses of 70 kDa and 68 kDa could be differentiated in both species. Beside somatostatin, both hydrolyzed several other neuropeptides with chain lengths between 8 and 30 amino acid residues. Cleavage sites were generally similar or identical, but some clear exceptions were observed for enzymes from both species which could be used to differentiate between the two proteases. The 68‐kDa protease cleaved somatostatin at three bonds (Asn5‐Phe6, Phe6‐Phe7 and Thr10‐Phe11) and neurotensin only at the Arg8‐Arg9 bond, whereas the 70‐kDa protease digested somatostatin at only two bonds (Phe6‐Phe7 and Thr10‐Phe11) and neurotensin as well as acetylneurotensin‐(8–13) additionally (pig protease) or almost exclusively (rat protease) at the Pro10‐Tyr11 bond. Relative rates for the digestions of various peptides were, however, more dependent on the species than on the type of protease. Cleavage sites for angiotensin II, bradykinin, dynorphin, gonadoliberin and substance P were, apart from different rates, identical for both proteases. In both species the 68‐kDa protease was found to be mainly, but not exclusively, soluble and not membrane‐associated, whereas the inverse was detected for the 70‐kDa protease. Based on distinct molecular and catalytic properties, the 68‐kDa protease is supposed to be congruent with the endopeptidase 24.15 (EC 3.4.24.15), the 70‐kDa protease with endopeptidase 24.16 (EC 3.4.24.16, neurotensindegrading endopeptidase). This investigation demonstrates that both proteases hydrolyze various neuropeptides with similar cleavage sites, but with species‐dependent activity. Species‐independent distinctions are the exclusive action of endopeptidase 24.16 on acetylneurotensin‐(8–13) and liberation of free Phe from somatostatin only by endopeptidase 24.15.
The effect of phospholipids, calcium ions and protein S on rate constants of human factor Va inactivation by activated human protein C
Authors: BAKKER Harry M. , TANS Guido , JANSSEN‐CLAESSEN Truus , THOMASSEN M. Christella L. G. D. , HEMKER H. Coenraad , GRIFFIN John H. , ROSING Jan
Published: March 2005

Rate constants for human factor Va inactivation by activated human protein C (APC) were determined in the absence and presence of Ca2+ ions, protein S and varying concentrations of phospholipid vesicles of different lipid composition. APC‐catalyzed factor Va inactivation in free solution (in the presence of 2 mM Ca2+) was studied under first‐order reaction conditions with respect to both APC and factor Va and was characterized by an apparent second‐order rate constant of 6.1 × 105 M−1 s−1. Stimulation of APC‐catalyzed factor Va inactivation by phospholipids was dependent on the concentration and composition of the phospholipid vesicles. Optimal acceleration (230‐fold) of factor Va inactivation was observed with 10 μM phospholipid vesicles composed of 20 mol% dioleoylglycerophosphoserine (Ole2GroPSer) and 80 mol% dioleoylglycerophosphocholine (Ole2GroPCho). At higher vesicle concentrations and at higher molar fractions of Ole2GroPSer some inhibition of APC‐catalyzed factor Va inactivation was observed.

Membranes that contained anionic phospholipids other than phosphatidylserine also promoted factor Va inactivation. The ability of different anionic lipids to enhance factor Va inactivation increased in the order phosphatidylethanolamine ≤ oleic acid ≤ phosphatidic acid ≤ phosphatidylglycerol ≤ phosphatidylmethanol ≤ phosphatidylserine. APC‐catalyzed factor Va inactivation in the presence of phospholipid vesicles could be saturated with respect to factor Va and the reaction obeyed Michaelis‐Menten kinetics. Both the Km for factor Va and the Vmax of factor Va inactivation were a function of the phospholipid concentration. The Km increased from 1 nM at 2.5 μM phospholipid (Ole2GroPSer/Ole2GroPCho 20:80, mol/mol) to 65 nM at 250 μM phospholipid. The Vmax increased from 20 mol factor Va inactivated · min−1· mol APC−1 at 2.5 μM phospholipid to 62 mol factor Va inactivated · min−1· mol APC−1 at 10 μM phospholipid and remained constant at higher phospholipid concentrations. Protein S appeared to be a rather poor stimulator of APC‐catalyzed factor Va inactivation. Protein‐S‐dependent rate enhancements were only observed in reaction mixtures that contained negatively charged phospholipid vesicles. Independent of the concentration and the lipid composition of the vesicles, protein S caused a twofold stimulation of APC‐catalyzed factor Va inactivation. This suggests that, in the human system, enhancement of APC binding to phospholipid vesicles by protein S is of minor importance. Considering that protein S is a physiologically essential antithrombotic agent it is likely that other factors or phenomena contribute to the in vivo antithrombotic action of protein S.
Experimental and modelling studies on the DNA cleavage by elsamicin A
Authors: PÁRRAGA Antonio , OROZCO Modesto , PORTUGAL José
Published: March 2005

The ability of elsamicin A, an antitumour antibiotic, to cleave DNA in the presence of ferrous iron and reducing agents, has been analysed using experimental and theoretical approaches. Experimentally, the antibiotic causes DNA breakage in the presence of ferrous ions and a reducing agent. The DNA‐cleaving activity appears to be partially blocked by the action of superoxide dismutase and catalase. These results indicate that the elsamicin aglycone moiety (chartarin) can be involved in the production of free radicals. We have performed a broad theoretical study based in the quantum‐mechanical framework, which allow us to determine the redox properties of elsamicin that lead to the generation of radical species. Our results clearly show that elsamicin acts as a true catalyst in the production of superoxide radicals. Moreover, it is suggested that the oxidation/reduction mechanism of the aglycone moiety of elsamicin (a lactone), leading to DNA breakage, is different from the mechanism followed by other well‐known anti‐cancer drugs, whose chromophore is a quinone.
Regulation of expression of a Xenopus borealis embryonic/larval α3 skeletal‐actin gene
Authors: BOARDMAN Mark , CROSS Gareth. S. , JONES Elizabeth A. , WOODLAND Hugh R.
Published: March 2005

We have isolated a genomic clone, related in sequence to the skeletal‐actin gene sub‐family. It is expressed in the skeletal muscle of embryos from the neurula stage onwards and in tadpoles, but not in adults. The equivalent Xenopus laevis gene is expressed as a major transcript in adult muscle, as well as at earlier stages. The intron/exon structure is typical of vertebrate skeletal‐actin genes, as is the possession of multiple copies of three serum‐response elements in the promoter of this gene. The Xenopus actin and β‐globin genes were fused in their second introns. This construct, which contained 2.4 kb of upstream sequence, was injected into fertilized eggs at the two‐cell stage. It showed the normal pattern of tissue‐specific transcription. Thus all of the information necessary for appropriate expression of this actin gene in the embryo is contained in the region that extends from a point 2.4 kb upstream of transcription initiation to the centre of the second exon. A series of enhancer constructs were made in which upstream regions of the actin gene were placed upstream of a X. laevisβ‐globin gene. The region immediately adjacent to the promoter, containing the three serum‐response elements, was able to drive muscle‐specific expression, and there was also a general enhancement of transcription by regions further upstream.
A reinvestigation of the pre‐steady‐state ATPase activity of the nitrogenase from Azotobacter vinelandii
Authors: MENSINK Richard E. , WASSINK Hans , HAAKER Huub
Published: March 2005

The pre‐steady‐state ATPase activity of nitrogenase has been reinvestigated. The exceptionally high burst in the hydrolysis of MgATP by the nitrogenase from Azotobacter vinelandii communicated by Cordewener et al. (1987) [Cordewener J., ten Asbroek A., Wassink H., Eady R. R., Haaker H. & Veeger C. (1987) Eur. J. Biochem. 162, 265–270] was found to be caused by an apparatus artefact. A second possible artefact in the determination of the stoichiometry of the pre‐steady‐state ATPase activity of nitrogenase was observed. Acid‐quenched mixtures of dithionite‐reduced MoFe or Fe protein of Azotobacter vinelandii nitrogenase and MgATP contained phosphate above the background level. It is proposed that due to this reaction, quenched reaction mixtures of nitrogenase and MgATP may contain phosphate in addition to the phosphate released by the ATPase activity of the nitrogenase complex.

It was feasible to monitor MgATP‐dependent pre‐steady‐state proton production by the absorbance change at 572 nm of the pH indicator o‐cresolsulfonaphthalein in a weakly buffered solution At 5.6°C, a pre‐steady‐state phase of H+ production was observed, with a first‐order rate constant of 2.2 s−1, whereas electron transfer occurred with a first‐order rate constant of 4.9 s−1. At 20.0°C, MgATP‐dependent H+ production and electron transfer in the pre‐steady‐state phase were characterized by observed rate constants of 9.4 s−1 and 104 s−1, respectively. The stopped‐flow technique failed to detect a burst in the release of protons by the dye‐oxidized nitrogenase complex.

It is concluded that the hydrolysis rate of MgATP, as judged by proton release, is lower than the rate of electron transfer from the Fe protein to the MoFe protein.
Inhibition of the GlcNAc transferase of the glycosylphosphatidylinositol anchor biosynthesis in African trypanosomes
Authors: MILNE Kenneth G. , FERGUSON Michael A. J. , MASTERSON Wayne J.
Published: March 2005

A wide variety of eukaryotic membrane proteins are anchored to the cell surface by a covalent linkage to glycosylphosphatidylinositol. One of the best characterised examples is the variant surface glycoprotein of the protozoan parasite, Trypanosoma brucei. The pathway for the formation of the glycosylphosphatidylinositol precursor has been previously described, with the first step being the transfer of GlcNAc, from UDP‐GlcNAc to endogenous phosphatidylinositol to form N‐acetyl‐glucosaminylphosphatidylinositol [Doering, T. L., Masterson, W. J., Hart, G. W. & Englund, P. T. (1989) J. Biol. Chem. 264, 11168–11173]. Here we report that low concentrations of sulphydryl alkylating reagents irreversibly inhibit this transferase in a trypanosome‐derived cell‐free system. The site of inactivation by N‐ethylmaleimide appears to be at, or close to, the enzyme active site, since incubation of the enzyme preparation with the donor molecule UDP‐GlcNAc substantially protects the enzyme from inactivation. The protection appears to be primarily dependent on the nucleotide portion of the molecule, since UMP and UDP can mimic the protection seen with UDP‐GlcNAc.
Changes in calpain and calpastatin mRNA induced by β‐adrenergic stimulation of bovine skeletal muscle
Authors: PARR Timothy , BARDSLEY Ronald G. , GILMOUR R. Stewart , BUTTERY Peter J.
Published: March 2005

Administration of β‐adrenergic agonists to mammals can produce skeletal muscle hypertrophy in some species and muscle types. The growth‐promoting effect appears to be due to suppression of protein breakdown rather than stimulation of synthesis, although evidence from turnover studies is equivocal. In ovine muscle, changes in the activity of the calcium dependent neutral proteinases (calpains I and II) and their specific inhibitor (calpastatin) accompany β‐agonist‐induced hypertrophy. These observations suggest that the calpain system is involved in myofibrillar protein turnover in some way. Alternatively, the relationship with hypertrophy may be indirect, since the calpains also interact with hormone and growth‐factor receptors, protein kinase C and transcription factors, in addition to a range of membrane, cytoskeletal and nuclear proteins.

In the present study, attempts have been made to determine if the β‐agonist‐induced effects on the calpain system are associated with corresponding changes in specific mRNA. The activity of both calpain isoforms and calpastatin was measured in bovine longissimus dorsi samples from trials in which test animals were treated with the β agonist cimaterol. Total RNA was extracted from the muscle samples. A cDNA probe for calpastatin mRNA was generated from bovine RNA by the polymerase chain reaction. This cDNA and a human calpain‐II large‐subunit cDNA were used to detect specific mRNA by Northern‐blot analysis.

β‐agonist treatment of Friesian steers caused significant longissimus dorsi hypertrophy. Increases in muscle mass (+ 37%, P < 0.005), calpain‐II specific activity (+ 27%, P < 0.05) and calpastatin‐specific activity (+ 76%, P < 0.05) were found in treated animals. Total RNA was unchanged, but there was a 96% overall increase in calpastatin mRNA and a 30% increase in calpain‐II large‐subunit mRNA in muscle from treated animals. The mRNA changes are similar in direction and degree to the activity changes. Both calpain‐II large subunit and inhibitor expression may therefore be stimulated by agonist action at the level of transcription or mRNA stabilisation. Multiple calpastatin mRNA species were detected in steers, as reported for other species. Differential changes in these messages, induced by the β agonist, suggest that expression or stability of alternative mRNA species may be a factor in calpastatin regulation.
Biosynthesis and intracellular transport of α‐2,6‐sialyltransferase in rat hepatoma cells
Authors: BOSSHART Herbert , BERGER Eric G.
Published: March 2005

We investigated biosynthesis, intracellular transport and release of β‐galactoside α‐2,6‐sialyltransferase in a dexamethasone‐inducible rat hepatoma cell line. Confluent cells were induced by 10 μM dexamethasone for 24 h, and metabolically labelled with [35S]methionine/cysteine, followed by immunoprecipitation of sialyltransferase and electrophoretic/fluorographic analysis. The 35S‐labelled enzyme was synthesized as a 46‐kDa precursor, converted to an intermediate 47‐kDa form after 1 h, and gradually to a mature form of 48 kDa within the following 3 h. By means of either tunicamycin inhibition of N‐glycosylation or cleavage of N‐glycans from isolated sialyltransferase using N‐glycosidase F, the sizes of the precursor and the mature form were reduced to 41 kDa and 43 kDa, respectively. After a 4‐h chase, treatment with endoglycosidase H revealed two distinct molecular forms of sialyltransferase, bearing either two N‐acetyllactosamine‐type or one oligomannose‐type and one N‐acetyllactosamine‐type N‐linked sugar chain. In addition, sialyltransferase became sensitive to neuraminidase digestion after a 4‐h chase. The half‐life of intracellular [35S]sialyltransferase was estimated at 3 h. A soluble form was detectable in the supernatant, 2 h after the pulse. Only 12% of the initially labelled sialyltransferase was found in the medium after 12 h, while 73% of the enzyme was degraded intracellularly. To characterize a possible intracellular degradation site, we studied intracellular transport in the presence of either secretion‐blocking or acidotropic agents or protease inhibitors. Degradation was significantly delayed by all treatments.

Our results show that sialyltransferase follows the secretory pathway as a membrane protein and is retained at a late Golgi stage. We suggest that the bulk of sialyltransferase in rat hepatoma cells is diverted to a post‐Golgi degradation pathway. This route contrasts with the post‐Golgi trafficking of β‐1,4‐galactosyltransferase in HeLa cells, which is constitutively secreted [Strous, G. J. A. M. & Berger, E. G. (1982) J. Biol. Chem. 257, 7623–7628].
Protein structure of pig liver 4‐aminobutyrate aminotransferase and comparison with a cDNA‐deduced sequence
Authors: DE BIASE Daniela , MARAS Bruno , BOSSA Francesco , BARRA Donatella , JOHN Robert A.
Published: March 2005

The amino acid sequence of pig liver 4‐aminobutyrate aminotransferase has been determined by gas‐phase sequencing of proteolytically derived peptide fragments. The sequence differs substantially from that predicted for the same enzyme on the basis of the sequence of cDNA derived from pig brain in recently published work [Kwon, O., Park, J. & Churchich, J. E. (1992) J. Biol. Chem. 267, 7215–7216]. Apart from a few minor differences, the two sequences are completely different in the segment of protein comprising the 36 residues at positions 107–142. Insertion of a cytosine between bases 402 and 403 in the cDNA sequence, together with deletion of the guanine at position 510, results in a DNA sequence which predicts exactly the amino acid sequence determined by peptide analysis in the present work. The mammalian enzyme has approximately 44% sequence identity with the same enzyme from two unicellular eukaryotes (Saccharomyces cerevisiae, Aspergillus nidulans) and 22% identity with that from Escherichia coli.
Basement‐membrane heparan sulfate proteoglycan binds to laminin by its heparan sulfate chains and to nidogen by sites in the protein core
Authors: BATTAGLIA Cristina , MAYER Ulrike , AUMAILLEY Monique , TIMPL Rupert
Published: March 2005

A large, low‐density form of heparan sulfate proteoglycan was isolated from the Engelbreth‐Holm‐Swarm (EHS) tumor and demonstrated to bind in immobilized‐ligand assays to laminin fragment E3, collagen type IV, fibronectin and nidogen. The first three ligands mainly recognize the heparan sulfate chains, as shown by inhibition with heparin and heparan sulfate and by the failure to bind to the proteoglycan protein core. Nidogen, obtained from the EHS tumor or in recombinant form, binds exclusively to the protein core in a heparin‐insensitive manner. Studies with other laminin fragments indicate that the fragment E3 possesses a unique binding site of laminin for the proteoglycan. A major binding site of nidogen was localized to its central globular domain G2 by using overlapping fragments. This allows for the formation of ternary complexes between laminin, nidogen and proteoglycan, suggesting a key role for nidogen in basement‐membrane assembly. Evidence is provided for a second proteoglycan‐binding site in the C‐terminal globule G3 of nidogen, but this interaction prevents the formation of such ternary complexes. Therefore, the G3‐mediated nidogen binding to laminin and proteoglycan are mutually exclusive.
Kinase inhibition by a phosphorylated peptide corresponding to the major insulin receptor autophosphorylation domain
Authors: CHAVANIEU Alain , CALAS Bernard , VAGLIO Philippe , GRIGORESCU Florin
Published: March 2005

We studied the inhibitory effect of non‐phosphorylated and triphosphorylated synthetic peptides, corresponding to amino acids 1143–1155 of the insulin proreceptor (domain 1151) on autophosphorylation and kinase of the insulin receptor. Tyrosine‐phosphorylated peptides were synthesized using the N(9‐fluorenylmethoxycarbonyl)‐O‐dibenzylphosphono‐l‐ tyrosine.

The triphosphorylated peptide (1151‐P3) and the non‐phosphorylated peptide (1151‐NP), respectively, inhibited insulin receptor autophosphorylation by 65% and 70%, in a dose‐dependent and additive manner. When the receptor was pre‐phosphorylated for 1 min with [γ‐32P]ATP, 1151‐P3 decreased autophosphorylation to 60% of maximun, whereas 1151‐NP had no further effect. In both non‐activated and preactivated receptors, 1151‐P3 inhibition of receptor autophosphorylation was prevented by adding 2 mM vanadate. Kinase activity towards exogenous substrate poly(Glu4, Tyr) was dose‐dependently inhibited by both analogues. This effect was independent of the state of receptor phosphorylation or the addition of vanadate. Since 1151‐P3 inhibited the exogenous kinase without altering receptor endogenous autophosphorylation after the addition of vanadate, we investigated 1151‐NP and 1151‐P3 competition for the phosphorylation of a resin‐immobilized 1151 peptide. While 1151‐NP (at 2 mM) was highly competitive, inhibiting phosphate incorporation by 70%, 1151‐P3 caused a four‐fold increase in the phosphorylation of 1151‐NP–resin. The receptor underwent conformational changes during autophosphorylation and an antibody directed against a peptide corresponding to amino acids 1314–1330 of the proreceptor (1322Ab) was previously shown to immunoprecipitate specifically the non‐phosphorylated receptor forms. Nevertheless, the 1322Ab immunoprecipitated a fully autophosphorylated receptor in the presence of 1151‐NP, but not of 1151‐P3, thus suggesting a conformational change induced by the non‐phosphorylated peptide.

In conclusion, kinase inhibition was still observed after the addition of phosphate groups to three 1151‐peptide tyrosines, but the peptide effect on receptor autophosphorylation, phosphorylation of homologous 1151‐NP–resin and conformational changes induced in the receptor was altered dramatically. These data may provide a basis for further understanding the role of tyrosine phosphorylation in insulin receptor kinase activation or regulation.
Dimerization and reactivation of triosephosphate isomerase in reverse micelles
Authors: GARZA‐RAMOS Georgina , TUENA DE GÓMEZ‐PUYOU Marietta , GÓMEZ‐PUYOU Armando , GRACY Robert W.
Published: March 2005

The reactivation of the homodimeric enzyme triosephosphate isomerase (TPI) was studied in reverse micelles. The enzyme was denatured in conventional aqueous mixtures with guanidine hydrochloride and transferred to reverse micelles formed with cetyltrimethylammonium bromide, hexanol, n‐octane and water. In the transfer step, denatured TPI monomers distributed in single micelles, and guanidine hydrochloride was diluted more than 100 times. Under optimal reactivation conditions, 100% of the enzyme activity could be recovered. The rate of appearance of the catalytic activity increased with the concentration of protein, which indicated that catalysis required the formation of the dimer. The rate of TPI reactivation also increased with increasing protein concentration in the system with denatured TPI covalently derivatized at the catalytic site with the substrate analogue 3‐chloroacetol phosphate. Thus, reactivation could take place via the formation of dimers composed of an inactive and an active subunit. Reactivation critically depended on the amount of water in the reverse micelles. The plot of the extent of reactivation versus the amount of water (2.5–7.0%) was markedly sigmoidal. Less than 20% reactivation took place with water concentrations below 3.5%, due to the formation (in less than 30 s) of stable inactive structures. The results indicate that reverse micelles provide a useful system to probe the events involved in the transformation of unfolded monomers to polymeric enzymes.
Involvement of a labile axial histidine in coupling electron and proton transfer in Methylophilus methylotrophus cytochrome c″
Authors: COSTA Helena S. , SANTOS Helena , TURNER David L. , XAVIER António V.
Published: March 2005

Methylophilus methylotrophus cytochrome c” is an unusual monohaem protein (15 kDa) undergoing a redox‐linked spin‐state transition [Santos, H. & Turner, D. L. (1988) Biochim. Biophys. Acta 954, 277–286]. The midpoint redox potential of cytochrome c” was measured over the pH range 4–10. The pH dependence of the midpoint redox potential was interpreted in terms of a model that considers the redox‐state dependence of the ionization of two distinct and non‐interacting protonated groups in the protein. This analysis led to the following pKa values within the pH range studied: pK0a1= 6.4, pKTa1= 5.4 and pKTa2= 8.1. Proton‐NMR spectroscopy was used to assist the characterization of the two ionizing groups responsible for the observed redox‐Bohr effect: the group ionizing with a lower pKTa was assigned to a haem propionic acid substituent and the other to the axial histidine ligand which becomes detached upon reduction, which has a pKOa too low to be measured. It is shown that M. methylotrophus cytochrome c” is able to couple electron and proton transfer in the physiological pH range through a mechanism involving reversible change in the haemiron coordination. Possible implications for the physiological role of the protein are discussed.
The primary structure of a protein containing a putative [6Fe‐6S] prismane cluster from Desulfovibrio vulgaris (Hildenborough)
Authors: STOKKERMANS Jack P. W. G. , PIERIK Antonio J. , WOLBERT Ronnie B. G. , HAGEN Wilfred R. , VAN DONGEN Walter M. A. M. , VEEGER Cees
Published: March 2005

The gene encoding a protein containing a putative [6Fe–6S] prismane cluster has been cloned from Desulfovibrio vulgaris (Hildenborough) and sequenced. The gene encodes a polypeptide composed of 553 amino acids (60 161 Da). The DNA‐derived amino acid sequence was partly confirmed by N‐terminal sequencing of the purified protein and of fragments of the protein generated by CNBr cleavage. Furthermore, the C‐terminal sequence was verified by digestion with carboxypeptidases A and B. The polypeptide contains nine Cys residues. Four of these residues are gathered in a Cys‐Xaa2‐Cys‐Xaa7‐Cys‐Xaa5‐Cys motif located towards the N‐terminus of the protein. No relevant sequence similarity was found with other proteins, including those with high‐spin Fe‐S clusters (nitrogenase, hydrogenase), with one significant exception: the stretch containing the first four Cys residues spans two submotifs, Cys‐Xaa2‐Cys and Lys‐Gly‐Xaa‐Cys‐Gly, separated by 11 residues, that are also present in high‐spin Fe‐S cluster containing CO dehydrogenase. Western‐blot analysis demonstrates cross‐reactivity of antibodies raised against the purified protein both in Desulfovibrio strains and other sulfate‐reducing bacteria. Hybridization of the cloned gene with genomic DNA of several other Desulfovibrio species indicates that homologous sequences are generally present in the genus Desulfovibrio.
Mechanistic studies of 3‐deoxy‐d‐manno‐2‐octulosonate‐8‐phosphate synthase from Escherichia coli
Authors: KOHEN Amnon , JAKOB Abla , BAASOV Timor
Published: March 2005

The anomeric specificity and the steady‐state kinetic mechanism of homogeneous 3‐deoxy‐d‐manno‐2‐octulosonate‐8‐phosphate (KDO8P) synthase were investigated. The open‐chain 4‐deoxy analogue of arabinose‐5‐phosphate (Ara5P), which is structurally prohibited from undergoing ring closure, was synthesized and tested as a substrate for the synthase. It was found that the analogue functions as a substrate with a similar kcat value to that of the original substrate. The kcat/Km value for the natural substrate is seven‐times greater than that of the 4‐deoxy analogue. However, taking into account the 9.5% and approximately 1% concentrations of the aldehyde forms of the 4‐deoxy analogue and Ara5P in solution, then the ‘true’Km values must be in the range 31.5 μM and 0.26 μM, respectively, requiring about a 3 kcal/mol contribution to the binding energy by the 4‐hydroxyl group of Ara5P. The data provides evidence that the enzyme acts upon the acyclic form of the natural substrate.

The steady‐state kinetic study of KDO8P synthase was analyzed via inhibition using the products KDO8P and inorganic phosphate, and d‐ribose‐5‐phosphate as a dead‐end inhibitor. First, intersecting lines in double‐reciprocal plots of initial‐velocity data at substrate concentrations in the micromolar range suggest a sequential mechanism for the enzyme‐catalyzed reaction. The inhibition by d‐ribose‐5‐phosphate is competitive for Ara5P and uncompetitive for phosphoenolpyruvate (P‐pyruvate). These inhibition patterns are consistent with the model wherein P‐pyruvate binding precedes that of Ara5P binding. Furthermore, this order of substrate binding was supported by the observations that KDO8P is a competitive inhibitor for P‐pyruvate binding, supporting the concept that KDO8P and P‐pyruvate bind to the same enzyme form, and noncompetitively with respect to Ara5P. In addition, the inhibition by inorganic phosphate is noncompetitive with respect to both P‐pyruvate and Ara5P, suggesting an apparent ordered release of products such that Pi first, followed by KDO8P. In conclusion, these data suggest a steady‐state kinetic mechanism for KDO8P synthase where P‐pyruvate binding precedes that of Ara5P, followed by the ordered release of inorganic phosphate and KDO8P.
Matrix NADH dehydrogenases of plant mitochondria and sites of quinone reduction by complex I
Authors: MENZ R. Ian , GRIFFITH Megan , DAY David A. , WISKICH Joseph T.
Published: March 2005

In order to distinguish the pathways involved in the oxidation of matrix NADH in plant mitochondria, the oxidation of NADH and nicotinamide hypoxanthine dinucleotide (reduced form) was investigated in submitochondrial particles prepared from beetroot (Beta vulgaris L. cv. Derwent Globe) and soybeans (Glycine max L. cv. Bragg). Nicotinamide‐hypoxanthine‐dinucleotide(reduced form)‐oxidase activity was more strongly inhibited by rotenone than the NADH‐oxidase activity but both of the rotenone‐inhibited activities could be stimulated by adding ubiquinone‐1. The corresponding ubiquinone‐1‐reductase activities were inhibited by rotenone (to 69%) and further inhibited by N,N′‐dicyclohexylcarbodiimide (to 79%), whilst the K3Fe(CN)6‐reductase activities were not sensitive to either rotenone or N,N′‐dicyclohexylcarbodiimide. Immunological analysis of mitochondrial proteins using an antiserum raised against purified beetroot complex I indicated very few differences between soybean and fresh and aged beetroot mitochondria, despite their varying sensitivities to rotenone.

We confirm that there are two dehydrogenases capable of oxidising internal NADH and that only one of these, namely complex I, is inhibited by rotenone. Further, we conclude that complex I has two potential sites of quinone reduction, both sensitive to N,N′‐dicyclohexycarbodiimide inhibition but only one of which is sensitive to rotenone inhibition.
Structural requirements for glycosyl‐phosphatidylinositol‐anchor attachment in the cellular receptor for urokinase plasminogen activator
Authors: MØLLER Lisbeth Birk , PLOUG Michael , BLASI Francesco
Published: March 2005

The urokinase‐plasminogen‐activator receptor (u‐PAR) is a glycosyl‐phosphatidylinositol(glycosyl‐PtdIns)‐anchored membrane protein. Using site‐directed mutagenesis, we have studied features in the u‐PAR sequence important for successful glycosyl‐PtdIns attachment. Two critical sequence elements were identified. In the sequence Ser282‐Gly283‐Ala284, simultaneous substitution of all of these residues prevented membrane anchoring. Individual substitution of each of the residues indicated that Gly283 is the more critical residue and the likely attachment site. However, it was unexpectedly found that mutation of this residue gave rise only to a partial impairment of glycosyl‐PtdIns attachment. We therefore propose that more than one residue within this sequence can be utilized as glycosyl‐PtdIns‐attachment site. In the last eight COOH‐terminal amino acids encoded in u‐PAR cDNA, deletion of this sequence (residues 306–313) completely prevented glycosyl‐PtdIns attachment. However, the remaining COOH‐terminal region proved still to possess a potential glycosyl‐PtdIns signal activity; it could be converted to a new functional glycosyl‐PtdIns signal by substitution of a single positively charged residue (Arg304). Substitution of Arg304 by Leu converted this truntaced u‐PAR to a glycosyl‐PtdIns‐anchored protein, indistinguishable from the wild type. Substitution of Arg304 by a negatively charged residue (Glu) led to a partial aquisition of the glycosyl‐PtdIns‐anchoring ability. These findings show that charged amino acids placed in the COOH‐terminus interfere negatively with glycosyl‐PtdIns‐anchoring, and, furthermore, that this effect is more pronounced for positively charged than for negatively charged amino acid residues.
Analysis of glycosaminoglycan chains from different proteoglycan populations in human embryonic skin fibroblasts
Authors: SCHMIDTCHEN Artur , FRANSSON Lars‐Åke
Published: March 2005

1. The structure of chondroitin/dermatan and heparan‐sulphate chains from various proteoglycan populations derived from cultured human skin fibroblasts have been examined. Confluent cell cultures were biosynthetically labelled with [3H]‐glucosamine and 35SO42‐, and proteoglycans were purified according to buoyant density, size and charge density [Schmidtchen, A., Carlstedt, I., Malmström, A. & Fransson, L.‐Å. (1990) Biochem. J. 265, 289–300]. Some proteoglycan fractions were further fractionated according to hydrophobicity on octyl‐Sepharose in Triton X‐100 gradients. The glycosaminoglycan chains, intact or degraded by chemical or enzymic methods were then analysed by gel chromatography on Sepharose CL‐6B, Bio‐Gel P‐6, ion exchange HPLC and gel electrophoresis.

2. Three types of dermatan‐sulphate chains were identified on the basis of disaccharide composition and chain length. They were derived from the large proteoglycan, two small proteoglycans and a cell‐associated proteoglycan with core proteins of 90 kDa and 45 kDa. Intracellular, free dermatansulphate chains were very similar to those of the small proteoglycans.

3. Heparan‐sulphate chains from different proteoglycans had, in spite of small but distinct differences in size, strikingly similar compositional features. They contained similar amounts of d‐glucuronate, l‐iduronate (with or without sulphate) and N‐sulphate groups. They all displayed heparin‐lyase‐resistant domains with average molecular mass of 10–15 kDa. The heparan‐sulphate chains from proteoglycans with 250‐kDa and 350‐kDa cores were the largest (> 50 kDa), containing an average of four or five domains, in contrast to heparan‐sulphate chains from the small heparan‐sulphate proteoglycans which had average molecular mass of 45 kDa and consisted of three or four such domains. Free, cell‐associated heparan‐sulphate chains were heterogeneous in size (5–45 kDa).

4. These results suggest that the core protein may have important regulatory functions with regard to dermatan‐sulphate synthesis. On the other hand, synthesis of heparan sulphate may be largely controlled by the cell that expresses a particular proteoglycan core protein.