bluuebunny/crossref_metadata_2025 huggingface dataset:
Optimization of yeast‐expressed human liver cytochrome P450 3A4 catalytic activities by coexpressing NADPH‐cytochrome P450 reductase and cytochrome b5
Authors: PEYRONNEAU Marie‐Anne , RENAUD Jean‐Paul , TRUAN Gilles , URBAN Philippe , POMPON Denis , MANSUY Daniel
Published: March 2005

Human liver P450 NF25 (CYP3A4) had been previously expressed in Saccharomyces cerevisiae using the inducible GAL10‐CYC1 promoter and the phosphoglycerate kinase gene terminator [Renaud, J. P., Cullin, C., Pompon, D., Beaune, P. and Mansuy, D. (1990) Eur. J. Biochem. 194, 889–896]. The use of an improved expression vector [Urban, P., Cullin, C. and Pompon, D. (1990) Biochimie 72, 463–472] increased the amounts of P450 NF25 produced/culture medium by a factor of five, yielding up to 10 nmol/l. The availability of recently developed host cells that simultaneously overexpress yeast NADPH‐P450 reductase and/or express human liver cytochrome b5, obtained through stable integration of the corresponding coding sequences into the yeast genome, led to biotechnological systems with much higher activities of yeast‐expressed P450 NF25 and with much better ability to form P450 NF25–iron‐metabolite complexes. 9‐fold, 8‐fold, and 30‐fold rate increases were found respectively for nifedipine 1,4‐oxidation, lidocaine N‐deethylation and testosterone 6β‐hydroxylation between P450 NF25‐containing yeast microsomes from the basic strain and from the strain that both overexpresses yeast NADPH‐P450 reductase and expresses human cytochrome b5. Even higher turnovers (15‐fold, 20‐fold and 50‐fold rate increases) were obtained using P450 NF25‐containing microsomes from the yeast just overexpressing yeast NADPH‐P450 reductase in the presence of externally added, purified rabbit liver cytochrome b5. This is explained by the fact that the latter strain contained the highest level of NADPH‐P450 reductase activity. It is noteworthy that for the three tested substrates, the presence of human or rabbit cytochrome b5 always showed a stimulating effect on the catalytic activities and this effect was saturable. Indeed, addition of rabbit cytochrome b5 to microsomes from a strain expressing human cytochrome b5 did not further enhance the catalytic rates. The yeast expression system was also used to study the formation of a P450‐NF25–iron‐metabolite complex. A P450 Fe(II)‐(RNO) complex was obtained upon oxidation of N‐hydroxyamphetamine, catalyzed by P450‐NF25‐containing yeast microsomes. In microsomes from the basic strain expressing P450 NF25, 10% of the starting P450 NF25 was transformed into this metabolite complex, whereas more than 80% of the starting P450 NF25 led to complex formation in microsomes from the strain overexpressing yeast NADPH‐P450 reductase. These results show that specific activities of yeast‐expressed P450 NF25 may be artificially low, owing to limiting amounts of the associated microsomal redox proteins and emphasize the importance of controlling the amounts of the different components of the monooxygenase complex in order to optimize these catalytic activities, especially when the expression system is to be used for demonstrating metabolic capacities towards new substrates.
The role of calcium and protein kinase C in the IgE‐dependent activation of phosphatidylcholine‐specific phospholipase D in a rat mast (RBL 2H3) cell line
Authors: LIN Peiyuan , GILFILLAN Alasdair M.
Published: March 2005

Our previous studies have suggested that phosphatidylcholine‐specific phospholipase D (PtdCho‐PLD) plays a role in IgE‐dependent diacylglycerol production, protein kinase C activation and mediator release in the RBL 2H3 mast cell line. We have extended these studies to examine the mechanisms by which PtdCho‐PLD may be regulated in these cells. RBL 2H3 cellular lipids were labeled with [14C]arachidonic acid or [3H]myristic acid, then PtdCho‐PLD activity was monitored by the formation of radiolabeled phosphatidylethanol when ethanol was included in the incubation medium. Trinitrophenol‐ovalbumin conjugate (10 ng/ml), when added to cells previously sensitized with anti‐(trinitrophenelated mouse IgE) (0.5 μg/ml), ionomycin (1 μM) and thapsigargin (0.1 μM), stimulated PtdCho‐PLD activation and mediator release in cells incubated in buffer containing 1.8 mM calcium, but not in cells incubated in calcium‐free, buffer. Phorbol 12‐myristate 13‐acetate (0.1 μM) activated PtdCho‐PLD in both buffers, but on its own did not trigger mediator release. When intracellular calcium was chelated with 5,5′‐dimethyl‐1,2‐bis(2‐aminophenoxy)ethane‐N,N,N′,N′‐tetraacetic acid, trinitrophenol‐ovalbumin conjugate failed to activate PtdCho‐PLD and histamine release. Similarly, down‐regulation of protein kinase C activity by long‐term exposure to the phorbol ester (0.1 μM) and preincubation of the cells with protein kinase inhibitors resulted in the loss of the trinitrophenol‐ovalbumin response on PtdCho‐PLD activity and histamine release. Taken together, the above results suggest that IgE‐dependent PtdCho‐PLD activation is dependent on both activation of protein kinase C and a rise in the intracellular free calcium concentration.
Differential regulation of the transcript levels of some nuclear‐encoded and mitochondrial‐encoded respiratory‐chain components in response to growth activation
Authors: LUCIAKOVA Katarina , LI Ronggui , NELSON B. Dean
Published: March 2005

Biogenesis of mammalian mitochondria requires the participation of both nuclear and mitochondrial genes. In order to study the expression and coordination of these two sets of genes, serumdeprived, quiescent NIH 3T3 cells were activated by serum addition. The steady‐state levels of the transcripts for two growth‐response genes (the mitochondrial adenine‐nucleotide translocator and non‐mitochondrial β‐actin), one nuclear‐encoded respiratory‐chain component (F1‐ATPase β‐subunit) and the mitochondrial‐encoded subunit I of cytochrome oxidase decreased significantly in quiescent cells and were rapidly restored with similar kinetics after addition of serum. The transcripts for two additional nuclear‐encoded mitochondrial genes (cytochrome c1 and cytochrome oxidase subunit IV) did not respond to serum deprivation or growth activation. These results imply that mitochondrial biogenesis is at least partially regulated through growth‐dependent mechanisms. Furthermore, the expression of nuclear genes encoding mitochondrial respiratory‐chain components does not appear to be tightly coordinated, suggesting the existence of multiple control circuits.
Autoactivation of proteinase A initiates activation of yeast vacuolar zymogens
Authors: van den HAZEL H. Bart , KIELLAND‐BRANDT Morten C. , WINTHER Jakob R.
Published: March 2005

The Saccharomyces cerevisiae PEP4 gene encodes proteinase A, an aspartyl protease. pep4 mutants are defective in the activation of many vacuolar hydrolases, including proteinase B. We have expressed a pep4 mutation which directs the accumulation of pro‐proteinase A with a defective active site. Co‐expression with PEP4 leads to normal processing, i.e. the mutant zymogen is functional as a substrate for the maturation reaction in trans. We conclude that wild‐type pro‐proteinase A has the ability to mediate its own activation. Elimination of the co‐expressed PEP4 gene did not effectively stop the processing of the mutant zymogen, owing to a strong, proteinase‐B‐dependent, phenotypic lag. In a proteinase‐B‐negative strain, processing of pro‐proteinase A led to an active form of a higher molecular mass than the normal mature form.
Transcriptional activation of human (2′–5′)oligoadenylate synthetase gene expression by the phorbol ester 12‐O‐tetradecanoyl‐phorbol 13‐acetate in type‐I‐interferon‐treated HL‐60 and HeLa cells
Authors: CHANG Chih‐Chao , BORELLI Thomas J. , WILLIAMS Bryan R. G. , WU Joseph M.
Published: March 2005

(2′–5′)Oligoadenylate [(2′–5′)(A)n] synthetase is a key enzyme in the interferon‐elicited antiviral response whose controlled expression in interferon‐treated cells has been only partially elucidated. In this investigation, we have compared the modulation of the (2′–5′)(A)n synthetase gene by interferon alone and by the combination of interferon and a second cellular effector, 12‐O‐tetradecanoyl‐phorbol 13‐acetate (TPA). Although TPA alone had no effect on (2′–5′)(A)n synthetase, it potentiated the induction of (2′–5′)(A)n of synthetase by interferon in HL‐60 and HeLa cells by increasing content of its mRNA and an immunoreactive 40‐kDa isoenzyme. Since TPA activates protein kinase C (PKC), other PKC‐activating phorbol‐ester analogues were tested and found to be effective, whereas the PKC inhibitor staurosporine reduced the potentiative activity of TPA. By using the (2′–5′)(A)n synthetase gene promoter linked to a reporter gene, chloramphenicol acetyltransferase (CAT), TPA and interferon were found to result in a doubling of CAT activity compared to cells treated with interferon alone. Moreover, when nuclear extracts prepared from control cells or cells treated with TPA and interferon (IFN), separately or together, were incubated with radioactively labeled oligodeoxynucleotides containing the interferon‐responsive element (IRE), TPA was shown to down‐regulate an IFN‐inducible IRE/protein complex. These data further suggest that TPA regulates (2′–5′)(A)n synthetase gene expression at the level of transcription.
Metabolism of inositol phosphates in the protozoan Paramecium
Authors: FREUND Wolf‐Dietrich , MAYR Georg W. , TIETZ Cornelia , SCHULTZ Joachim E.
Published: March 2005

Basal and stimulated levels of inositol phosphates were determined in the protozoan Paramecium labelled with myo‐[3H]inositol. Under resting conditions, intracellular InsP6 (phytic acid), InsP5 and InsP4 concentrations were 140, 10 and 2 μM, respectively. InsP5 was comprised of 56% Ins(1,2,3,4,5)P5 and/or Ins(1,2,3,5,6)P5, 40% Ins(1,2,4,5,6)P5 and/or Ins(2,3,4,5,6)P5 and small amounts of Ins(1,3,4,5,6)P5 and Ins(1,2,3,4,6)P5. InsP4 was mainly Ins(1, 4, 5, 6)P4 and/or Ins(3, 4, 5, 6)P4. Other inositol phosphates were not detected at a detection limit of 50–85 nM. Using various depolarizing and hyperpolarizing stimuli, no significant changes in level of inositol phosphates were observed in vivo, indicating that in the ciliate a contribution of inositol phosphates to signal‐transduction mechanisms is unlikely. In homogenates prepared from myo‐[3H]inositol‐labelled cells, a marked relative increase in InsP3 and InsP4 over the concentrations in vivo was observed. These inositol phosphates were identified as degradation products of endogenous InsP6. A novel separation methodology for inositol phosphates was established to allow unequivocal assignment of phosphate locations of all dephosphorylated InsP6‐derived products. The dephosphorylation was catalyzed by a phytase‐like enzyme with a molecular mass of 240 kDa, most likely of a hexameric structure. The enzyme had a pH optimum of 7.0 and did not require divalent cations for activity. Substrate concentrations above 300 μM were inhibitory. Dephosphorylation of InsP6 by the Paramecium enzyme differs from that of phytases from plants in that it proceeds via a sequential release of phosphate groups from positions 6, 5, 4 and 3 of the myo‐inositol ring or/and positions 4, 5, 6 and 1.
A correlation‐coefficient method to predicting protein‐structural classes from amino acid compositions
Authors: CHOU Kuo‐Chen , ZHANG Chun‐Ting
Published: March 2005

A protein is usually classified into one of the following four structural classes: all α, all β, (α+β) and α/β. In this paper, based on the maximum correlation‐coefficient principle, a new formulation is proposed for pridicting the structural class of a protein according to its amino acid composition. Calculations have been made for a development set of proteins from which the amino acid compo‐sitions for the standard structural classes were derived, and an independent set of proteins which are outside the development set. The former can test the self consistency of a method and the latter can test its extrapolating effectiveness. In both cases, the results showed that the new method gave a considerably higher rate of correct prediction than any of the previous methods, implying that a significant improvement has been achieved by implementing the maximum‐correlation‐coefficient principle in the new method.
Characterisation of a haem active‐site mutant of horseradish peroxidase, Phe41 → Val, with altered reactivity towards hydrogen peroxide and reducing substrates
Authors: SMITH Andrew T. , SANDERS Stephen A. , THORNELEY Roger N. F. , BURKE Julian F. , BRAY Robert R. C.
Published: March 2005

A horseradish peroxidase variant ([F41V] HRP‐C*), in which Val replaces the conserved Phe at position 41 adjacent to the distal His, has been constructed. Its composition and spectroscopic, catalytic and substrate‐binding properties were compared with those of the wild type recombinant (HRP‐C*) and plant (HRP‐C) enzymes. Presteady‐state kinetic measurments of the rate constant for compound I formation (k1) revealed an eightfold decrease in the reactivity of the Phe41→Val variant towards H2O2, in comparison with HRP‐C or HRP‐C*. Measurement of the remaining rate constants, K2 and k3, for the two single‐electron reduction reactions of [F41V] HRP‐C with para‐aminobenzoic acid as reducing substrate, showed that the were 2.5‐fold and 1.3‐fold faster, respectively. In contrast, analysis of data from steady‐state assays with 2,2′‐azinobis(3‐ethylbenzthiazoline‐6‐sulphonate) as reducing substrate, showed decreased reactivity of the mutant enzyme to this compound, indicating a change in substrate specificity. Over the substrate range studied, the data for HRP‐C* and for [F41V] HRP‐C conformed to a simple modification of the accepted peroxidase mechanism in which first‐order step (ku), assumed to be product dissociation, becomes rate‐limiting under our standard assay conditions. Calculation of rate constants from steady‐state data yielded values of k1 for both enzyme forms in adequate agreement with those from pre‐steady state measurements. They showed, furthermore, that both k3 for 2,2′‐azinobis(3‐ethylbenzthizzoline‐6‐sulphonate) and ku were substantially decreased, fivefold and tenfold, respectively in the mutant. Analogous to the decrease in ku, we observed a towfold increase in the affinity of the mutant variant for the inhibitor benzhydroxamic acid. The coordination‐state equilibrium of the haem iron also appeared shifted towards the hexacoordinate high‐spin form. These observations indicate that in additions to affecting reactivity to H2O2, mutations in the distal region and close to the haem iron also affect reactivity towards different reducing substrates, inducing perturbations in the neighbourhood of the aromatic‐substrate‐binding site, known to be 0.8–1.2 nm from the haem iron.
Structural studies by proton‐NMR spectroscopy of plant horseradish peroxidase C, the wild‐type recombinant protein from Escherichia coli and two protein variants, Phe41 → Val and Arg38 → Lys
Authors: VEITCH Nigel C. , WILLIAMS Robert J. P. , BRAY Robert C. , BURKE Julian F. , SANDERS Stephen A. , THORNELEY Roger N.F. , SMITH Andrew T.
Published: March 2005

Wild‐type recombinant horseradish peroxidase isoenzyme C and two protein variants, Phe41 → Val anfd Arg38 → Lys, have been characterised using both one‐ and two‐dimensional NMR spectroscopy. Proton NMR spectra recorded in both resting and cyanide‐ligated states of the proteins were compared with those of the corresponding plant peroxidase. The latter contains 18% carbohydrate in eight N‐linked oligosaccharide side chains whereas the recombinant proteins are expressed in non‐glycosylated form. The spectra of the plant enzyme and refolded recombinant protein are essentially identical with the exception of carbohydreate‐linked resonances in the former indicating that their solution structures are highly similar. This comparison also identifies classes of carbohydrate resonances in the plant enzymes which provides new information on the local environment and mobility of the oligosaccharide side chains.

Comparison of the spectra of the Cyanide‐ligated states of the two variants and those of plant horseradish peroxidase C indicated that there were significant differences with respect to haem and haem‐linked resonances. These could not be rationalised simply on the basis of the local perturbation expected from a single‐site substitution. The two substititions made to residues on the distal side of the haem apparently influenced the degree of imidazolate character of the proximal His170 imidazole ring thus perturbing the magnetic environment of the haem group. Inspection of the spectra of the Phe41 → Val varient also showed that the resonances of a phenylalanine residue in the haem pocket had been incorrectly made for the Phe41 → Val varient were also a basis to investigate the structure of the complex formed with the aromatic donor molecule, benzhydroxamic acid.
Insulin Stimulates Phosphatidylinositol‐3‐Kinase activity in rat adipocytes
Authors: GOIRGETTI Sophie , BALLOTTI Robert , KOWALSKI‐CHAUVEL Aline , CORMONT Mireille , VAN OBBERGHEN Emmanuel
Published: March 2005

Phosphatidylinositol (PtdIns) 3‐kinase is thought to participate in the signal transduction pathways initiated by the activation of receptor tyrosine kinases including the insulin receptor. To approach the physiological relevance of this enzme in insulin signaling, we studied the activation of PtdIns‐3‐Kinase in adipocytes, a major insulin traget tissure for glucose trasnsport and utilisation. To analyze possible interactions of enzyme with cellular proteins, immunoprecipitations with the following antibodies were performed: (a) anti‐phosphotyrosine antibodies, (b) two antibodies to the 85‐kDa subunit of PtdIns‐3‐Kinase (p85) and (c) an antibody to the 185‐kDa major insulin receptor substrate (p185). We show that in cell extreacts from adipocytes exposed to insulin, and after immunoprecipitation with an anti‐phosphotyrosine antibody and an antibody to p85, we are able to detect a PtdIns‐3‐Kinase activity stimulated by the hormone. Similarly, after immunoprecipitation with an antibody to p185, an increase in the PtdIns‐3‐Kinase activity could be demonstrated. Taken together these results suggest that, upon insulin stimulation of fat cells, PtdIns‐3‐kinase itself is tyrosine phosphorylated and/or associated with an insulin receptor substrrate, such as p185, which could function as a link between the insulin receptor and PtdIns‐3‐kinase.The PtdIns‐3‐Kinase was activated within 1 min of exposure to insulin, and the half‐maximal effect was reached at the same concentration, i.e. 3nM, as for stimulation of the insulin receptor kinase. Subcellular fractionation showed that PtdIns‐3‐Kinase activity was found both in the membranes and in the cytosol. Further, immunoprecipitation with an antibody to p85, which possesses the capacity to activate PtdIns‐3‐Kinase, suggests that the presence of the enzyme in the membrane may be due to an insulin‐induced recuitment of the PtdIns‐3‐Kinase from the cytosol to the membrane. Finally, we used isproterenol, which exerts antogonistic effects on insulin action. This drug was found to inhibit both the PtdIns‐3‐Kinase and the insulin receptor activation by insulin, suggesting that the activation of the PtdIns‐3‐Kinase was closely regulated by the insulin receptor tyrosine kinase. The occurrence of an insulin‐stimulated PtdIns‐3‐kinase in adipocytes leads us to propose that this enzyme might be implicated in the generation of metabolic responses induced by insulin.
Detailed structural analysis of asparagine‐linked oligosaccharides of the nicotinic acetylcholine receptor from Torpedo californica
Authors: SHOJI Hiroki , TAKAHASHI Noriko , NOMOTO Hiroshi , ISHIKAWA Masami , SHIMADA Ichio , ARATA Yoji , HAYASHI Kyozo
Published: March 2005

The structures of the major oligosaccharide moieties of the nicotinic acetylcholine receptor (AcChoR) protein from Torpedo californica have been reported [Nomoto, H., Takahashi, N., Nagaki, Y., Endo, S., Arata, Y. and Hayashi, K. (1986) Eur. J. Biochem. 157, 233–242] to be high‐mannose types. Here we report detialed analysis of the structure of the remaining oligosaccharides in this receptor.

The sialylated oligosaccharides released by glycopeptidase (almond) digestion were separated according to the number of sialic acid residues using high‐performance anion‐exchange chromatography with pulsed amperometric detection. After removal of sialic acid from each fraction, the resulting neutral oligosaccharides were separately pyridylaminated and were analyzed by a combination of sequential exoglycosidase digestion and HPLC, then identified on a two‐dimensional sugar map. The structures of two desialylated pyridylamino‐oligosaccharides were further analyzed by high‐resolution proton NMR.

Each oligosaccharide was composed of species containing varying numbers of sialic acids. The desialylated complex‐type oligosaccharides of AcChoR consisted of ten, eight and one different biantennary, triantennary tetraantennary oligosaccharide, respectively. The biantennary oligosaccharides were divided into two groups; oligosaccharides with fucose at the proximal N‐acetylglucosamine (six varities) and oligosaccharides without fucose (four varieties). Each group consisted of species differing in the number of terminal galactose residues. The major component of the biantennary oligosaccharides had two galactose residues at the non‐reducing termini. The terminal α‐galactose residue(s) linked to C3 of β‐galactose were found in the fucose‐containing biantennary oligosaccharides (two varieties). The triantennary oligosaccharides were also divided into two groups; oligosaccharides with (four varieties) and without (four varieties) besecting N‐acetylglucosamine. These groups were composed of species differing in the number of terminal galactose residues. The major component of the triantennary oligosaccharides was fully galactosylated with three galactose residues. An unusual group, Galβ1‐3GlcNAc, was present in low levels in the triantennary oligosaccharides. In contrast, the tetraantennary oligosaccharide was composed of only species, which is fully galactosylated with four galactose residues.
In vivo and in vitro characterization of overproduced colicin E9 immunity protein
Authors: WALLIS Russell , REILLY Ann , ROWE Arthur , MOORE Geoffrey R. , JAMES Richard , KLEANTHOUS Colin
Published: March 2005

We report the overproduction of the immunity protein for the DNase colicin E9 and its characterization both in vivo and in vitro. The genes for colicin immunity proteins are normally co‐expressed from Col plasmids with their corresponding colicins. In the context of the enzymatic colicins, the two proteins form a complex, thereby protecting the host bacterium from the antibiotic activity of the colicin. This complex is then released into the medium, whereupon the colicin alone translocates (through the appropriate receptor) into sensitive bacterial strains, resulting in bacterial cell death. The immunity protein for colicin E9 (Im9) has been overproduced in a bacterial host in the absence of its colicin, to enable sufficient material to be isolated for structural studies. As a prelude to such studies, the in‐vivo and in‐vitro properties of overproduced Im9 were analysed. Electrospray mass spectrometry verified the molecular mass of the purified protein and analytical ultracentrifugation indicated that the native protein approximates a symmetric monomer. Fluorescense‐enhancement and gel‐filtration experiments show that purified Im9 binds to colicin E9 in a 1:1 molar ratio and that this binding neutralizes the DNase activity of the colicin. These results lay the foundations for a full biophysical and structural characterization of the colicin E9 DNase inhibitor protein, Im9.
Photochemical trapping of a bacteriopheophytin anion in site‐specific reaction‐center mutants from the photosynthetic bacterium Rhodobacter sphaeroides
Authors: GRAY Kevin A. , WACHTVEITL Josef , OESTERHELT Dieter
Published: March 2005

The mutant YY in the rection center of Rhodobacter sphaeroide, in which Phe181 on the L chain has been replaced by Tyr, and the double mutant FY, With Tyr210 on th M chain replaced by Phe and Phe181 on the L chain replaced by Tyr, have been constructed by site‐directed mutagenesis. The studies described here were performed to complement a previous mutational analysis of mutant FF with Tyr210 replaced by Phe. Both new strains grow photohererotrophically. The optical absorption spectra of reaction centers isolated from these mutants have band shifts attributable to the monomer bacteriochlorophylls in the vicinity of the substitutions. Photochemical trapping of the bacteriopheophytin anion (I−) indicates that the bacteriopheophytin on the B branch is reduced to a much greater extent in FF and FY as compared to YY and wild‐type YF. Low temperature (77K) absorption spectra clearly show that in the wild‐type (YF) and YY rection centers only the 545‐nm‐absorbing bacteriopheophytin is reduced while in the FF and FY reaction centers both the 535‐nm and 545‐nm‐absorbing bacteriopheophytins are reduced. A simple kinetic analysis is used to ecplain these results. This analysis suggests that, in order for the observed trapping results to occur, a decrease in the ‘cycling’ time must take place, that is changes in the rate(s) of charge recombination must accompany the already known decrease in the forward election transfer rate.
The protein of Mr 21 000 constituting the prosome‐like particle of duck erythroblasts is homologous to apoferritin
Authors: COUX Olivier , CAMOIN Luc , NOTHWANG Hans‐Gerd , BEY Fayçal , SILVA PEREIRA Ildinete , KEITH Gérard , STROSBERG Arthur‐Donny , SCHERRER Klaus
Published: March 2005

In duck erythroblasts, two major populations of untranslated messenger (m) RNP can be separated by sucrose gradient centrifugation in low ionic strength. One of these contains globin mRNA associated to protein factors, among them the prosomes. The other, sedimenting in the 35S zone, contains non‐globin mRNA. From this ‘35S’ mRNP, a new RNP particle called the prosome‐like particle was isolated and characterized [Akhayat, O., Infante, A. A., Infante, D., Martins de Sa, C., Grossi de Sa, M.‐F. & Scherrer, K. (1987) Eur. J. Biochem. 170, 23–33]. The PLP is a multimer of a protein of Mr 21000, and contains small RNA species. The particle is tightly associated with repressed mRNA and inhibits in vitro protein synthesis. We show here that the protein of Mr 21000, constituting the prosome‐like particle, is apoferritin. Different approaches confirm the RNP character of this particle and provide evidence that some of its RNA species are tRNA. The hypothesis is discussed as to whether (apo–)ferritin might serve other functions in addition to iron storage.
Biogenesis of the yeast vacuole (lysosome)
Authors: HIRSCH Hans H. , SCHIFFER Hans H. , WOLF Dieter H.
Published: March 2005

The vacuolar proteinase yscB (PrB) has been implicated in the final maturation of procarboxypeptidase yscY (pro‐CpY) to the mature wild‐type form CpYb of 61 kDa. In PrB‐deficient mutants, only the proteinase yscA processed form CpYa of 62 kDa is found [Mechler, B., Müller, H. & Wolf, D. H. (1987) EMBO J. 6, 2157–2163]. We report now that, akin to CpY, two forms of mature proteinase yscA (PrA) can be distinguished. In PrB‐deficient mutant cells, PrAa, migrating at about 43 kDa in SDS/PAGE, is found, whereas PrAb, found in wild‐type cells, had the known molecular mass of 42 kDa. In the PrB‐deficient strain, pro‐PrA and pro‐CpY matured only to the higher‐molecular‐mass forms, PrAa and CpYa, and the maturation of both precursors was slower than in the isogenic wild‐type strain. Pulse‐labeling experiments indicated that the mature forms, PrAb or CpYb, are generated directly in the PrB‐containing wild‐type strain in vivo. In vitro experiments showed that PrB is able to trigger maturation of its 42‐kDa pro‐PrB precursor to mature PrB in the absence of PrA. Mature PrB and its proteolytic activity, however, shows a higher stability in the presence of mature PrA. The data indicate a molecular and kinetic participation of proteinase yscB in vacuolar hydrolase precursor maturation.
α1(VIII)‐collagen gene transcripts encode a short‐chain collagen polypeptide and are expressed by various epithelial, endothelial and mesenchymal cells in newborn mouse tissues
Authors: MURAGAKI Yasuteru , SHIOTA Chiyo , INOUE Masaya , OOSHIMA Akira , OLSEN Bjorn Reino , NINOMIYA Yoshifumi
Published: March 2005

Type‐VIII collagen is a major constituent of Descemet's membrane and contains two genetically distinct α chains, α1(VIII) and α2(VIII). We have previously cloned the human α1(VIII) and α2(VIII) genes and determined that they are located on chromosomes 3 and 1, respectively. Comparison of the α1(VIII) and α2(VIII) genes with the α1(X)‐collagen gene showed that the structure of the three genes and the sequence of their collagen polypeptides were strikingly similar. Therefore, we have grouped the three genes in a common subclass of collagens which we have named the short‐chain collagens because of the relatively small size of their triple‐helical domains. In the present study, we have isolated and characterized a mouse gene fragment encoding the entire mouse α1(VIII)‐collagen chain and determined the complete primary structure of the polypeptide chain. The size of mouse α1(VIII)‐collagen mRNA, as estimated by Northern‐blot analysis, was 4.2 kb, compared with 2.8 kb previously reported for the corresponding rabbit mRNA. By cloning and sequencing of four overlapping cDNA, we demonstrate that this larger size of mouse α1(VIII) mRNA is due to a larger 3′ untranslated region in the mouse transcript. Using the gene fragment as a probe, we performed Northern‐blot hybridization analysis of RNA prepared from newborn mice and demonstrated that α1(VIII) collagen mRNA is expressed at high levels in the calvarium, eye and skin. In situ hybridization revealed that α1(VIII) RNA is present in skin keratinocytes, corneal epithelial and endothelial cells, lens epithelial cells, as well as mesenchymal cells surrounding cartilage and calvarial bone and in the meninges surrounding the brain.
Structural and functional characterisation of recombinant human haemoglobin A expressed in Saccharomyces cerevisiae
Authors: COGHLAN David , JONES Gareth , DENTON Katharine A. , WILSON Michael T. , CHAN Bernard , HARRIS Roy , WOODROW John R. , OGDEN Jill E.
Published: March 2005

Recombinant human HbA, produced by co‐expressing α‐globin and β‐globin chains in the yeast Saccharomyces cerevisiae, has been characterised extensively both physically and functionally. Structural studies using N‐terminal sequence analysis, peptide mapping, amino acid composition analysis and electrospray MS demonstrated that the recombinant protein was identical to standard HbA purified from erythrocytes. The functional properties of the recombinant protein were assessed using equilibrium and kinetic measurements of oxygen and carbon monoxide binding. The oxygen‐binding studies demonstrated that the yeast‐derived HbA behaved as a fully functional, cooperative tetramer (Hill coefficient, 2.9), exhibited a normal Bohr effect and response to phosphate, and displayed a rate of oxygen dissociation identical to that of the native human molecule. The recombinant protein also showed the same characteristics of carbon monoxide combination as the standard protein. These studies demonstrate that yeast provides an ideal system for the production of Hb for structural and functional analysis and a potentially useful source of HbA for formulation into a Hb‐based oxygen carrier.
Penicillin‐binding protein 2x of Streptococcus pneumoniae
Authors: LAIBLE Götz , KECK Wolfgang , LURZ Rudi , MOTTL Harald , FRÈRE Jean‐Marie , JAMIN Marc , HAKENBECK Regine
Published: March 2005

A 2.5‐kb DNA fragment including the structural gene coding for the penicillin‐binding protein 2x (PBP 2x) of Streptococcus pneumoniae has been cloned into the vector pJDC9 and expressed in Escherichia coli. Mapping of RNA polymerase binding sites by electron microscopy indicated that the pbpX promoter is well recognized by the E. coli enzyme. However, high‐level expression occurred mainly under the control of the lac promoter upstream of the pJDC9 multiple cloning site. After induction with isopropyl β‐d‐thiogalactopyranoside, PBP 2x was expressed as one of the major cellular proteins. PBP 2x produced in E. coli corresponded to the pneumococcal PBP 2x in terms of electrophoretic mobility, fractionation with the cytoplasmic membrane, and penicillin‐binding capacity. Deletion of 30 hydrophobic N‐terminal amino acid residues at positions 19–48 resulted in high‐level expression of a cytoplasmic, soluble PBP 2x derivative (PBP 2x*) which still retained full β‐lactam‐binding activity. A two‐step procedure involving dye affinity chromatography was established for obtaining large amounts of highly purified enzymatically active PBP 2x*.
A model system of coupled activity of co‐immobilized creatine kinase and myosin
Authors: ARRIO‐DUPONT Martine , BÉCHET Jean‐Jacques , d'ALBIS Anne
Published: March 2005

Myosin and creatine kinase were co‐immobilized onto Immunodyne films to mimic the behaviour of creatine kinase bound to the M‐line of myofilaments. The Mg‐ATPase activity of bound myosin was studied by a coupled enzymatic assay, which detects Mg‐ADP in the bulk solution by means of pyruvate kinase and lactate dehydrogenase. The competition for Mg‐ADP between pyruvate kinase and creatine kinase either free in solution or co‐immobilized with myosin was studied at various creatine phosphate concentrations. Bound creatine kinase competed efficiently when present in very low amounts, corresponding to an activity ratio higher than 1:20 000 between creatine kinase and pyruvate kinase and a molar ratio higher than 1:1000 between creatine kinase and myosin. The Mg‐ADP produced by myosin ATPase in the vicinity of the film did not diffuse into the bulk solution but, in the presence of creatine phosphate, was recycled into Mg‐ATP by the neighbouring creatine kinase. The existence of an unstirred layer near the surface of the film is sufficient to explain the channeling of ADP (or ATP) between co‐immobilized myosin and creatine kinase, without direct interaction or ‘intimate coupling’ between the enzymes. The problem now is to determine the importance of this kind of facilitated diffusion in the myofilaments in vivo.
Regulation of the mammalian carbamoyl‐phosphate synthetase II by effectors and phosphorylation
Authors: SHAW Sean M. , CARREY Elizabeth A.
Published: March 2005

We have measured the ‘core’ mammalian carbamoyl‐phosphate synthetase II (CPSII) activity, using NH4Cl as the nitrogen‐donating substrate and trapping carbamoyl phosphate as urea through its reaction with ammonium ions. When ATP and magnesium ion concentrations are close to those found in the cell, the substrate saturation curves for ammonia and bicarbonate are hyperbolic, giving Km (NH3) values of 166 μM at high ATP concentrations and 26 μM at low ATP concentrations, while the Km (bicarbonate) is 1.4 mM at both ATP concentrations used. These values for the Km (NH3) are lower than previously reported for CPS II, and closer to the values for the mitochondrial counterpart. The Km for ammonia and bicarbonate are not altered by phosphorylation of the multienzyme polypeptide CAD, which contains the first three enzyme activities of pyrimidine biosynthesis. The CPS II activity is lower with an excess of either ATP or magnesium ions, causing the apparently sigmoid dependence of actviity upon ATP concentration to be enhanced at low concentrations of free magnesium ions. The feedback inhibitor, UTP, acts by stabilising a state with a low affinity for magnesium ions and for ATP. In the presence of the activator, 5‐phosphoribosyl diphosphate (PRibPP), the enzyme has a higher affinity for magnesium ions and thus the ATP dependence of the activity is hyperbolic. Phosphorylation of CAD similarly activates the CPS II enzyme by increasing the affinity for magnesium ions and by pushing the equilibrium away from the low‐affinity UTP‐stabilised state. Using our improved assay procedure, we observe a very large activation by PRibPP of carbamoylphosphate synthesis at low concentrations of magnesium ions, and we find that unlike UTP, the activator PRibPP is able to act on the phosphorylated enzyme.
Stimulation of a membrane tyrosine phosphatase activity by somatostatin analogues in rat pancreatic acinar cells
Authors: COLAS Begona , CAMBILLAU Chantal , BUSCAIL Louis , ZEGGARI Mustapha , ESTEVE Jean‐Pierre , LAUTRE Valérie , THOMAS Francçois , VAYSSE Nicole , SUSINI Christiane
Published: March 2005

A phosphoryl protein tyrosine phosphatase (PTPase) activity has been characterized in rat pancreatic acinar membranes using 32P‐labeled poly(Glu,Tyr) as substrate. Acinar membranes exhibited a high affinity for the substrate, with an apparent Km of 0.46 μM and an apparent Vmax of 0.9 nmol · mg protein−1· min−1. Acinar membrane PTPase activity displayed specific characteristics of other PTPases; it was inhibited by the inhibitors Zn2+, orthovanadate and by the divalent cations Mn2+ and Mg2+, and was stimulated by the reducing‐agent dithiothreitol. It was also inhibited by soybean trypsin inhibitor and stimulated by trypsin. Gel permeation of pancreatic acinar membranes gave a single peak of enzyme activity with an apparent molecular mass of 70 000 Da. Further purification by HPLC on DEAE revealed two peaks of PTPase activity at 120 mM and 180 mM NaCl. These two peaks reacted in a Western‐blot procedure with anti‐(peptide) serum directed towards conserved domain of PTPase as a common 67‐kDa form associated with lower‐molecular‐mass proteolytic fragments (31–56 kDa). Incubation of pancreatic acini with somatostatin analgoues, SMS 201–995 or BIM 23014, resulted in a stimulation of membrane PTPase activity. The stimulation was rapid and transient, with a maximal level reached within 15 min of addition. The two analogs stimulated PTPase activity in a dose‐dependent manner with half‐maximal activation occurring at 7 pM and 37 pM and maximal activation at 0.1 nM and 0.1–1 nM for SMS 201–995 and BIM 23014, respectively. The stimulated‐membrane PTPase activity also eluted at an apparent molecular mass of 70 kDa in gel‐permeation chromatography. The two analogs inhibited the binding of [125I‐Tyr3]SMS 201–995 to pancreatic acinar membranes with similar relative potencies to that observed on stimulation of PTPase activity. We conclude that pancreatic acinar membranes possess a low‐molecular‐mass PTPase which is stimulated by somatostatin analogs at concentrations involving activation of membrane somatostatin receptors.
Purification and characterization of a thermostable proteinase isolated from Thermus sp. strain Rt41A
Authors: PEEK Keith , DANIEL Roy M. , MONK Colin , PARKER Lynne , COOLBEAR Tim
Published: March 2005

Thermus sp. strain Rt41A produces an extracellular thermostable alkaline proteinase. The enzyme has a high isoelectric point (10.25–10.5) which can be exploited in purification by using cation‐exchange chromatography. The proteinase was purified to homogeneity and has a molecular mass of 32.5 kDa by SDS/PAGE. It is a glycoprotein, containing 0.7% carbohydrate as glucose equivalents, and has four half‐cystine residues present as two disulphide bonds. Maximum proteolytic activity was observed at pH 8.0 against azocasein and greater than 75% of this activity was retained in the pH range 7.0–10.0. Substrate inhibition was observed with casein and azocasein. The enzyme was stable in the pH range 5.0–10.0 and maximum activity, in a 10‐min assay, was observed at 90°C with 5 mM CaCl2 present. No loss of activity was observed after 24 h at 70°C and the half‐lives at 80°C and 90°C were 13.5 h and 20 min, respectively. Removal of Ca2+ reduced the temperature for maximum proteolytic activity against azocasein to 60°C and the half‐life at 70°C was 2.85 min. The enzyme was stable at low and high ionic strength and in the presence of denaturing reagents and organic solvents.

Rt41A proteinase cleaved a number of synthetic amino acid p‐nitrophenol esters, the kinetic data indicating that small aliphatic or aromatic amino acids were the preferred residue at the P1 position. The kinetic data for the hydrolysis of a number of peptide p‐nitroanilide substrates are also reported. Primary cleavage of the oxidized insulin B chain occurred at sites where the P1′ amino acid was aromatic. Minor cleavage sites (24 h incubation) were for amino acids with aliphatic side chains at the P1′ position. The esterase and insulin cleavage data indicate the specificity is similar for both the P1 and P1′ sites.
Glycosphingolipids in cestodes
Authors: DENNIS Roger D. , BAUMEISTER Stefan , GEYER Rudolf , PETER‐KATALINIC Jasna , HARTMANN Rudolf , EGGE Heinz , GEYER Egbert , WIEGANDT Herbert
Published: March 2005

The presence of glycosphingolipids in the metacestodes of the fox tapeworm, Taenia crassiceps, has been established. The normal‐phase TLC pattern of the neutral‐fraction glycolipids revealed groups of bands corresponding to homologous components of increasing sugar chain length. The three simplest glycolipid components have been isolated and their chemical constitution determined as being of the neogala series: Galβ1Cer, Galβ6Galβ1Cer and Galβ6Galβ6Galβ1Cer. The ceramide tetrasaccharide fraction has been found to consist of a mixture of neogalatetraosylceramide, as an elongation of the neogala series, Galβ6Galβ6Galβ6Galβ1Cer and the component Galα4Galβ6‐Galβ6Galβ1Cer (both occurring in approximately equimolar proportions). The long‐chain bases of the ceramide monogalactoside, digalactoside, trigalactoside and tetragalactosides contain, as well as small amounts of sphingosine, predominantly dihydrosphingosine/phytosphingosine in the approximate ratios 1.7:1, 1.4:1, 1:1 and 2.3:1, respectively. The major ceramide fatty acids have particularly long chains, with hexacosanoic and octacosanoic acids predominating. Upon reverse‐phase TLC, the glycolipid components ceramide monogalactoside, digalactoside and trigalactoside were each separable into five component bands. Parent glycolipid components therefore show component band distributions comparable to one another in being governed by similar ceramide constitutions.
Sequence analysis of photoaffinity‐labelled peptides derived by proteolysis of photosystem‐2 reaction centres from thylakoid membranes treated with [14C]azidoatrazine
Authors: WHITELEGGE Julian P. , JEWESS Philip , PICKERING Margaret G. , GERRISH Christopher , CAMILLERI Patrick , BOWYER John R.
Published: March 2005

Photosystem‐2 reaction centres were prepared from pea thylakoid membranes that had been photoaffinity labelled with [14C]‐azidoatrazine (2‐azido‐4‐ethylamino‐6‐isopropylamino‐s‐triazine), a derivative of the herbicide atrazine which binds to the secondary plastoquinone electron‐acceptor site of photosystem 2. SDS/PAGE of the 14C‐labelled reaction centres followed by fluorography revealed photoaffinity‐labelled proteins of apparent molecular masses 30 kDa and 55 kDa, which corresponded to the D1 polypeptide and to an SDS‐stable heterodimer of the D1 and D2 polypeptides, respectively. To obtain sequence information on the site of photoaffinity labelling, an 8‐kDa photoaffinity‐labelled peptide, generated by proteolysis of the reaction‐centre material with trypsin, was isolated and purified to apparent homogeneity using reverse‐phase and size‐exclusion HPLC techniques. The amino terminus of the photoaffinity‐labelled peptide was determined to be Leu‐Gly‐Met‐Arg‐Pro‐Xaa‐Ile‐Ala‐Val‐Ala‐Tyr by Edman sequencing. This corresponds to the amino terminus of a predicted tryptic peptide of D1 and confirms that azidoatrazine photolabels the D1 polypeptide of photosystem 2 in the region Leu137 – Arg225. Chymotrypsin/trypsin digestion of photoaffinity‐labelled reaction centres followed by reverse‐phase HPLC was used to isolate a smaller photoaffinity‐labelled peptide. On Edman sequencing, Ser‐Ala were identified as the first two residues and 14C was released on the third cycle, after which further degradation was blocked. The two potential peptide fragments with Ser‐Ala at the amino terminus in the region Leu137 – Arg225 are Ser148‐Ala‐Pro and Ser212‐Ala‐Met. Proline is an unlikely target for reaction with the nitrene of the photoactivated azidoatrazine, and the data are thus consistent with Met214 as the site of photoaffinity labelling on D1 when thylakoid membranes are illuminated with ultraviolet irradiation in the presence of [14C]azidoatrazine.
The effect of amino acid substitution at position 219 of Citrobacter freundii cephalosporinase on extension of its substrate spectrum
Authors: TSUKAMOTO Kikuo , OHNO Reiko , NUKAGA Michiyoshi , SAWAI Tetsuo
Published: March 2005

The cephalosporinase of Citrobacter freundii GN346 is a class‐C β‐lactamase comprising 361 amino acids. The substitution of the glutamic acid at position 219 in the enzyme by lysine was previously shown to broaden its subrate specificity to unfavorable substrates such as oxyimino cephalosporins [Tsukamoto, K., Ohno, R. & Sawai, T. (1990) J. Bacteriol. 172, 4348–4351]. To investigate the cause of this phenomenon, Glu219 was changed to glutamine, cysteine or tryptophan. All the resultant enzymes showed higher cefuroxime‐hydrolytic activities than the wild type, the order of increasing cefuroxime‐hydrolytic activity being as follow: Trp > Lys > Cys > Gln > Glu. The rate of hydrolysis of cefuroxime by the Trp219 enzyme was approximately 3×104 times that of the wild‐type enzyme. The order of increasing cefuroxime hydrolysis was approximately proportional to the molecular volume of the amino acid substituted and independent of the ionic character of the amino acid. The cysteine residue at position 219 in the Cys219 enzyme allowed its complete reaction with an SH‐blocking reagent, 4‐chloromercuriphenylsulfonic acid. The modified enzyme with the bulkier residue showed a 45% higher cefuroxime‐hydrolytic activity than the untreated enzyme. These results suggested that extension of the substrate spectrum may be attributed to alteration in the configuration of the enzyme around position 219.
A method for the generation of monoclonal antibodies against rare cell‐surface molecules
Authors: KAYYEM Jon F. , ROMAN Janet M. , VON BOXBERG Ysander , SCHWARZ Uli , DREYER William J.
Published: March 2005

Monoclonal antibodies provide a powerful tool for the identification and analysis of novel cell‐surface molecules. We present here a method for antigen preparation and an immunization protocol that facilitates generation of mAb reactive with cell‐surface molecules of low abundance and/or low antigenicity. The procedure involves isolation and extensive fractionation of cell‐surface and detergent‐soluble extracellular‐matrix molecules prior to immunization. Cell‐surface proteins on intact tissue are biotin‐labeled using a reagent that does not penetrate cells. Avidin affinity chromatography is then used to purify these biotinylated molecules. Size‐exclusion HPLC is used to separate these surface molecules on the basis of apparent molecular mass. Finally, immunization with antigen coupled to keyhole‐limpet hemocyanin is combined with long‐term booster immunizations to generate a hyperimmune response resulting in high‐affinity IgG.

A test application of this approach was aimed at the generation of mAb against cell‐surface molecules of approximately 135 kDa in the developing chicken retinotectal system. Immunochemical analyses using antibodies produced by this approach which showed restricted patterns of tissue staining reveal that mAb were generated against all previously identified immunoglobulin superfamily molecules of this size in this system. Furthermore, we produced many additional antibodies that labeled retinotectal tissue in novel staining patterns. In the two cases analyzed in detail, these new patterns reflect the distributions of previously uncharacterized members of the immunoglobulin superfamily. The success of this initial study suggests that this method may represent a broadly applicable approach towards the preparation of extensive libraries of antibodies against cell‐surface molecules expressed on cells from numerous sources.