bluuebunny/crossref_metadata_2025 huggingface dataset:
The complete primary structure of the human α1(VIII) chain and assignment of its gene (COL8A1) to chromosome 3
Authors: MURAGAKI Yasuteru , MATTEI Marie‐Genevieve , YAMAGUCHI Noriko , OLSEN Bjorn R. , NINOMIYA Yoshifumi
Published: March 2005

Type VIII collagen molecules, expressed by corneal and vascular endothelial cells, appear to be heterotrimers composed of two genetically distinct polypeptides, α1(VIII) and α2(VIII), in the ratio of 2:1. Characterization of the rabbit α1(VIII) gene has demonstrated that it consists of only four exons, one of which is large and encodes the entire triple‐helical and carboxyl non‐triple‐helical domains. A similar exon organization has been found for the chicken α(X) and the mouse and human α2(VIII) collagen genes. The genes encoding α1(VIII), α2(VIII), and α(X) collagen chains are therefore homologous members of a unique class of genes within the collagen superfamily. In the present paper we describe for the first time the primary structure of the human α1(VIII) collagen chain, based on isolation and sequencing of a genomic DNA fragment. The results indicate that the human α1(VIII) chain has the same domain structure as the rabbit protein. We also demonstrate that the gene is localized on the long arm of the human chromosome 3. The availability of genomic DNA encoding the human α1(VIII) collagen chain and information about its chromosomal location should make it possible to examine whether hereditary diseases are linked to abnormalities in the structure or expression of the α1(VIII) gene.
Yeast carboxypeptidase Y requires glycosylation for efficient intracellular transport, but not for vacuolar sorting, in vivo stability, or activity
Authors: WINTHER Jakob R. , STEVENS Tom H. , KIELLAND‐BRANDT Morten C.
Published: March 2005

Functions of the carbohydrate side chains of the yeast vacuolar enzyme carboxypeptidase Y (CPY) were investigated by removal, through site‐directed mutagenesis, of the sequences which act as signals for N‐linked glycosylation. The mutant forms of the enzyme were analysed with respect to activity and intracellular sorting, and the stabilities in vivo and in vitro were studied. It was found that carbohydrate was not important for accurate vacuolar targeting of CPY, but that the rate of transport of the unglycosylated CPY through the secretory pathway to the vacuole was reduced. Tunicamycin, which inhibits the formation of asparagine‐linked glycosylation, had a similar effect on the transport of CPY at 23°C. However, the absence of N‐linked carbohydrate in general had the more dramatic result of blocking the transport of CPY altogether at an increased temperature (37°C). The unglycosylated mutant CPY was not temperature sensitive for transport in the absence of tunicamycin. Analysis of mutant enzymes containing a single glycosyl residue at each of the four positions showed that the residue at position 87 was particularly important for transport. There was no decrease in the intracellular stability of the completely unglycosylated enzyme, and in vitro the rate of heat inactivation of this species was not increased.
Primary structure of a 15‐kDa protein from rat intestinal epithelium
Authors: KANDA Tatsuo , ODANI Shoji , TOMOI Masaaki , MATSUBARA Yoichi , ONO Teruo
Published: March 2005

An abundant and novel cytosolic protein was purified from the rat intestinal epithelium by gel filtration, ionexchange and hydroxylapatite chromatography. The protein was eluted into two different positions (fractions 1 and 2) on DEAE‐cellulose chromatography. We have completed the primary structure of the protein of fraction 1 by Edman degradation. The protein (144 565 Da) contains 127 amino acid residues and has an acetylated alanine at its NH2‐terminus. Comparison of the primary structure of the protein with porcine gastrotropin [Walz, A. D., Wider, M. D., Snow, J. W., Dass, C. & Desiderio, D. M. (1988) J. Biol. Chem. 263, 14189–14195] and rat hepatic fatty‐acid‐binding protein revealed that identical residues within these proteins are found in 90 and 54 out of a total of 127 positions, respectively.

Bioactivity studies demonstrated that neither the protein nor liver and intestinal fatty‐acid‐binding proteins influence gastric acid secretory activity in rats with gastric fistulas compared to pentagastrin. The protein showed very low affinity for palmitic‐acid‐binding in vitro assay system and only trace amounts of endogenous fatty acids were detected from the protein.

The protein, rat intestinal 15‐kDa protein is considered to be a new member of the fatty‐acid‐binding protein family based on its structural features.
On the FAD‐induced dimerization of apo‐lipoamide dehydrogenase from Azotobacter vinelandii and Pseudomonas fluorescens
Authors: van BERKEL Willem J. H. , BENEN Jacques A. E. , SNOEK Marja C.
Published: March 2005

The apoenzymes of lipoamide dehydrogenase from pig heart and from Pseudomonas fluorescens were prepared at pH 2.7 and pH 4.0, respectively, using a hydrophobic interaction chromatography procedure recently developed for lipoamide dehydrogenase from Azotobacter vinelandii and other flavoproteins [Van Berkel et al. (1988) Eur. J. Biochem. 178, 197–207].

The apoenzyme from pig heart, having 5% of residual activity, shows an equilibrium between the monomeric and dimeric species. Both the yield and the degree of reconstitution of dimeric holoenzyme is 75% of starting material under optimal conditions. The kinetics of reconstitution of pig heart apoenzyme differ slightly from that obtained with the apoenzyme prepared by acid ammonium sulfate precipitation at pH 1.5 [Kalse, J. F. and Veeger, C. (1968) Biochem. Biophys. Acta 159, 244–256].

The apoenzyme from P. fluorescens is in the monomeric state and shows negligible residual activity. The yield and degree of reconstitution of the dimeric holoenzyme is more than 90% of startng material.

Reconstitution of the apoenzymes from A. vinelandii and P. fluorescens involves minimally a two‐step sequential process. Initial flavin‐binding results in regaining of full dichloroindophenol activity, quenching of tryptophan fluorescence and strong increase of FAD fluorescence polarization. In the second step, dimerization occurs as reflected by regain of lipoamide activity, strongly increased FAD fluorescence and increased hyperchroism of the visible absorption spectrum.

The kinetics of FAD‐induced dimerization are strongly dependent on the apoenzyme used. At 0°C, the monomeric apoenzyme‐FAD complex is either stabilized (P. fluorescens) or only transiently detectable (A. vinelandii). Dimerization of P. fluorescens enzyme is strongly stimulated in the presence of NADH.
Assembly and disulfide rearrangement of recombinant surfactant protein A in vitro
Authors: HASS Claus , VOSS Tilman , ENGEL Jürgen
Published: March 2005

The surfactant‐associated protein, protein A, produced by transgenic Chinese hamster ovary cells exhibits a heterogeneous population of structures. Electron microscopy reveals lollipop‐shaped monomers consisting of a collagenous triple helix and a globular domain as well as oligomers in which two, three or more protomers are connected by their collagenous stalks. Each protomer consists of three α‐chains (36 kDa) but under non‐reducing conditions few free α‐chains are observed by SDS/PAGE. Instead γ‐components (three chains), γ2 (six chains) and higher components are observed which are derived from intra‐and inter‐protomer disulfide cross‐linking. Complete reduction at low temperature dissociates the oligomers, but preserves the intact structure of monomers as demonstrated by electron microscopy and trypsin digestion. Circular dichroism revealed an unfolding of the collagen triple helices of fully reduced protein A at 26 °C and of the unreduced protein A around 41.5°C. Reoxidation of the fully reduced protein A re‐established mainly the disulfide bonds within the triple helix but not between monomers. Very few higher assembly forms were reformed even at high protein A concentrations. Cellular in vivo systems must possess an efficient assembly mechanism which cannot be simulated by an in vitro system.
Regulation of hepatic energy metabolism by epidermal growth factor
Authors: RASHED Sahar M. , PATEL Tarun B.
Published: March 2005

Employing the non‐recirculating perfused rat liver preparation, we have investigated the regulation of hepatic gluconeogenesis, and metabolic fluxes through the tricarboxylic acid cycle and 2‐oxoglutarate dehydrogenase reaction by epidermal growth factor (EGF) which mimics the actions of both insulin and Ca2+‐mobilizing hormones (e.g. vasopressin). As monitored by the rate of 14CO2 production from [2‐14C]pyruvate (0.5 mM), EGF (10 nM) transiently stimulated the activity of the tricarboxylic acid cycle. EGF also transiently stimulated hepatic gluconeogeneiss from pyruvate. The transient stimulation of tricarboxylic acid cycle activity and gluconeogenesis were accompanied by an increase in perfusate Ca2+ content indicating that EGF also altered hepatic Ca2+ fluxes. EGF‐elicited stimulation of gluconeogenesis was, at least in part, the result of a transient (50%) inhibition of pyruvate kinase activity. Likewise, EGF‐mediated stimulation of tricarboxylic acid cycle activity can, in part, be attributed to EGF‐elicited stimulation of metabolic flux through the mitochondrial, Ca2+‐sensitive, 2‐oxoglutarate dehydrogenase reaction. The regulation of hepatic metabolism by EGF appears to be the manifestation of alteration in cellular Ca2+ content since in experiments performed under conditions known to abolish the ability of EGF to alter cytosolic free‐Ca2+ concentrations, i. e. in livers of pertussis‐toxin‐treated rats, EGF did not alter either perfusate Ca2+ content or any of the metabolic parameters monitored. Additionally, experiments involving pulsatile infusion of either EGF or phenylephrine into livers demonstrated that, unlike the α1‐adrenergic receptor, homologous desensitization of the EGF receptor occurs. Such a homologous desensitization of the EGF receptor can explain the transient nature of EGF‐elicited stimulation of various metabolic processes. Since protein kinase C activation by EGF can lead to receptor desensitization, experiments were performed with phorbol esters which either activate or do not alter protein kinase C activity. While the inactive phorbol ester 4α‐phorbol 12,13‐didecanoate did not modulate the hepatic actions of EGF, activation of protein kinase C by 4β‐phorbol 12‐myristate 13‐acetate (70 nM) abolished the ability of EGF to stimulate gluconeogenesis, tricarboxylic acid cycle activity and metabolic flux through the 2‐oxoglutarate dehydrogenase complex.
Cloning, nucleotide sequence and expression of the cytochrome c‐552 gene from Hydrogenobacter thermophilus
Authors: SANBONGI Yoshihiro , YANG Jun‐Hao , IGARASHI Yasuo , KODAMA Tohru
Published: March 2005

A cytochrome c‐552 gene from a thermophilic hydrogen‐oxidizing bacterium, Hydrogenobacter thermophilus, was cloned by using two oligonucleotide probes, which had been synthesized based on the known amino acid sequence of the protein. A 780‐bp PstI –SphI fragment of the cloned DNA was sequenced and found to contain the entire structural gene coding for cytochrome c‐552 bracketed by apparent Escherichia coli consensus sequences for initiation and termination of transcription. Cytochrome c‐552 is synthesized in vivo as a precursor having an N‐terminal signal sequence consisting of 18 amino acid residues. The cloned cytochrome c‐552 gene without its own signal sequence was introduced into the pKK223‐3 vector and expressed in E. coli upon induction with isopropyl β‐d‐thiogalactoside. An expressed cytochrome c‐552 protein had a methionine residue at the N‐terminus since an initiation signal was introduced before the first amino acid residue of the mature cytochrome c‐552. The heme c was attached to apo‐type cytochrome c‐552 in the cytoplasm of E. coli and the holoprotein had spectral properties, similar to the authentic cytochrome c‐552 from H. thermophilus.
Cellobiose dehydrogenases of Sporotrichum (Chrysosporium) thermophile
Authors: CANEVASCINI Giorgio , BORER Peter , DREYER Jean‐Luc
Published: March 2005

Both cellobiose dehydrogenases of Sporotrichum (Chrysosporium) thermophile, ATCC 42464, obtained after fractionation with DEAE‐Trisacryl chromatography and named cellobiose dehydrogenase I and II have been purified to homogeneity by different chromatographic techniques. Both enzymes are slightly glycosylated flavocytochrome‐b proteins with similar catalytic properties but with distinct molecular masses (91 kDa and 192 kDa for enzymes I and II, respectively) and isoelectric point (4.1 versus 3.45). Examination by SDS/PAGE clearly showed that the larger enzyme II is a homodimer, whose subunit is close to, but different from dehydrogenase I which is homogeneous by this technique. After limited digestion of both enzymes with papain, two main fractions with residual activity are formed, one carrying the heme, the other being the flavin component; each fraction is characterized by its particular chromatographic behaviour. The flavin carrying component shows an atypical (for flavoprotein) three‐banded spectrum indicative of the presence of a flavin derivative. Both enzymes react very slowly with oxygen clearly forming some superoxide radicals and possibly hydrogen peroxide. Cellobiose and other cellodextrins are oxidized at their reducing glycosyl moiety to the corresponding aldonic acid. With the use of the autooxidable phenazinemethosulphate, cellulose (either in a hydrated form or crystalline) is also oxidized at free reducing ends so that appreciable amounts of cellobionic acid are released upon enzymatic hydrolysis.
The chloroplast transcription apparatus from mustard (Sinapis alba L.)
Authors: TILLER Kai , EISERMANN Andrea , LINK Gerhard
Published: March 2005

A chloroplast protein fraction with σ‐like activity [Bülow, S. & Link, G. (1988) Plant Mol. Biol. 10, 349–357], was further purified and characterized. Chromatography on heparin‐Sepharose, DEAE‐Sepharose and Sephacryl S‐300 led to the separation of three σ‐like factors (SLF) polypeptides with Mr 67000 (SLF67), 52000 (SLF52) and 29000 (SLF29). None of these polypeptides bind to DNA itself, but each one confers enhanced binding and transcriptional activity when added to Escherichia coli RNA‐polymerase core enzyme and DNA fragments carrying a chloroplast promoter. SLF67, SLF52, and SLF29 differ in their ionic‐strength requirements for activity. They each mediate the binding to promoters of the chloroplast genes psbA, trnQ, and rps16, with different efficiencies. It is suggested that chloroplast transcription in vivo might be controlled at least in part by these functionally distinct factors.
Isolation and characterization of the human pyruvate kinase M gene
Authors: TAKENAKA Masaru , NOGUCHI Tamio , SADAHIRO Shigeki , HIRAI Haruhiko , YAMADA Kazuya , MATSUDA Tamiko , IMAI Enyu , TANAKA Takehiko
Published: March 2005

Genomic clones containing the human pyruvate kinase M (PKM) gene, which encodes the M1‐type and M2‐type isozymes, were isolated and their exon sequences were determined. The gene is approximately 32 kb and consists of 12 exons and 11 introns. Exons 9 and 10 contain sequences specific to the M1 and M2 types, respectively, indicating that the human isozymes are produced from the same gene by alternative splicing as in the case of the rat gene. The exon‐intron structure of the human PKM gene is identical to that of the rat gene, and the introns of both genes interrupt the exons at the same points. Introns 6 and 7 begin with GC dinucleotide instead of the consensus GT, but the other exon‐intron boundaries are consistent with the GT‐AG rule. The gene is transcribed from multiple start sites. The 5′‐flanking region of the gene contains putative Sp1‐binding sites, but no TATA box or CAAT box, and shows high sequence similarity to that of the rat M gene. Bacterial chloramphenicol acetyltransferase assay revealed that the upstream region between positions −493 and −51 contained a cis‐acting element(s) that was essential for expression of the M gene in HeLa cells. Long stretches of conserved regions were found in the introns around the M1‐specific and M2‐specific exons, suggesting that these regions may be involved in the alternative splicing machinery.
Differentiation of ovine brown adipocyte precursor cells in a chemically defined serum‐free medium
Authors: CASTEILLA Louis , NOUGUÈS Jean , REYNE Yves , RICQUIER Daniel
Published: March 2005

The rapid apparent conversion of brown adipose tissue into white adipose tissue in newborn offspring of large mammals, such as sheep and cattle is not explained at the cellular level. To study the differentiation of lamb brown adipocyte, a genomic fragment corresponding to the uncoupling protein was cloned from an ovine DNA library. Stromal vascular fibroblasts isolated from the perirenal adipose tissue of newborn lambs completely differentiated into brown adipocytes expressing the uncoupling protein gene, in a chemically defined serum‐free medium. Dexamethasone was necessary for the expression of the uncoupling protein gene. When stromal vascular fibroblasts were isolated from 3‐week‐old lambs, the glucocorticoid analog still promoted in vitro differentiation of adipocytes. However those adipocytes were unable to express uncoupling mRNA and could be considered as white adipocytes.

The data indicate that dexamethasone is necessary but not sufficient clone for the complete differentiation of brown adipocytes, and that the preadipocytes are committed to differentiation into brown or white adipocytes before culture.
Inactivation of Escherichia coli outer‐membrane phospholipase A by the affinity label hexadecanesulfonyl fluoride
Authors: HORREVOETS Anton J. G. , VERHEIJ Hubertus M. , de HAAS Gerard H.
Published: March 2005

The Escherichia coli outer‐membrane phospholipase A (OM PLA) is a membrane‐bound acyl hydrolase with a broad substrate specificity. In order to obtain more insight into the mechanism of action of this enzyme, we designed an active‐site‐directed inhibitor for OM PLA on the basis of the known substrate specificity as a first step in the elucidation of the catalytic mechanism of this enzyme. The inhibitor, hexadecanesulfonyl fluoride, consists of a long hydrocarbon chain for high‐affinity binding by the enzyme and a sulfonyl fluoride moiety as a reactive group. The kinetics of the inactivation of OM PLA by hexadecanesulfonyl fluoride were studied in Triton X‐100 micelles. Inactivation is very fast, specific and shows the same characteristics with respect to acyl specificity, pH profile and metal ion requirement as the activity of OM PLA on substrates. Incubation of OM PLA with a stoichiometric amount of hexadecanesulfonyl fluoride leads to a total and irreversible loss of enzyme activity, resulting from the sulfonylation of Ser144. This Ser144, which we suggest to be the active‐site serine of OM PLA, is part of the sequence HDSNG, whereas in the water‐soluble serine proteases and lipases the structural motif GXSXG is normally encountered. On the basis of the kinetics of inactivation of OM PLA by hexadecanesulfonyl fluoride, we discuss a possible catalytic mechanism of the enzyme.
Genetic engineering of Escherichia coli inorganic pyrophosphatase
Authors: LAHTI Reijo , SALMINEN Tiina , LATONEN Sari , HEIKINHEIMO Pirkko , POHJANOKSA Katariina , HEINONEN Jukka
Published: March 2005

Two tyrosines are supposed to be essential for the activity and to participate in the stabilization of Escherichia coli inorganic pyrophosphatase (PPiase) against heat denaturation [Samejima, T., Tamagawa, Y., Kondo, Y., Hachimori, A., Kaji, H., Takeda, A. and Shiroya, Y. (1988) J. Biochem. (Tokyo) 103, 766–772]. To locate these two tyrosines in the amino acid sequence, we substituted all the eight tyrosines of E. coli PPiase with phenylalanine and studied the properties of these YF mutant PPiases.

Interestingly, substitution of the tyrosines (Tyr51, Tyr55 and Tyr141) conserved with the amino acid sequence of yeast PPiase [Lahti, R., Kolakowski, L. F., Heinonen, J., Vihinen, M., Pohjanoksa, K. and Cooperman, B. (1990) Biochim. Biophys. Acta 1038, 338–345] exerted the most drastic effects on the structure and activity of E. coli PPiase. PPiase variants YF51, YF55 and YF141 had 64%, 7% and 22% of the wild‐type PPiase activity, respectively. Furthermore, PPiase variant YF141 had an increased sensitivity to heat denaturation, whereas mutant PPiase YF55 displayed a profound conformational change, as demonstrated by the binding of the fluorescent dye 9‐(diethylamino)‐5H‐benzo(α) phenoxazine‐5‐one (Nile red) that monitors the hydrophobicity of protein surfaces. None of the tyrosines of E. coli PPiase seem to be essential for catalysis, but Tyr55 and Tyr141 are important for the structural integrity of E. coli PPiase.
NMR study of Galeorhinus japonicus myoglobin
Authors: YAMAMOTO Yasuhiko , IWAFUNE Koji , NANAI Norishige , OSAWA Akemi , CHÛJÔ Riichirô , SUZUKI Tomohiko
Published: March 2005

The molecular structure of the active site of myoglobin from the shark, Galeorhinus japonicus, has been studied by 1H‐NMR. Some hyperfine‐shifted amino acid proton resonances in the met‐cyano form of G. japonicus myoglobin have been unambiguously assigned by the combined use of various two‐dimensional NMR techniques; they were compared with the corresponding resonances in Physter catodon myoglobin. The orientations of ThrE10 and IleFG5 residues relative to the heme in G. japonicus met‐cyano myoglobin were semiquantitatively estimated from the analysis of their shifts using the magnetic susceptibility tensor determined by a method called MATDUHM (magnetic anisotropy tensor determination utilizing heme methyls) [Yamamoto, Y., Nanai, N. & Chûjô, R. (1990) J. Chem. Soc., Chem. Commun., 1556—1557] and the results were compared with the crystal structure of P. catodon carbonmonoxy myoglobin [Hanson, J. C. & Schoenborn, B. P. (1981) J. Mol. Biol. 153, 117–124]. In spite of a substantial difference in shift between the corresponding amino acid proton resonances for the two proteins, the orientations of these amino acid residues relative to the heme in the active site of both myoglobins were found to be highly alike.
Primary and secondary structural patterns in eukaryotic cytochrome P‐450 families correspond to structures of the helix‐rich domain of Pseudomonas putida cytochrome P‐450cam
Authors: OUZOUNIS Christos A. , MELVIN William T.
Published: March 2005

An extensive sequence analysis of the eukaryotic cytochrome P‐450 (P‐450) protein families was conducted with a view to identifying conserved regions that might be related to secondary structural features in the Pseudomonas putida camphor hydroxylase (P‐450cam). All sequences available on‐line were collected, classified and aligned within families. Distinctively different sequences were chosen from each of seven eukaryotic families, and an unbiased multi‐alignment was constructed. Profile patterns of the most conserved regions were generated and screened against the sequence of P‐450cam, the structure of which has been elucidated by X‐ray crystallography. While some of these profiles did not map on the P‐450cam sequence, the structurally most important helices were clearly identified and the correlations were found to be statistically significant. Our analysis suggests that the helix‐rich domain with the cysteine pocket and the oxygen‐binding site is conserved in all P‐450 forms. Helices I and L from P‐450cam can be easily identified in all eukaryotic P‐450 forms. Other helices which seem to exist in all P‐450 forms include helices C, D, G and J, K. In the helix‐poor domain of P‐450cam, only structures b3/b4 seem to have been conserved. The obvious sequence conservation throughout the helix‐rich domain of the P‐450cam protein might be expected for a molecular class whose overall topology is preserved. Additional support for the conservation of structure between eukaryotic cytochromes P‐450 and P‐450cam comes from secondary structure prediction of the eukaryotic sequences.
New assays for the tyrosine hydroxylase and dopa oxidase activities of tyrosinase
Authors: WINDER Alison J. , HARRIS Henry
Published: March 2005

New assays for the tyrosine hydroxylase and dopa oxidase activities of tyrosinase (EC 1.14.18.1) have been developed. The tyrosine hydroxylase assay uses L‐[carboxy‐14C]tyrosine as the substrate. 14CO2 is released from the products of the hydroxylation and further metabolism of L‐[carboxy‐14C]tyrosine by incubation with ferricyanide, and measured radiometrically. D‐Dopa is a preferable cofactor to L‐dopa for the assay. Dopa oxidase activity is measured spectrophotometrically. Dopaquinone, produced on the oxidation of L‐dopa, reacts with Besthorn's hydrazone (3‐methyl‐2‐benzothiazolinone hydrazone) to form a pink pigment with an absorbance maximum at 505 nm. Details of the optimisation of conditions for the assays and their specificities for the two enzyme activities are described.
Effect of mutagenesis at each of five histidine residues on enzymatic activity and stability of ribonuclease H from Escherichia coli
Authors: KANAYA Shigenori , KATAYANAGI Katsuo , MORIKAWA Kosuke , INOUE Hideo , OHTSUKA Eiko , IKEHARA Morio
Published: March 2005

To examine the role of histidine residues in ribonuclease H from Escherichia coli, kinetic parameters for the enzymatic activity and conformational stabilities against guanidine hydrochloride denaturation of mutant enzymes, in which each of the five histidine residues was replaced with alanine, were determined and compared with the wild‐type enzyme. The mutation of His83 resulted in a marked increase in Km along with an increase in kcat. The mutation of His114 caused a large reduction in both the free energy of unfolding in water, ΔGH2O, and the mid‐point of the unfolding curve, [D]1/2. These results indicate that His83, which is one of the four well‐exposed histidine residues in the crystal structure, is located close to a substrate‐binding site, and His114, which is buried inside the protein molecule, contributes to the conformational stability, probably through the formation of a hydrogen bond with a main‐chain carbonyl group. None of the histidine residues is required for activity.
Purification, characterisation and mutagenesis of highly expressed recombinant yeast pyruvate kinase
Authors: MURCOTT Toby H. L. , McNALLY Teresa , ALLEN Simon C. , FOTHERGILL‐GILMORE Linda A. , MUIRHEAD Hilary
Published: March 2005

Recombinant yeast pyruvate kinase has been purified from a strain of Saccharomyces cerevisiae expressing the enzyme to very high levels. Expression was from a multicopy plasmid under the control of the yeast phosphoglycerate kinase promoter. The gene was expressed in the absence of the genomically encoded pyruvate kinase, using a strain of yeast in which the pyruvate kinase gene has been disrupted by the insertion of the yeast Ura3 gene. The purification procedure minimised proteolytic artefacts and enabled the covenient purification of 15–20 mg enzyme from 1 l culture. The purified enzyme was characterised by a high specific activity and by a lack of proteolytic degradation. Two active‐site mutants of yeast pyruvate kinase have been produced, expressed and characterised in this system and preliminary results are described.
Characterization of a novel type of chain‐terminator Galβ1–6Galβ1–4)GlcNAc in an oligosaccharide related to N‐glycosylated protein glycans isolated from GM1 the urine of patients with gangliosidosis
Authors: MICHALSKI Jean Claude , LEMOINE Jérôme , WIERUSZESKI Jean Michel , FOURNET Bernard , MONTREUIL Jean , STRECKER Gérard
Published: March 2005

Two new oligosaccharides were isolated from the urine of a patient with GM1 gangliosidosis. Final purification of the oligosaccharides was accomplished by capillary supercritical fluid chromatography. Structural analysis was by chemical analysis, chemical‐ionization mass spectrometry and 400‐MHz 1H‐NMR spectroscopy, leading to two primary structures. The first is derived from a classical triantennary N‐acetyllactosamine‐type glycan:

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The second is unusual with a terminal disaccharide Galβ1‐6Gal, which had not yet been described for glycans of the N‐acetyllactosamine type:
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Single‐step purification and structural characterization of human interleukin‐6 produced in Esherichia coli From a T7 RNA polymerase expression vector
Authors: ARCONE Rosaria , PUCCI Pietro , ZAPPACOSTA Francesca , FONTAINE Véronique , MALORNI Antonio , MARINO Gennaro , CILIBERTO Gennaro
Published: March 2005

Human interleukin‐6 or B‐cell stimulatory factor‐2 is a cytokine involved in acute phase and immune response. Cloning of cDNA for human interleukin‐6 in the pT7.7 expression plasmid under the control of a bacteriophage T7 RNA polymerase promoter system allows rapid production of the cytokine in Escherichia coli. Upon cell induction with isopropyl thiogalactopyranoside, recombinant human interleukin‐6 is overexpressed and forms insoluble inclusion bodies. Solubilization of the protein with 6 M guanidine hydrochloride and refolding in the presence of a reduction/oxidation system results in a quantitative recovery of recombinant human interleukin‐6. This material is already 70% pure and can be further purified to homogeneity with a single passage over a weak anionic‐exchange column. Extended structural characterization of the purified protein by electrospray mass spectrometry, automated Edman degradation and peptide mapping by high‐pressure liquid chromatography/fastatom‐bombardment mass spectrometry demonstrates that recombinant human interleukin‐6 is identical to the natural protein both in amino acid sequence and S‐S bridge content. However, it contains a minor component accounting for about 20% of the entire translated protein which exhibits a Met‐Ala dipeptide extension at the N‐terminus. Purified recombinant human interleukin‐6 is biologically active because it is able to induce at least 70‐fold the human C‐reactive promoter transfected in human hepatoma Hep 3B cells and is stable for several months in 10% glycerol at 4°C. The expression system described in the present work has the main advantage of producing a high yield of recombinant human interleukin‐6 (about 25 mg/l) combined with a very simple purification scheme.
Isolation and characterization of cDNAs encoding viscotoxins of mistletoe (Viscum album)
Authors: SCHRADER Gesine , APEL Klaus
Published: March 2005

Viscotoxins have been isolated from leaf homogenates of European mistletoe (Viscum album L.) and purified to apparent homogeneity. Antisera raised against these polypeptides were used to screen a cDNA expression library in λgt11. Two positive clones have been isolated, one encoding a full‐length preprotein of viscotoxin A3 and the other encoding the precursor of viscotoxin B. Besides the viscotoxin domain the precursor contained a signal sequence and an acidic polypeptide domain. Similar higher molecular mass precursor proteins have been described for thionins of leaves and seeds of barley. Even though the acidic part of the viscotoxin precursor is much shorter than the corresponding domain of the precursors of the leaf and seed thionins of barley, both the negative charge and the number and the relative position of cysteine residues have been conserved within the acidic domain. This result is consistent with our proposal that the acidic domain of the thionin precursor may play an important role in keeping the thionin inactive within the plant cell.
Cytosolic l‐alanine:4,5‐dioxovalerate transaminase differs from the mitochondrial form
Authors: SINGH Nishi Kant , TYAGI Rakesh Kumar , DATTA Kasturi
Published: March 2005

l‐Alanine:4,5‐dioxovalerate transaminase was detected in the kidney cytosolic fraction with a lower specific activity than the mitochondrial enzyme. The enzyme was purified from the cytosol to homogeneity with a yield of 32%, and comparative analysis with the mitochondrial form was performed. Both forms of the enzyme have identical pH and temperature optima and also share common antigenic determinants. However, differences in their molecular properties exist. The molecular mass of the native cytoplasmic enzyme is 260 kDa, whereas that of the mitochondrial enzyme is 210 kDa. In addition, the cytoplasmic l‐alanine:4,5‐dioxovalerate transaminase had a homopolymeric subunit molecular mass of 67 kDa compared to a subunit molecular mass of 50 kDa for the mitochondrial l‐alanine:4,5‐dioxovalerate transaminase. This is the first report of two forms of l‐alanine:4,5‐dioxovalerate transaminase. The different responses of cytosolic and mitochondrial l‐alanine:4,5‐dioxovalerate transaminases to hemin supplementation both in vitro and in vivo was demonstrated. Maximum inhibition of mitochondrial l‐alanine:4,5‐dioxovalerate transaminase activity was demonstrated with hemin injected at a dose of 1.2 mg/kg body mass, whereas the same dose of hemin stimulated the cytosolic enzyme to 150% of the control. A one‐dimensional peptide map of partially digested cytosolic and mitochondrial l‐alanine:4,5‐dioxovalerate transaminase shows that the two forms of the enzymes are structurally related. Partial digestion of the cytosolic form of the enzyme with papain generated a fragment of 50 kDa which was identical to that of the undigested mitochondrial form (50 kDa). Moreover, papain digestion resulted in a threefold increase in cytosolic enzyme activity over the native enzyme, and such enhancement was comparable to the activity of the mitochondrial form of the enzyme. Therefore, we conclude that the cytosolic form of l‐alanine:4,5‐dioxovalerate transaminase is different from the mitochondrial enzyme.

Furthermore, immunoblot analysis indicated that the mitochondrial enzyme has antigenic similarity to the cytosolic enzyme as well as to the papain‐digested cytosolic enzyme 50‐kDa fragment.
Isolation and structure determination of a novel compatible solute from the moderately halophilic purple sulfur bacterium Ectothiorhodospira marismortui
Authors: GALINSKI Erwin A. , OREN Aharon
Published: March 2005

The halophilic phototrophic bacterium Ectothiorhodospira marismortui produces three organic osmolytes to counterbalance the osmotic pressure of the surrounding medium: glycine betaine, sucrose, and a novel compound. This new compound, which accounts for approximately 30% of the cells' compatible solutes, was isolated and identified by mass spectrometry and nuclear magnetic resonance. It was characterized as Nα‐carbamoyl‐l‐glutamine 1‐amide, an unusual amino acid derivative with no previous reference in the chemical literature. The relatively high cytoplasmic concentration of this compound (∼ 0.5 M) observed at all growth conditions suggests that it may serve a vital function as an osmoticum and/or protectant for Ectothiorhodospira marismortui in a saline environment.
113Cd‐NMR investigation of a cadmium‐substituted copper, zinc‐containing superoxide dismutase from yeast
Authors: KOFOD Pauli , BAUER Rogert , DANIELSEN Eva , LARSEN Erik , BJERRUM Morten J.
Published: March 2005

113Cd nuclear magnetic resonance spectroscopy has been used to investigate the metal binding sites of cadmium‐substituted copper,zinc‐containing superoxide dismutase from baker's yeast. NMR signals were obtained for 113Cd(II) at the Cu site as well as for 113Cd(II) at the Zn site. The two subunits in the dimeric enzyme were found to have identical coordination properties towards 113Cd(II) as only two 113Cd signals were found in 113Cd2113Cd2SOD. The difference between the chemical shifts of 113Cd(II) at the Zn site when no copper is coordinated at the Cu site, and when Cu(I) or Cd(II) is coordinated, were found to be very small indicating that 113Cd(II) must be bound to the same number and type of ligands in both cases. Furthermore, the spectra show that the rate of exchange of protein‐bound 113Cd(II) and free 113Cd2+ is slow on the NMR time scale also at the Cu site. The present study suggests an explanation for the discrepancy in the literature regarding 113Cd‐NMR investigations of bovine superoxide dismutase.
Isolation and characterization of authentic Phe‐Met‐Arg‐Phe‐NH2 and the novel Phe‐Thr‐Arg‐Phe‐NH2 peptide from Nereis diversicolor
Authors: BARATTE Blandine , GRAS‐MASSE Hélène , RICART Guy , BULET Philippe , DHAINAUT‐COURTOIS Nicole
Published: March 2005

Immunocytochemical studies have shown that peptides like Phe‐Met‐Arg‐Phe‐NH2 (FMRFamide) are widely distributed throughout the nervous system of three Nereidae. In Nereis diversicolor we have isolated these peptides from an extract of total worms by affinity chromatography and two steps of reversed‐phase high‐performance liquid chromatography. The sequences of the purified peptides have been determined by amino acid sequencing and on the basis of their reactivity with an anti‐FMRFamide serum specific for the determinant Arg‐Phe‐NH2. Two primary structures have been established: Phe‐Thr‐Arg‐Phe‐NH2 (FTRFamide) and Phe‐Met‐Arg‐Phe‐NH2 (FMRFamide). Furthermore a methionine sulfoxide derivative of the FMRFamide has been identified. We have synthesized the FTRFamide peptide and in all cases, the native peptides were indistinguishable from the synthetic counterparts. The structure of the two native peptides and of the methionyl sulfoxide derivate have been confirmed by fast‐atom‐bombardment and tandem mass spectrometry.
Modulation by oxygen of the glucagon‐dependent activation of the phosphoenolpyruvate carboxykinase gene in rat hepatocyte cultures
Authors: HELLKAMP Josef , CHRIST Bruno , BASTIAN Helge , JUNGERMANN Kurt
Published: March 2005

In liver phosphoenolpyruvate carboxykinase (PCK) activity, protein and mRNA are localized predominantly in the periportal zone. The activation of the PCK gene by glucagon was studied in primary rat hepatocyte cultures under physiological arterial and venous oxygen tensions [16% and 8% (by vol.)]. PCK gene expression was monitored on the level of transcription, mRNA abundance and enzyme activity as well as enzyme synthesis and degradation.

Transcription of the PCK gene was increased by 10 nM glucagon maximally after 0.5 h; it reached nearly basal levels again after 2 h. The increase in transcription was 45% lower under 8% oxygen than under 16% oxygen.

PCK mRNA was maximally increased after 2 h under 16% oxygen and after 4 h under 8% oxygen; it subsequently declined to twice the basal values after 8 h. The maximal increase after 2 h was 50% lower under 8% oxygen than under 16% oxygen.

PCK enzyme activity was maximally increased after 4–6 h. The maximal enhancement after 4 h was 50% lower under 8% oxygen than under 16% oxygen.

The increase in PCK enzyme activity was due to an enhanced synthesis rate of PCK protein. The rate increase after 3 h was 35% lower under 8% oxygen than under 16% oxygen.

The degradation of PCK protein was equal under both oxygen tensions.

The results show that in cultured rat hepatocytes the induction of PCK gene expression is modulated by physiological concentrations of oxygen. The modulation occurred at the level of gene transcription, mRNA abundance, enzyme protein synthesis and enzyme activity. The periportal to perivenous oxygen gradient could be the major factor responsible for the predominant expression of the PCK gene in the periportal zone.
Sphingolipid biosynthesis in cultured neurons
Authors: MANDON Elisabet C. , van ECHTEN Gerhild , BIRK Rolf , SCHMIDT Richard R. , SANDHOFF Konrad
Published: March 2005

Addition of exogenous sphingosine homologues (D‐erythro configuration) with different alkyl chain lengths (12 and 18 carbon atoms) to the medium of primary cultured cerebellar cells resulted in a decrease of serine palmitoyltransferase activity in a time‐ and concentration‐dependent manner. This enzyme catalyzes the first committed step in sphingolipid biosynthesis. Half‐maximal reduction of enzyme activity occurred after a 4‐h treatment with 25 μM sphingoid bases. Maximal decrease (approx. 80%) was obtained after treating the cells for 4–8 h with 50 μM long‐chain bases. When a biosynthetically inert sphingoid, azidosphingosine (10–50 μM), was fed to the cells, decrease of 3‐ketosphinganine formation was much slower, reaching its maximum (approx. 80%) after 24 h. In contrast to D‐erythro‐sphingosine, L‐threo‐C18‐sphingosine did not yield any decrease of serine palmitoyltransferase activity when added to the cells under identical experimental conditions.

Decrease of serine palmitoyltransferase activity was fully reversible after removal of the long‐chain bases from the culture medium. Activities of other enzymes of lipid metabolism, ceramide synthase, long‐chain acyl‐CoA synthase and choline phosphotransferase, were not affected by the addition of sphingoid bases, indicating that the down regulation of serine palmitoyltransferase is quite specific.
Purification and properties of the phosphofructokinase from Lactobacillus bulgaricus
Authors: LE BRAS Gisèle , DEVILLE‐BONNE Dominique , GAREL Jean‐Renaud
Published: March 2005

Phosphofructokinase, the enzyme which catalyzes the conversion of fructose 6‐phosphate into fructose 1,6‐bisphosphate in Lactobacillus bulgaricus (Lactobacillus delbrueckii, subspecies bulgaricus) has been purified to homogeneity and some of its structural and functional properties have been studied. The enzyme is a tetramer composed of four 35‐kDa subunits. Its N‐terminal sequence determined on 38 residues is homologous to those of the major allosteric enzymes from Escherichia coli and Bacillus stearothermophilus, suggesting that the three proteins have closely related structures.

The maximum velocity of the enzyme from L. bulgaricus increases with pH according to the ionization of a group with a pK of 6.2. At all pH values, the saturation by fructose 6‐phosphate is hyperbolic. At the optimum pH of 8.2, the maximum velocity and the affinities for the ATP and fructose 6‐phosphate substrates are not modified by the presence of ADP or GDP nor by phosphoenolpyruvate. Partial inhibition by phosphoenolpyruvate exists at acidic pH, but is not related to an allosteric mechanism similar to that in E. coli. This inhibition results from a shift from 6.2 to 7.1 of the pK of an ionizable group which controls Vmax. Protection against thermal denaturation shows that the enzyme binds phosphoenolpyruvate and not GDP. The phosphofructokinase from L. bulgaricus appears as a structural analog of the E. coli enzyme which does not undergo an allosteric transition between two states R and T, but instead remains in a unique conformational state, intermediate between the R and T states; the active sites have an R‐like conformation since they bind fructose 6‐phosphate, whereas the regulatory sites have a T‐like conformation since they bind phosphoenolpyruvate and not GDP.
Structural characterization of the lipid A component of Pseudomonas aeruginosa wild‐type and rough mutant lipopolysaccharides
Authors: KULSHIN Vladimir A. , ZÄHRINGER Ulrich , LINDNER Buko , JÄGER Karl‐Erich , DMITRIEV Boris A. , RIETSCHEL Ernst Th.
Published: March 2005

The structure of the lipid A component of lipopolysaccharides isolated from two wild‐type strains (Fisher 2 and 7) and one rough mutant (PAC 605) of Pseudomonas aeruginosa was investigated using chemical analysis, methylation analysis, combined gas‐liquid chromatography/mass spectrometry, laser‐desorption mass spectrometry and NMR spectroscopy. The lipid A backbone was found to consist of a pyranosidic β1,6‐linked d‐glucosamine disaccharide [β‐d‐GlcpN‐(1 → 6)‐d‐GlcpN], phosphorylated in positions 4′ and 1. Position 6′ of the β‐d‐GlcpN‐(1 → 6)‐d‐GlcpN disaccharide was identified as the attachment site of the core oligosaccharide and the hydroxyl group at C‐4 was not substituted. Lipid A of the three P. aeruginosa strains expressed heterogeneity with regard to the degree of acylation: a hexaacyl as well as a pentaacyl component were structurally characterized. The hexaacyl lipid A contains two amide‐bound 3‐O‐acylated (R)‐3‐hydroxydodecanoic acid groups [12:0(3‐OH)] at positions 2 and 2′ of the GlcN dissacharide and two ester‐bound (R)‐3‐hydroxydecanoic acid groups [10:0(3‐OH)] at positions 3 and 3′. The pentaacyl species, which represents the major lipid A component, lacks one 10:0(3‐OH) residue, the hydroxyl group in position 3 of the reducing GlcN residue being free. In both hexa‐ and pentaacyl lipid A the 3‐hydroxyl group of the two amide‐linked 12:0(3‐OH) residues are acylated by either dodecanoic (12:0) or (S)‐2‐hydroxydodecanoic acid [12:0(2‐OH)], the lipid A species with two 12:0(2‐OH) residues, however, being absent. The presence of only five acyl residues in the major lipid A fraction may account for the low endotoxic activity observed with P. aeruginosa lipopolysaccharide.
Ribosome‐bound EF‐1α‐like protein of yeast Saccharomyces cerevisiae
Authors: DIDICHENKO Svetlana A. , TER‐AVANESYAN Michael D. , SMIRNOV Vladimir N.
Published: March 2005

The SUP2 (SUP35) omnipotent suppressor gene encodes the EF‐1α‐like polypeptide, intimately involved in the control of translational ambiguity in the yeast Saccharomyces cerevisiae. The present study is devoted to the immunological characterization of the Sup2 protein.

The SUP2 gene was fused to the Escherichia coli lacZ gene and a polyclonal antibody against the corresponding Sup2–β‐galactosidase hybrid protein was obtained. This antibody identified a 79‐kDa protein that was absent in those cells where the SUP2 gene was disrupted, and an abundance of this protein was observed in cells overexpressing the SUP2 gene. The localization of this protein was studied in subcellular fractionation experiments. The SUP2 gene product proved to be uniformly distributed throughout ribosome‐enriched samples, i.e. free polysomes, crude microsomes and rough endoplasmic reticulum. It was not found in the cytoplasm and smooth endoplasmic reticulum. The SUP2‐encoded protein was fully ribosome associated and less abundant than the ribosomal protein L3. Also, in a sucrose gradient, Sup2 preferentially cosedimented with the 40S ribosomal subunit, but not with the 60S subunit. The functional significance of this association is discussed.
Sorbitol dehydrogenase: cDNA coding for the rat enzyme
Authors: KARLSSON Christina , JÖRNVALL Hans , HÖÖG Jan‐Olov
Published: March 2005

Two separate cDNA‐clones, together coding for rat sorbitol dehydrogenase, have been isolated from a liver cDNA library in Δgt11 by screening with oligonucleotide probes. One clone contained a 1020‐bp fragment starting at the codon for amino acid residue 104 and ending with a 261‐bp 3′ non‐coding region, the second encompassed the entire 5′ region and ended with a 3′ truncation corresponding to amino acid residue 315. The coding region consists of 356 amino acid residues, one more than in the human and sheep enzymes. The presence of the extra residue at position 3, a proline, can be explained by a shifted splice point in the mRNA. The primary structure of rat sorbitol dehydrogenase allows triplet comparisons of three distinct rat‐ungulate‐human enzymes differing in quaternary structure and metal content within the zinc‐containing alcohol dehydrogenase family. The variability of sorbitol dehydrogenase (tetramer with one zinc atom/subunit; no activity towards ethanol) is large (18%), exactly like that for the class I alcohol dehydrogenase (dimer with two zinc atoms/subunit; no activity towards sorbitol), differing threefold from that of the class III alcohol dehydrogenase/glutathione‐dependent formaldehyde dehydrogenase (dimer with two zinc atoms/subunit; 6% variability) suggesting that the distinct extents of variability within this protein family are independent of substrate specificity, metal content and quaternary structure.