bluuebunny/crossref_metadata_2025 huggingface dataset:
Glucose oxidase from Penicillium amagasakiense
Authors: Kiess Michael , Hecht Hans‐Jürgen , Kalisz Henryk M.
Published: March 2003

The complete amino acid sequence of glucose oxidase from Penicillium amagasakiense was determined by Edman degradation and mass spectrometry of peptide fragments derived from three different specific proteolytic digests and a cyanogen bromide cleavage. The complete sequence of each monomer comprises 587 amino acid residues, contains three cysteine residues, and seven potential N‐glycosylation sites, of which at least five were confirmed to be glycosylated. Glucose oxidase from P. amagasakiense shows a high degree of identity (66 %) and 79 % similarity to glucose oxidase from Aspergillus niger, and is a member of the glucose‐methanol‐choline (GMC) oxidoreductase family. The tertiary structures of glucose oxidase from A. niger and cholesterol oxidase from Brevibacterium sterolicum were superimposed to provide a template for the sequence comparison of members of the GMC family. The general topology of the GMC oxidoreductases is conserved, with the exception of the presence of an active site lid in cholesterol oxidase and the insertion of additional structural elements in the substrate‐binding domain of alcohol oxidase. The overall structure can be divided into five distinct sequence regions : FAD‐binding domain, extended FAD‐binding domain, flavin attachment loop and intermediate region, FAD covering lid, and substrate‐binding domain. The FAD‐binding and the extended FAD‐binding domains are composed of several separate sequence regions. The other three regions each comprise a single contiguous sequence. Four major consensus patterns have been identified, including the nucleotide‐binding consensus sequence close to their N‐termini. The functions of the two motifs recently selected by the Genetics Computer Group, Madison, Wisconsin, as additional signature patterns of the GMC oxidoreductases are discussed. The other consensus patterns belong to either the FAD‐binding or the extended FAD‐binding domain. In addition, the roles of conserved residues are discussed wherever possible.
Localization of the 23‐kDa subunit of the oxygen‐evolving complex of photosystem II by electron microscopy
Authors: Boekema Egbert J. , Nield Jon , Hankamer Ben , Barber James
Published: March 2003

A dimeric photosystem II light‐harvesting II super complex (PSII‐LHCII SC), isolated by sucrose density gradient centrifugation, was previously structurally characterized [Boekema, E. J., Hankamer, B., Bald, D., Kruip, J., Nield, J., Boonstra, A. F., Barber, J. & Rögner, M. (1995) Proc. Natl Acad. Sci. USA 92, 175−179]. This PSII‐LHCII SC bound the 33‐kDa subunit of the oxygen‐evolving complex (OEC), but lacked the 23‐kDa and 17‐kDa subunits of the OEC. Here the isolation procedure was modified by adding 1 M glycine betaine (1‐carboxy‐N,N,N‐trimethylmethanaminium hydroxide inner salt) to the sucrose gradient mixture. This procedure yielded PSII‐LHCII SC that contained both the 33‐kDa and the 23‐kDa subunits and had twice the oxygen‐evolving capacity of the super complexes lacking the 23‐kDa polypeptide. Addition of CaCl2 to PSII‐LHCII SC with the 23‐kDa subunit attached did not increase the oxygen‐evolution rate. This suggests that the 23‐kDa subunit is bound in a functional manner and is present in significant amounts. Over 5000 particle projections extracted from electron microscope images of negatively stained PSII‐LHCII SC, isolated in the presence and absence of glycine betaine, were analyzed using single‐particle image‐averaging techniques. Both the 23‐kDa and 33‐kDa subunits could be visualized in top‐view and side‐view projections. In the side view the 23‐kDa subunit is seen to protrude 0.5−1 nm further than the 33‐kDa subunit, giving the PSII particle a maximal height of 9.5 nm. Measured from the centres of the masses, the two 33‐kDa subunits associated with the dimeric PSII‐LHCII SC are separated by 6.3 nm. The corresponding distance between the two 23‐kDa subunits is 8.8 nm.
Functional expression of recombinant N‐methyl‐	D‐aspartate receptors in the yeast Saccharomyces cerevisiae
Authors: Li Ziyu , Becker Jürgen , Noe Christian R.
Published: March 2003

The yeast Saccharomyces cerevisiae was used for expressing the genes encoding the ionotropic N‐methyl‐
D‐aspartate (NMDA) receptor subunits from rats (NR1a, NR2A, NR2C) and mice (NR2B). Four plasmids were constructed by cloning the different NMDA receptor genes in the two multi‐copy yeast‐Escherichia coli shuttle vectors pMB01 (←NR1a gene) and pMB02 (←NR2A‐2C genes). The protease‐deficient S. cerevisiae strain cI3‐ABYS‐86 (leu−, ura−) was transformed or co‐transformed with the resulting plasmids pMBNR1a (leu+) or pMBNR1a/pMBNR2A−C (ura+) respectively.

Western blotting analysis with antibodies raised against amino acid sequences at the C‐termini of the respective subunits revealed that the recombinant receptor proteins were differently expressed and only partially glycosylated in the cell membranes of the recombinant yeast strains.

The expression and localization of the recombinant NMDA receptor proteins were also proved by immunofluorescence microscopy which indicated a distinct expression of the different NMDA receptor subunits in the plasma membrane of the transformed yeast cells.

Pharmacological characterization of crude membrane preparations of the recombinant yeast cells showed saturable binding of the glycine antagonist [3H]MDL105,519 with Kd values of 56.9 ± 6.19 nM (NR1a/NR2A), 26.72 ± 2.13 nM (NR1a/NR2B), and 21.22 ± 1.64 nM (NR1a/NR2C), and bound capacities of 17.94 ± 1.24 pmol/mg membrane protein (NR1a/NR2A), 11.45 ± 0.67 pmol/mg (NR1a/NR2B), and 16.15 ± 0.86 (NR1a/NR2C) pmol/mg. The [3H]MDL105,519 binding was inhibited by the glycine antagonist 5,7‐dichlorokynurenate, ethyl‐2‐carboxy‐4,6‐dichloro‐3‐indoleacetate, and itself, but not by glycine,
D‐serine or 1‐amino‐cyclopropanecarboxylic acid. Specific binding of [3H]glycine or the NMDA channel blocker [3H]dizolcipine were not observed.
Activation of the precursor of human stromelysin 2 and its interactions with other matrix metalloproteinases
Authors: Nakamura Hiroyuki , Fujii Yutaka , Ohuchi Eiko , Yamamoto Etuhide , Okada Yasunori
Published: March 2003

Matrix metalloproteinases (MMP) are synthesized as inactive zymogens (proMMP) and subsequently activated by many factors to degrade the extracellular matrix (ECM). In the present study, we have examined the intermolecular activation mechanisms of proMMP by MMP‐10 (stromelysin 2). ProMMP‐10 was purified from the culture media of OSC‐20 human oral squamous carcinoma cells stimulated with 12‐O‐tetradecanoylphorbol 13‐acetate. The final products are partially activated (≈38 % of the full activity) during the purification steps and contain proMMP‐10 of Mr 56 000 with minor protein bands of Mr 47 000, 24 000 and 22 000. The zymogen is activated by 4‐aminophenylmercuric acetate and processed to the active forms of Mr 47 000 and 24 000. The NH2‐terminal sequence of the 47 000‐ and 24 000‐Mr species is Phe82‐Ser‐Ser‐Phe‐Pro‐Gly, which is identical to that of stromelysin 2. ProMMP‐9 (progelatinase B) is activated by MMP‐10 to its full activity and processed to the low‐Mr species of Mr 81 000, 65 000, 57 000 and 55 000, the former two of which show proteolytic activity on a gelatin zymography. The NH2‐terminal sequence analysis indicates that the 81 000‐, 65 000‐ and 57 000‐Mr species have the identical sequence of Phe88‐Gln‐Thr‐Phe‐Glu‐Gly, suggesting the cleavage of the Arg87−Phe88 peptide bond for activation and both NH2‐terminal and COOH‐terminal truncation in the 65 000‐ and 57 000‐Mr forms. MMP‐10 also activates proMMP‐7 (promatrilysin) up to about 60 % of the full activity and generates the same active species of Mr 19 000 as that obtained by activation with 4‐aminophenylmercuric acetate. Incubation of proMMP‐2 (progelatinase A) or proMMP‐3 with MMP‐10 does not result in activation of these proMMP. These results indicate that in addition to the previously reported activation of proMMP‐1 (tissue procollagenase) and proMMP‐8 (neutrophil procollagenase), MMP‐10 can also activate proMMP‐9 and proMMP‐7, and suggest the possibility that MMP‐10 may replace a role of MMP‐3 in the ECM degradation in concert with other MMP under various pathological conditions.
Repression of phosphoenolpyruvate carboxykinase gene activity by insulin is blocked by 3‐aminobenzamide but not by PD128763, a selective inhibitor of poly(ADP‐ribose) polymerase
Authors: Yau Lorraine , Elliot Tracy , Lalonde Chantal , Zahradka Peter
Published: March 2003

Expression of the phosphoenolpyruvate carboxykinase (PEPCK) gene is induced by 3‐aminobenzamide, an inhibitor of poly(ADP‐ribose) polymerase. Synthesis of PEPCK mRNA is repressed by insulin, but remains detectable in H4IIE hepatoma cells exposed simultaneously to both 3‐aminobenzamide and insulin. This capability of 3‐aminobenzamide to block the inhibitory actions of insulin suggests that ADP‐ribosylation is required for the regulation of PEPCK gene expression by insulin. Furthermore, neither changes in chromatin condensation nor cell growth status were linked to these events. The inability of 3,4‐dihydro‐5‐methylisoquinolinone (PD128763), a selective inhibitor of poly(ADP‐ribose) polymerase, to impede insulin‐dependent repression of PEPCK gene expression, however, indicated that 3‐aminobenzamide does not operate by inhibiting poly(ADP‐ribosyl)ation. The potential involvement of mono(ADP‐ribosyl)ation, a process that is also inhibited by 3‐aminobenzamide, in the regulation of PEPCK gene activity was then evaluated. Analysis of poly(ADP‐ribose) polymerase activity and poly(ADP‐ribosyl)ation confirmed that there were no significant changes in response to insulin, while microsomal mono(ADP‐ribosyl)transferase activity was elevated approximately fourfold. An increase in protein hydroxylamine‐sensitive mono(ADP‐ribosyl)ation was observed following insulin treatment. The sensitivity of the mono(ADP‐ribosyl)transferase activity to 3‐aminobenzamide but not PD128763 makes it plausible that mono(ADP‐ribosyl)ation rather than poly(ADP‐ribosyl)ation contributes to the regulation of PEPCK gene expression.
Chemical modifications of the vasoconstrictor peptide angiotensin II by nitrogen oxides (NO, HNO2, HOONO)
Authors: Ducrocq Claire , Dendane Maryvonne , Laprévote Olivier , Serani Laurent , Das Bhupesh C. , Bouchemal‐Chibani Nadia , Doan Bich‐Thuy , Gillet Brigitte , Karim Abdelhak , Carayon Alain , Payen Didier
Published: March 2003

Nitric oxide (NO) and angiotensin II are natural regulators of blood pressure. Under aerobic conditions, NO is transformed into its higher oxides (N2O4, NO2, NO/NO2 or N2O3) and oxoperoxonitrate (currently named peroxynitrite) by coupling with superoxide. Previous studies have shown that these reactive nitrogen species should be involved in vivo in the transformation of cysteine and tyrosine into the corresponding nitrosothiol and 3‐nitrotyrosine. In the present study, attention has been focused on the relative reactivities of HNO2, peroxynitrite, and NO in the presence of dioxygen, towards the arginine and tyrosine residues of the peptide angiotensin II. Nitration of the tyrosine residue is clearly the main reaction with peroxynitrite. By contrast, besides 20 % of nitration of the tyrosine residue, NO in the presence of dioxygen leads to nitrosation reactions with the arginine residue similar to those observed with HNO2 at pH 5, possibly through the intermediate N2O3 reactive species. Angiotensin II is converted for the most part to peptides having lost either a terminal amine function or the whole guanido group, leading respectively to citrulline‐containing angiotensin II or to a diene derivative. Identification established mainly by tandem mass spectrometry of peptidic by‐products allows us to propose a cascade of nitrosations of all the amine functions of the arginine residue. Further in vivo studies show that transformations of the arginine residue in angiotensin II do not alter its vasoconstrictive properties, whereas nitration of the tyrosine residue totally inhibits them.
Processing of vertebrate box C/D small nucleolar RNAs in plant cells
Authors: Leader David J. , Clark Gillian P. , Boag Jillian , Watters Jennifer A. , Simpson Craig G. , Watkins Nicholas J. , Maxwell E. Stuart , Brown John W. S.
Published: March 2003

The recent isolation of a number of plant box C/D small nucleolar (sno)RNAs demonstrates the conservation in plants of sequence and structural elements of processed box C/D snoRNAs. Boxes C and D, and terminal inverted repeats are known to be essential for accumulation and processing in vertebrates and yeast. Processing of vertebrate box C/D snoRNAs was examined by expression of various mouse hsc70 intron 5‐U14 constructs in tobacco protoplasts. Full‐length U14 and internally deleted U14 accumulated in the plant cells. Human U3 and U8 fragments, consistent with processing to internal box C/C′ sequences, also accumulated in the plant cells. The similarity of processing behaviour of the vertebrate box C/D constructs in tobacco protoplasts and Xenopus oocytes suggests the mechanism of processing, involving recognition and association of proteins, is conserved in plants.
Chlorophyll a synthesis upon interruption and deletion of por coding for the light‐dependent NADPH :protochlorophyllide oxidoreductase in a photosystem‐I‐less/chlL− strain of Synechocystis sp. PCC 6803
Authors: He Qingfang , Brune Daniel , Nieman Ronald , Vermaas Wim
Published: March 2003

The gene coding for the light‐dependent NADPH :protochlorophyllide oxidoreductase (POR) was interrupted or deleted in a Synechocystis sp. PCC 6803 strain lacking photosystem I (PS I) as well as ChlL, which takes part in light‐independent catalysis of protochlorophyllide reduction. Interruption of por by a kanamycin‐resistance cartridge between the codons for M263 and V264 (about 83 % into the coding region) did not abolish POR activity, but resulted in a decrease in the protochlorophyllide‐(PChlide)‐binding capacity of POR. Deletion of por in the PS I‐less/chlL− strain generated a mutant [PS I‐less/chlL−/por (del)] which accumulated both monovinyl‐PChlide and divinyl‐PChlide and excreted PChlides into the medium. This mutant also synthesized small amounts of protochlorophyllide dihydrogeranylgeraniol ester (protochlorophyll) when it was grown under light‐activated heterotrophic growth conditions. However, the mutant was still able to synthesize small amounts of normal chlorophyll a under weak continuous illumination, even though the quantum yield of chlorophyll a formation was reduced. Either protochlorophyll or PChlide reduction by an unspecific reductase or by a ChlB/ChlN complex could account for chlorophyll a synthesis in the PS I‐less/chlL−/por (del) strain. Functional photosystem II (PS II) was assembled in this mutant, but the PS II/chlorophyll ratio was fourfold lower than in the PS I‐less strain with normal chlorophyll synthesis. The PS I‐less/chlL−/por (del) mutant had a 77‐K fluorescence emission maximum at 685 nm but no peak or shoulder at 695 nm when the cells were excited at 435 nm. Much of the chlorophyll in the PS I‐less/chlL−/por (del) mutant therefore seems to be associated with components other than PS II.
Different forms of the mRNA encoding the heat‐shock transcription factor are expressed during the life cycle of the parasitic helminth Schistosoma mansoni
Authors: Lantner Frida , Ziv Etty , Ram Daniela , Schechter Israel
Published: March 2003

Several cDNAs and a gene encoding the heat‐shock transcription factor (HSF) of schistosome were cloned, and multiple forms of the mRNA were found at different developmental stages of the parasite. The encoded protein contained a DNA‐binding domain with expected sequence identity (39−58 %) to other HSF molecules, and two leucine zipper motifs (LZ123 and LZ4) involved in the oligomerization of HSF. Adult worms express three isoforms of HSF mRNA generated by alternative splicing inside the coding region that contains in‐frame splice signals. Introns are not involved in the process since the deleted segments (36 bp or 45 bp) are not flanked by any intron in the gene. Structural variations generated by alternative splicing (insertion of 3 amino acids or 15 amino acids) are continual with LZ4 and added hydrophobic residues are in register with the hydrophobic heptad repeats of LZ4. Structural diversity at the C‐terminus of LZ4 may affect the strength of LZ4 interaction with the oligomerization domain (LZ123) and thus modulate the DNA‐binding activity of HSF. The conservation of this mechanism in mouse and schistosome may reflect evolutionary pressure to generate multiple HSF species exhibiting functional diversity and capable of responding to different stress signals and physiological signals. Adult worms express HSF mRNA of 2.5 kb, in agreement with the size of the cDNA, while cercariae (developmental stage preceding adult worm) show multiple bands in the range 2.5−3 kb. Available data indicate that the HSF mRNAs of cercariae are inactive. We propose that these mRNA species are generated by an alternative splicing that incorporates introns, which inactivate the mRNA by the insertion of termination codons and/or by shifting of the reading frame. Parasite HSF protein produced in bacteria showed DNA sequence recognition similar to that of HSF in parasite extracts, i.e. the recombinant HSF reacted bettter with a variant heat‐shock element (HSE; one base change in the third NGAAN pentamer of the ideal HSE consensus sequence) than with the ideal HSE. The size of the HSF gene is 12 kb and it is composed of ten exons and nine introns. Excluding the introns, the gene and cDNA show 100 % sequence identity. A plant HSF gene contains only a single intron, which matches with the position of intron I2 of schistosome. That the position of this intron is conserved in remote species is indicative of an important function during evolution of the HSF gene.
Pyrrole‐2‐carboxylate decarboxylase from Bacillus megaterium PYR2910, an organic‐acid‐requiring enzyme
Authors: Omura Hironori , Wieser Marco , Nagasawa Toru
Published: March 2003

Inducible pyrrole‐2‐carboxylate decarboxylase, which catalyzes the decarboxylation of pyrrole‐2‐carboxylate to pyrrole and CO2 in stoichiometric amounts, was purified from Bacillus megaterium PYR2910. The purity of the enzyme was shown by SDS/PAGE and gel‐permeation HLPC. The enzyme has a molecular mass of approximately 98 kDa and consists of two identical subunits. It is highly specific for pyrrole‐2‐carboxylate, and also catalyzes the reverse reaction, the carboxylation of pyrrole. A unique feature of this enzyme is its requirement of an organic acid, such as acetate, propionate, butyrate or pimelate. A possible catalytic mechanism including a cofactor function of organic acid is discussed.
Structural analysis of the phase‐variable lipooligosaccharide from Haemophilus somnus strain 738
Authors: Cox Andrew D. , Howard Michael D. , Brisson Jean‐Robert , van der Zwan Mark , Thibault Pierre , Perry Malcolm B. , Inzana Thomas J.
Published: March 2003

The structure of the phase variable lipooligosaccharide (LOS) from Haemophilus somnus strain 738 was elucidated. The LOS was subjected to a variety of degradative procedures. The structures of the purified products were established by monosaccharide and methylation analyses, NMR spectroscopy and mass spectrometry. The following structures for the two major components were determined on the basis of the combined data from these experiments.
Kdo
PCho
2
P∣∣∣
6
4
4
β‐Gal‐1‐3‐β‐GlcNAc‐1‐3‐β‐Gal‐1‐4‐β‐Glc‐1‐4‐α‐Hep‐1‐5‐α‐Kdo‐2‐6‐β‐GlcN‐1‐6‐α‐GlcN‐1‐P
3
∣
1
α‐Hep‐6‐PEtn
Kdo
PCho
2
P∣∣∣
6
4
4
β‐Gal‐1‐3‐β‐GlcNAc‐1‐3‐β‐Gal‐1‐3‐β‐Glc‐1‐4‐β‐Glc‐1‐4‐α‐Hep‐1‐5‐α‐Kdo‐2‐6‐β‐GlcN‐1‐6‐α‐GlcN‐1‐P
3
∣
1
α‐Hep‐6‐PEtn
  In the structures Kdo is 3‐deoxy‐
D‐manno‐octulosonic acid, PEtn is phosphoethanolamine, PCho is phosphocholine, Hep is
L‐glycero‐
D‐manno‐heptose, and the remaining glucose units have the
D configuration. The elucidation of these structures has increased our understanding of the relationship between the phase‐variable LOS and the pathogenic potential of this organism.
Thylakoid membranes contain a high‐conductance channel
Authors: Hinnah Silke C. , Wagner Richard
Published: March 2003

Ion channels in the thylakoid membrane were investigated by direct patch clamping on swollen thylakoids. A preparation method has been developed in order to release osmotically swollen intact thylakoids from pea protoplasts derived from cotyledons of young Pisum sativum plants. The swollen thylakoids with typical diameters between 10 μm and 20 μm formed reproducibly high‐resistance seals with patch pipettes. We observed a potassium channel with a main conductant state of @KL ≈ 40 pS and a conductance of @KL ≈ 90 pS (in asymmetric 20/100 mM KCl) for the fully open channel. Surprisingly, the thylakoid membranes also contained a high‐conductance channel with a main conductant state of @KL ≈ 620 pS (in asymmetric 20/100 mM KCl), revealing also higher and lower conductant states. With a different experimental approach we showed that thylakoids are able to accumulate transiently the membrane impermeant fluorescent dye Lucifer Yellow which likewise suggests the presence of a pore‐like channel with a diameter large enough to allow permeation of Lucifer Yellow.
Molecular cloning, expression and characterization of an Ascaris inhibitor for pepsin and cathepsin E
Authors: Kageyama Takashi
Published: March 2003

Molecular cloning of a cDNA for a pepsin inhibitor in the round worm, Ascaris suum, was achieved. The ORF was found to encode a 20‐residue potential signal peptide and a 149‐residue inhibitor moiety. Northern analysis showed the mRNA for the inhibitor to be expressed in the body wall and not in the viscera. To obtain the active inhibitor, we constructed a yeast expression vector, pYES2API, containing the inducible galactosidase promoter and a DNA fragment encoding a fusion protein of the yeast α‐factor leader and the Ascaris pepsin inhibitor. The active inhibitor was secreted in the culture medium, the yield being approximately 3 mg l−1 day−1, and purified by a two‐step procedure that included HPLC. The inhibitor inactivated pepsin A and cathepsin E almost completely at amounts equimolar with the enzymes, but was 100‐fold less effective against pepsin C and did not act on cathepsin D and renin. Ki values for the inhibition of pepsin A and cathepsin E were in the nanomolar range below pH 5. Since the inhibitor activity was lost by modification of specific Lys residues, including Lys110, an electrostatic interaction between these Lys residues and Asp/Glu residues of pepsin A or cathepsin E was thought to be essential for the inhibition.
Nitrone spin traps and their pyrrolidine analogs in myocardial reperfusion injury : hemodynamic and ESR implications
Authors: Pietri Sylvia , Liebgott Thibaut , Fréjaville Claudine , Tordo Paul , Culcasi Marcel
Published: March 2003

Formation of free radicals during reperfusion of the isolated ischemic heart has often been demonstrated by detecting hydroxyl radical spin adducts of the nitrone 5,5‐dimethyl‐1‐pyrroline N‐oxide (DMPO) in coronary effluents. However, questions still remain regarding (a) whether the reported cardiovascular effects of nitrone perfusion may affect the formation of spin adducts, and (b) the primary generation of superoxide (O.es.cf1.˙.cf5.−.rb.ei2.rb), because of the short persistency of O.es.cf1.˙.cf5.−.rb.ei2.rb/DMPO spin adduct. We therefore compared the effects of perfusing 5 mM of two nitrones, DMPO and 5‐(diethoxyphosphoryl)‐5‐methyl‐1‐pyrroline N‐oxide (DEPMPO) or the two structurally related pyrrolidines, diethyl (2‐methyl‐2‐pyrrolidinyl) phosphonate (DEPMPH) and pyrrolidine (PyH), on postischemic functional recovery of rat hearts subjected to 10 min of low‐flow ischemia, 30 min of global ischemia and 60 min of reperfusion. All compounds were added to the perfusate before ischemia, throughout low‐flow ischemia and during the initial 10 min of reflow. In one additional group, hearts received DEPMPO only at reflow. Hemodynamic and in vitro ESR evidence is presented indicating that the phosphonate group of DEPMPO and DEPMPH confers these molecules with an enhanced cardioprotective efficacy, unrelated to radical scavenging, acting in synergy with the intrinsic radical trapping effects of the nitronyl group. Continuous‐flow ESR spin trapping using 5.7 mM DEPMPO administered at reflow, but not before ischemia, demonstrated for the first time extended formation of O.es.cf1.˙.cf5.−.rb.ei2.rb in the reperfused myocardium.
Biochemical and genetic characterization of the membrane‐associated malate dehydrogenase (acceptor) from Corynebacterium glutamicum
Authors: Molenaar Douwe , van der Rest Michel E. , Petrović Sonja
Published: March 2003

In addition to a cytoplasmic, NAD‐dependent malate dehydrogenase ( EC1.1.1.37), Corynebacterium glutamicum possesses a highly active membrane‐associated malate dehydrogenase (acceptor) (
EC1.1.99.16). This enzyme also takes part in the citric acid cycle. It oxidizes
L‐malate to oxaloacetate and donates electrons to ubiquinone‐1 and other artificial acceptors or, via the electron transfer chain, to oxygen. NAD is not an acceptor and the natural direct acceptor for the enzyme is most likely a quinone. The enzyme is therefore called malate :quinone oxidoreductase, abbreviated to Mqo. Mqo is a peripheral membrane protein and can be released from the membrane by addition of chelators. The solubilized form was partially purified and characterized biochemically. FAD is probably a tightly but non‐covalently bound prosthetic group, and the enzyme is activated by lipids.

A C. glutamicum mutant completely lacking Mqo activity was isolated. It grows poorly on several substrates tested. The mutant possesses normal levels of cytoplasmic NAD‐dependent malate dehydrogenase. A plasmid containing the gene from C. glutamicum coding for Mqo was isolated by complementation of the Mqo‐negative phenotype. It leads to overexpression of Mqo activity in the mutant. The nucleotide sequence of the mqo gene was determined and is the first sequence known for this enzyme. The derived protein sequence is similar to hypothetical proteins from Escherichia coli, Klebsiella pneumoniae, and Mycobacterium tuberculosis.
Endosome‐lysosome transfer of insulin and glucagon in a liver cell‐free system
Authors: Chauvet Geneviève , Tahiri Khadija , Authier François , Desbuquois Bernard
Published: March 2003

The endosome‐lysosome transfer of in vivo internalized insulin and glucagon has been studied in a rat liver cell‐free system and compared to that of galactosylated bovine serum albumin (GalBSA), a ligand of the asialoglycoprotein receptor. Density‐gradient analysis of a postmitochondrial supernatant isolated 8 min after injection of [125I]iodoinsulin showed that the membrane‐associated radioactivity (55 % of the total) migrated as a single peak at the position of galactosyltransferase, a Golgi marker (1.08−1.10 g/ml). After incubation at 37 +C in the presence of ATP, an additional peak of radioactivity (12 %) was detected at the position of acid phosphatase, a lysosomal marker (1.12−1.14 g/ml). No such peak was observed in a lysosome‐depleted fraction. An ATP‐dependent conversion of [125I]iodoinsulin to trichloroacetic‐acid‐soluble products occurred during incubation (20 %) but this was unaffected by lysosome depletion. Gel‐filtration and HPLC analysis of acid extracts of gradient fractions isolated after injection of [125I]iodoinsulins selectively labeled at tyrosine residues A14 or B26 revealed the presence of components which differed from intact iodoinsulins by size and/or hydrophobicity. Low molecular‐mass components were less abundant and, conversely, intact iodoinsulin and/or high molecular‐mass components more abundant in lysosomal fractions than in endosomal fractions. In vivo internalized [125I]iodoglucagon and [125I]iodogalBSA underwent a greater lysosomal transfer (17−21 %) and lesser degradation (8−11 %) than [125I]iodoinsulin. Glycyl‐
L‐phenylalanine 2‐naphtylamide and methionine O‐methyl ester, two lysosome‐disrupting enzyme substrates, partially released the radioactivity associated with lysosomal fractions (GalBSA > insulin = glucagon) but caused little or no release of that associated with endosomal fractions. Analysis of the α and β subunits of the insulin receptor by cross‐linking to [125I]iodoinsulin and Western immunoblotting, respectively, revealed a partial lysosomal transfer of these subunits during endosome‐lysosome interaction. We conclude that an endosome‐lysosome transfer of insulin and glucagon occurs in a liver cell‐free system and suggest that the low recovery of these peptides in lysosomal fractions in vivo results from their rapid degradation within endosomes.
Synthetic peptide‐containing surfactants
Authors: Nilsson Gunhild , Gustafsson Magnus , Vandenbussche Guy , Veldhuizen Edwin , Griffiths William J. , Sjövall Jan , Haagsman Henk P. , Ruysschaert Jean‐Marie , Robertson Bengt , Curstedt Tore , Johansson Jan
Published: March 2003

Pulmonary surfactant contains two hydrophobic proteins, SP‐B and SP‐C. With the aim of identifying synthetic SP‐B and SP‐C substitutes for replacement therapy of respiratory distress syndromes, we have studied two transmembrane peptides and two amphipathic peptides that are located in the plane of a phospholipid bilayer. One amphipathic peptide was designed by changing the amino acid sequence, but not the composition or size, of the 21‐residue peptide KL4. This peptide, designated KL2.3 from its spacing of nonpolar and polar residues, exhibited similar α‐helical content as KL4 but was oriented along a phospholipid bilayer plane, in contrast to the transmembrane orientation of KL4 in the same environment. The second amphipathic peptide analyzed was succinyl‐LLEKLLEWLK‐amide (WMAP10). KL4 more efficiently accelerated the spreading of a mixture of 1,2‐dipalmitoyl‐sn‐glycero‐3‐phosphocholine (Pam2GroPCho)/phosphatidylglycerol (PtdGro)/palmitic acid (PamOH), 68 : 22 : 9 (by mass), at an air/water interface than did any of the amphipathic peptides. Similarly, KL4, but not KL2.3, when present in an interfacial monolayer composed of Pam2GroPCho/1‐palmitoyl‐2‐oleoyl‐sn‐glycero‐3‐phosphoglycerol, 7 : 3 (by mass), increased lipid insertion from subphase vesicles.

An SP‐C analogue, SP‐C(Leu), with all helical valyl residues in native SP‐C replaced with Leu and the palmitoylcysteines at positions 5 and 6 replaced with Ser, but otherwise with essentially the same primary structure as the native peptide, was analyzed. SP‐C(Leu) exhibited similar α‐helical content as native SP‐C and a transmembrane orientation and, in contrast to poly‐valyl‐containing synthetic peptides, it folds into a helical conformation after acid‐induced denaturation. SP‐C(Leu) accelerated the spreading of Pam2GroPCho/PtdGro/PamOH, 68 : 22 : 9 (by mass), almost identically to native SP‐C, and lowered the surface tension during rapid cyclic film compressions in a pulsating bubble surfactometer to near zero and 43 mN/m at minimum and maximum bubble size, respectively. Airway instillation of 2 % (by mass) SP‐C(Leu) combined with Pam2GroPCho/PtdGro/PamOH in preterm rabbit fetuses improved dynamic lung compliance by about 30 % compared with untreated controls.
The Saccharomyces cerevisiae Pet309 protein is embedded in the mitochondrial inner membrane
Authors: Manthey Glenn M. , Przybyla‐Zawislak Beata D. , McEwen Joan E.
Published: March 2003

The nuclear PET309 gene of Saccharomyces cerevisiae is necessary for expression of the mitochondrial COX1 gene, which encodes subunit I of cytochrome c oxidase. In a pet309 null mutant, there is a defect both in accumulation of COX1 pre‐RNA, if it contains introns, and in translation of COX1 RNAs [Manthey, G. M. & McEwen, J. E. (1995) EMBO J. 14, 4031−4043]. To facilitate identification and intracellular localization of the protein Pet309p, that is encoded by the PET309 gene, Pet309p was tagged at the carboxy terminus with an epitope from the human c‐myc protein. A monoclonal antibody against the c‐myc epitope detected a 98‐kDa protein in mitochondria of yeast cells that expressed the PET309−c‐myc fusion protein from a high copy number plasmid. This protein was not detectable in cells that did not express the fusion protein, or that expressed it from a single copy centromeric vector. Additional analyses of mitochondrial subfractions demonstrated that the PET309−c‐myc fusion protein is localized specifically in the inner mitochondrial membrane. It could not be extracted by alkaline sodium carbonate, yet it was susceptible to proteinase K digestion in mitoplasts (mitochondria with a disrupted outer membrane). These results indicate that Pet309p spans the inner membrane, with domain(s) exposed to the intermembrane space side of the membrane. How Pet309p may function in concert with other gene products necessary for COX1 RNA translation or accumulation, such as Mss51p or Nam1p, respectively, is discussed.
The chemical structures of the capsular polysaccharides from Streptococcus pneumoniae types 32F and 32A
Authors: Karlsson Camilla , Jansson Per‐Erik , Sørensen Uffe B.
Published: March 2003

The structures of the capsular polysaccharides from Streptococcus pneumoniae types 32F and 32A have been determined by means of NMR spectroscopy as the principal method. It is concluded that both polysaccharides are composed of tetrasaccharide repeating units with a phosphorylcholine (PCho) group linked to the 3‐position of the 4‐substituted β‐
L‐rhamnose (Rha) residue. Both polysaccharides are substituted with one O‐acetyl group at the 2‐position of the same β‐
L‐rhamnose residue. In addition, the type‐32A polysaccharide is substituted with another O‐acetyl group at the 4‐position of the 2,3‐disubstituted α‐
D‐glucose residue, i.e. the branch‐point residue. An unusual detail in the structure is that the side chain is composed of a rhamnosyl phosphate.

R O‐PCho
∣∣
4
3
←4)‐β‐
D‐Glcp‐(1←3)‐α‐
D‐Glcp‐(1←4)‐β‐
L‐Rhap2Ac‐(1←
2
∣α‐
L‐Rhap‐P‐O
32F  R=H
32A
R=Ac
In the type‐32F polysaccharide R=H, and in the type‐32A polysaccharide R=Ac. The structure of C‐polysaccharide found in our preparations of type‐32F and type‐32A capsular polysaccharides is in agreement with that published previously for the pneumococcal common antigen C‐polysaccharide [Fischer, W., Behr, T., Hartmann, R., Peter‐Katalinic, J. & Egge, H. (1993) Eur. J. Biochem. 215, 851−857; Kulakowska, M., Brisson, J.‐R., Griffith, D. W., Young, N. M. & Jennings, H. J. (1993) Can. J. Chem. 71, 644−648].
Androgen‐sensitive human prostate cancer cells, LNCaP, produce both N‐terminally mature and truncated prostate‐specific antigen isoforms
Authors: Herrala Annakaisa , Kurkela Riitta , Vihinen Mauno , Kalkkinen Nisse , Vihko Pirkko
Published: March 2003

To characterize prostate‐specific antigen (PSA) produced by cancer cells, different isoforms of PSA secreted by the human prostate cancer cells, LNCaP, were purified. LNCaP‐PSA production was induced by synthetic androgen, R1881. LNCaP‐PSA was separated into four pools. The molecular mass of LNCaP‐PSA isoforms in these pools was 34 kDa under reducing conditions and 29 kDa under non‐reducing conditions on SDS/PAGE. pI of LNCaP‐PSA isoforms varied from 6.8 to 8.2. Pool A had the highest specific activity, 37 nmol/(min×mg). All the pools formed stable complexes with α1‐antichymotrypsin and α2‐macroglobulin. The pools contained 10−60 % of N‐terminally correctly processed LNCaP‐PSA isoforms. According to the molecular modelling, the addition or deletion of two or four N‐terminal amino acids could affect the three‐dimensional structure and thereby remarkably reduce the enzyme activity of LNCaP‐PSA.
Spatial structure of the M3 transmembrane segment of the nicotinic acetylcholine receptor α subunit
Authors: Lugovskoy Alexey A. , Maslennikov Innokenty V. , Utkin Yuri N. , Tsetlin Victor I. , Cohen Jonathan B. , Arseniev Alexander S.
Published: March 2003

The three‐dimensional structure of a synthetic peptide corresponding to the putative transmembrane segment M3 (amino acid residues 277−301) of the α subunit of the nicotinic acetylcholine receptor from Torpedo californica has been studied by means of two‐dimensional 1H‐NMR spectroscopy in a chloroform/methanol (1 : 1) mixture containing 0.1 M LiClO4. Complete resonance assignment has been performed using double‐quantum‐filtered COSY (DQF‐COSY), TOCSY and NOESY spectra. The spatial structure has been calculated using the Diana program on the basis of integrated intensities of NOESY spectra, HN‐CαH and HCα‐CβH spin‐spin coupling constants. Residues 279−297 of M3 form a right‐handed helix (root mean square deviation is 0.032 nm for backbone atoms and 0.088 nm for all heavy atoms). The conformations of the 17 side chains have been unambiguously determined. The obtained structure is in accord with the photolabeling pattern of the membrane nicotinic acetylcholine receptor (nAChR) which suggests α‐helical structure of M3 in the labeled portion [Blanton, M. P. & Cohen, J. B. (1994) Biochemistry 33, 2859−2872].
The ubiquityl‐calmodulin synthetase system from rabbit reticulocytes : isolation of the ubiquitin‐binding first component, a ubiquitin‐activating enzyme
Authors: Majetschak Matthias , Laub Markus , Meyer Helmut E. , Jennissen Herbert P.
Published: March 2003

Ubiquitin is often implicated as a specific tag for protein degradation via the ubiquitin system although only a limited number of physiological proteins have been shown to be degraded in their native tissues via this pathway in vivo. Ubiquitin may also, however, have other functions of a regulatory nature (non‐catabolic ubiquitylation). The ubiquitylation of calmodulin appears to fall into this category. Ubiquitin is linked to free calmodulin in the presence of the second messenger Ca2+ by the enzyme ubiquitin−calmodulin ligase (uCaM synthetase;
EC6.3.2.21) and there is no evidence that this step is followed by degradation of calmodulin via the ATP‐dependent 26‐S protease. Due to a lack of natural substrates and sufficient tissue material, only a few components of the ubiquitin system have been obtained in truly homogenous form from reticulocytes. We therefore decided to attempt this for the calmodulin ligase. The enzymic components of the uCaM synthetase system copurified over several steps and could be highly enriched by a novel sample displacement technique on an ion‐exchange resin. A fractionation of the synthetase components by affinity chromatography on ubiquitin−Sepharose and calmodulin−Sepharose yielded two essentially inactive components : a ubiquitin−Sepharose binding fraction (uCaM Syn‐F1)and a calmodulin−Sepharose binding fraction (uCaM Syn‐F2). The full activity of uCaM synthetase can be reconstituted when these two fractions are reunited. uCaM Syn‐F1 could then be separated from all other enzymes of ubiquitin metabolism and, employing the second component with the natural substrate calmodulin, could be purified over 3500‐fold to homogeneity. The ability to catalyze its own thiol labile ubiquitylation identified it as a member of the ubiquitin‐activating enzyme family (E1). The homogenous preparation contained a single protein of molecular mass 213 ± 21 kDa (mean ± SEM) as determined by gel filtration. The molecular mass of the monomer was determined by electrospray ion mass spectrometry to 112 140 ± 47 Da (mean ± SD). N‐terminal sequence analysis (20 amino acids) led to a single N‐terminal peptide beginning at residue 57 of the known rabbit cDNA sequence. No ragged N‐terminus was detected, as would be expected by the action of an aminopeptidase or other peptidases of low specificity. The monomer molecular mass calculated from the cDNA sequence (Arg57−Arg1058) is 111 975 Da, characterizing this enzyme from reticulocytes as a homodimer of 224 kDa.
A serine protease from the bovine duodenal mucosa, chymotrypsin‐like duodenase
Authors: Sokolova Elena A. , Starkova Natalia N. , Vorotyntseva Tatyana I. , Zamolodchikova Tatyana S.
Published: March 2003

Chymotrypsin‐like duodenase (ChlD), a new protease from the bovine duodenum mucosa was isolated and purified. The enzyme molecule is a single chain (25 kDa); the native enzyme is a monomer with an isoelectric point > 10.0. ChlD displays the chymotrypsin‐like activity and cleaves 4‐nitroanilide substrates of chymotrypsin, chymases and cathepsin G. ChlD hydrolyzes its best substrate 2‐N‐succinylvalylprolylphenylalanine 4‐nitroanilide with kcat of 2.8 s−1 and catalytic efficiency kcat/Km of 2300 M−1 s−1. The enzyme is stable with a pH range of 3−10 and exhibits the maximum activity at pH 8−10. ChlD is irreversibly inhibited by diisopropylphosphofluoridate and phenylmethanesulfonyl fluoride, which is indicative of an active‐site serine in this protease. α‐N‐tosyl‐
L‐phenylalanine chloromethane, a specific reagent for a catalytically active His, markedly inhibited ChlD. The enzyme activity was strongly inhibited by several natural inhibitors of serine proteases (from soybean, potato, Lima bean, kidney bean). The N‐terminal sequence of the native ChlD (23 amino acids) shows high similarity, but not identity, to those of duodenase, granzymes, chymases and cathepsin G.
Probing of the neuropeptide Y−Y1‐receptors interaction with anti‐receptor antibodies
Authors: Wieland Heike A. , Eckard Christophe P. , Doods Henri N. , Beck‐Sickinger Annette G.
Published: March 2003

The Y1 receptor, which belongs to the family of rhodopsin‐like GTP‐binding protein‐coupled, seven‐transmembrane helix‐spanning receptors, binds the 36‐mer neuromodulator neuropeptide Y (NPY) with nanomolar affinity. Synthetic fragments of the N‐terminus, extracellular loops and C‐terminus of the Y1 receptor were used to generate 18 anti‐receptor antibodies; ten of them recognize the receptor expressed on intact cells as well as on membranes that have been prepared (with the exception of one antibody raised against the intracellular C‐terminus) as investigated by ELISA. SDS/PAGE of solubilized membranes, subsequent Western blotting and staining with the antibodies revealed two proteins of 73 kDa and 51 kDa for both, the rat and the human receptor.

Competition with neuropeptide Y showed that the binding of seven antibodies is strongly inhibited in the presence of the native ligand. Using photoactivatible analogues, it could be demonstrated that the competition efficiency strongly depends on the position of the crosslinker within the ligand. Based on these studies, a model for the ligand‐receptor interaction is suggested. These antibodies represent novel tools for the structural characterization of the Y1 receptor and its interaction with NPY and antagonists as well as for localization studies.
The third serine proteinase with chymotrypsin specificity isolated from Atlantic cod (Gadus morhua) is a type‐II elastase
Authors: Ásgeirsson Bjarni , Leth‐Larsen Rikke , Thórólfsson Matthías , Nedertoft Morten M. , Højrup Peter
Published: March 2003

Preparations of chymotrypsin from Atlantic cod are heterogeneous and previously gave rise to two active peaks when subjected to pH‐gradient chromatography. Extension of the pH‐gradient resolved a third protein peak with benzoyltyrosine ethylester hydrolytic activity. The first two peaks have been characterized as chymotrypsin variants and designated A and B, whereas the identity of the third peak was not clear. Analysis of this protein by Edman sequencing and mass spectrometry has now confirmed a high degree of identity with the predicted protein sequence from a recently described cDNA clone. That sequence was named elastase B by sequence comparison. As the present elastase deviates in 16 positions from that of elastase B, we have named it elastase C. The elastase C was active in hydrolysing typical substrates used by chymotrypsin, namely benzoyl‐ L‐tyrosine ethylester and succinyl‐Ala‐Ala‐Pro‐Phe‐p‐nitroanilide, but inactive against the typical elastase substrates succinyl‐Ala‐Ala‐Ala‐p‐nitroanilide and orcein‐elastin. Comparison of the kinetic properties of the cod elastase C with bovine chymotrypsin and cod chymotrypsin variants A and B, using succinyl‐Ala‐Ala‐Pro‐Phe‐p‐nitroanilide, showed a lower catalytic efficiency of elastase C. The effects of several inhibitors on cod elastase C were identical to effects on chymotrypsins variants A and B, but dissimilar when compared with porcine pancreatic elastase. On the basis of the specificity and amino acid sequence, we conclude that the enzyme under study is most correctly classified as a type‐II elastase.
The catalytic mechanism of adenosylhomocysteine/methylthioadenosine nucleosidase from Escherichia coli
Authors: Allart Brigitte , Gatel Marie , Guillerm Danielle , Guillerm Georges
Published: March 2003

The substrate and inhibitory specificity of Escherichia coli adenosylhomocysteine (AdoHcy)/methylthioadenosine (MeSAdo) nucleosidase has been explored with several MeSAdo analogues modified on the sugar moiety at the 2′, 3′ and 5′ positions. Alteration at C3′ or at C2′ and C3′ positions in MeSAdo abolished substrate activity. However, the 2′‐deoxy analogue of MeSAdo is effective as a substrate; this result provides evidence against a possible general‐base catalysis involving the anchimeric assistance of the 2′‐α‐hydroxy group and the formation of a 1,2‐epoxide as an intermediate in the catalytic process. The results of a study of the interaction of an 8,5′‐cyclo analogue of MeSAdo with the enzyme indicate the importance of the glycosidic conformation of the substrate for binding to the active site. The enzyme discriminates against methanol attack from the solvent during catalysis. This implies the participation of an enzyme‐directed water nucleophile. A poor solvent kinetic deuterium‐isotope effect was measured (0.93) on the Vmax. Plots of log Vmax and log (Vmax/Km) for MeSAdo as a function of pH values from 5.0 to 8.5 are similar, with two presumably essential ionisable groups for catalysis with apparent pKa values of 5.6 and 8.2, whereas Km is independent of pH. When the 2′‐α‐hydroxy group of MeSAdo is substituted by fluorine, a significant decrease (28 500‐fold) in the Vmax for enzyme‐catalysed hydrolysis of the modified substrate is observed. This result indicates a transition state with a substantial oxocarbenium character. From these data, the reaction mechanism for AdoHcy/MeSAdo nucleosidase is discussed.
800 MHz 1H NMR solution structure refinement of oxidized cytochrome c7 from Desulfuromonas acetoxidans
Authors: Assfalg Michael , Banci Lucia , Bertini Ivano , Bruschi Mireille , Turano Paola
Published: March 2003

The solution structure of Desulfuromonas acetoxidans cytochrome c7 has been refined by using 1H‐NMR spectra recorded at 800 MHz and by using pseudocontact shifts in the final energy minimization procedure. The protein, composed of 68 amino acids, contains three paramagnetic heme moieties, each with one unpaired electron. The largely distributed paramagnetism broadens the lines in several protein parts. The structure is now relatively well resolved all over the backbone by the use of 1315 meaningful NOEs and 90 pseudocontact shifts. The statistical analysis of the structure indicates its satisfactory quality. The protein‐fold is quite similar to that of the analogous four‐heme cytochromes c3 for those parts which can be considered homologous. The solvent accessibility and the electrostatic potential surfaces surrounding the three hemes have been analyzed in terms of their reduction potentials. The resulting magnetic susceptibility anisotropy data obtained from pseudocontact shifts are analyzed in terms of structural data.