bluuebunny/crossref_metadata_2025_split huggingface dataset:
Functional differentiation in trematode hemoglobin isoforms
Authors: Rashid Aftab K. , Weber Roy E. 
Published: March 2003

The Hbs and the major electrophoretic Hb components (isoHbs) were isolated from three species of the trematodes, Explanatum explanatum (Ee), Gastrothylax crumenifer (Gc) and Paramphistomum epiclitum (Pe), that parasitise the common Indian water buffalo Bubalus bubalis. The Hbs are monomeric and resemble the so‐called nonfunctional mutant hemoglobins that have Tyr at B10 or E7 positions (replacing Leu and the His residues, respectively). However, they are capable of binding with O2 and CO. O2 equilibrium studies of trematode Hb isoforms reveal extremely high O2 affinities, with half‐saturation O2 tension (P50) values up to 800 times lower than those of human hemoglobins. This correlates with Tyr residues at B10 and at the distal position (E7) that decrease the O2 dissociation rate by contributing hydrogen bonds (H‐bonds) to the bound O2. These substitutions also increase the O2 association rates either due to orientation of E7‐Tyr towards the solvent and/or by sterically hindering the entry of water molecules into the heme pocket. The latter may account for the low rate of autoxidation of trematode Hbs. The Hbs and their isoforms from different species exhibited pronounced variation in O2 affinity, which may relate to subtle differences in the structure of the heme pocket. The O2 affinities of the composite (unfractionated) Hbs were intermediate to those of the individual Hb isoform. The P50 values of Hbs here obtained by direct O2 equilibrium measurements differed from those calculated from kinetic data already published [Kiger, L., Rashid, A. K., Griffon, N., Haque, M., Moens, L.,Gibson, Q. H., Poyart, C., & Marden, M. C. (1998). Biophys. J. 75, 990–998.] Intermediate state(s) due to slow reorientation of E7‐Tyr may account for this difference. Some Hb isoforms showed slight (either normal or reverse) Bohr effects. The hyperbolic O2 equilibrium curve, Hill coefficient (n) values near unity accord with a monomeric nature of trematode Hbs. In marked contrast to vertebrate Hbs, CO does not seem to compete effectively with O2 in trematode Hbs, as evident from partition coefficient values (M) below 1.
Critical relationship between glycosylation of recombinant lutropin receptor ectodomain and its secretion from baculovirus‐infected insect cells
Authors: Pajot‐Augy Edith , Bozon Véronique , Remy Jean‐Jacques , Couture Laurence , Salesse Roland 
Published: March 2003

The lutropin receptor ectodomain overexpressed under the control of the powerful polyhedrin promoter in baculovirus‐infected Sf9 insect cells, is mainly found in an inactive, intracellularly‐aggregated form. It is secreted in an active form under the control of the P10 promoter, a somewhat weaker and earlier promoter, at the price of a lower production. The apparent molecular masses of the two species encoded by the same cDNA are 48 kDa and 60–68 kDa, respectively. The relationship between the extent and type of glycosylation and the extracellular targeting for the recombinant lutropin receptor ectodomains was investigated precisely with endoglycosidases, lectins of various specificities, and a glycosylation inhibitor, and tested with monoclonal and polyclonal antibodies. The results indicate that the strong polyhedrin promoter probably overwhelms the processing capacity of the ER in Sf9 cells, so that only a high‐mannose precursor is expressed in large amounts. Only a minute amount of protein is secreted, which has been processed by Sf9 exoglycosidases/glycosyltransferases and bears complex/hybrid oligosaccharides. The weaker P10 promoter allows secretion of a mature and active receptor ectodomain, bearing complex glycosylation. An important O‐linked glycosylation is also added post‐translationally on this species. In particular, β‐galactose and sialic acid residues were specifically detected in the secreted species, evidence of the induction of the corresponding glycosyltransferases or of their genes. These results suggest that Sf9 cells should eventually be engineered with chaperones and glycosyltransferases in order to improve the production of demanding glycoproteins such as the porcine lutropin ectodomain, so as to open the way to resolution of the three‐dimensional structures of these receptors.
Pen c 1, a novel enzymic allergen protein from Penicillium citrinum
Authors: Su Ning‐Yuan , Yu Chia‐Jung , Der Shen Hong‐ , Pan Fu‐Ming , Chow Lu‐Ping 
Published: March 2003

A 33‐kDa alkaline serine protease secreted by Penicillium citrinum strain 52‐5 is shown to be an allergenic agent in this fungus. The protein, designated Pen c 1, was purified by sequential DEAE‐Sepharose and carboxymethyl (CM)‐Sepharose chromatographies. Pen c 1 has a molecular mass of 33 kDa and a pI of 7.1. The caseinolytic enzyme activity of this protein was studied. The protein binds to serum IgE from patients allergic to Penicillium citrinum.The cDNA encoding Pen c 1 is 1420 bp in length and contains an open reading frame for a 397‐amino‐acid polypeptide. Pen c 1 codes for a larger precursor containing a signal peptide, a propeptide and the 33‐kDa mature protein. Sequence comparison revealed that Pen c 1 possesses several features in common with the alkaline serine proteases of the subtilisin family. The essential Asp, His, and Ser residues that make up the catalytic triad of serine proteases are well conserved. Northern blots demonstrated that mRNAs transcribed from this gene are present at early stages of culture. The allergen encoded by Pen c 1 gene was expressed in Escherichia coli as a fusion protein bearing an N‐terminal histidine‐affinity tag. The protein, purified by affinity chromatography with a yield of 130 mg of pure protein per liter of culture, was able to bind to both a monoclonal anti‐Pen c 1 antibody and IgE from the serum of patients allergic to Penicillium. Recombinant Pen c 1 can therefore be expressed in E. coli in large quantities and should prove useful as a standardized specific allergen for immuno‐diagnosis of atopic disorders. In addition, full caseinolytic enzyme activity could be generated in the purified recombinant protein by sulfonation and renaturation, followed by removal of the affinity tag, indicating that the refolded protein can assume the same conformation as the native protein.
An endogenous activator of renal glutamic acid decarboxylase
Authors: Turský Timotej , Bandžuchová Elena 
Published: March 2003

The renal glutamic acid decarboxylase (GAD) differs from the brain and pancreatic enzyme by its strong binding to membranes that is not influenced by detergents. After centrifugation of freshly prepared homogenate of the rat renal cortex, only 10–15% of GAD activity was found in supernatants and 15–30% in pellets. The majority of the GAD activity was lost. The bound GAD was found in the pellet. A thermolabile activator was present in the supernatant, which was not lost on dialysis. Approximately 55% of the total GAD activity was solubilized in homogenates stored for 24 h at 4 °C without detergent, whereas in homogenates stored with Triton X‐100, the solubilized GAD increased to 80%. This solubilization was decreased by inhibitors of thioproteases such as leupeptin, antipain and trans‐epoxysuccinyl‐l‐leucylamido‐(4‐guanidino)butane (E‐64). Solubilized GAD was applied to DEAE Toyopearl resin and the GAD activator was eluted with 35 mm Pi. GAD was eluted with 250 mm Pi. The effect of ATP on the activity of renal GAD was also different to its effect on brain GAD. ATP is a strong inhibitor of the brain enzyme at physiological concentrations. ATP (and Pi), together with chlorides (another brain GAD inhibitor), stabilize the renal GAD. However, renal GAD was inhibited by ATP in the presence of leupeptin in freshly prepared homogenates. Similarly, ATP inhibits solubilized GAD from homogenates stored without Triton X‐100 for 24 h at 4 °C, but Pi retains its stabilizing effect in this preparation. A significant finding of the work presented here is the obligatory requirement of an endogenous activator for renal GAD activity. Whether this activator is an enzyme converting the inactive GAD to active enzyme (as hypothesized for brain GAD), or whether it is a protein affecting the activity of renal GAD by binding (as observed for GAD in some plants) remains to be established.
The half‐life of human procathepsin S
Authors: Nissler Karl , Strubel Wolfgang , Kreusch Stefan , Rommerskirch Winfried , Weber Ekkehard , Wiederanders Bernd 
Published: March 2003

Two processes, synthesis and degradation, contribute to the intracellular concentration of a protein. As most malignant tumors or tumor cell lines show elevated levels of proteinases, we studied the half‐life of a cysteine proteinase, procathepsin S, in order to determine whether tumor cells can regulate their cathepsin concentration via changing the degradation rate of the enzyme. The following procathepsin S species were examined: wild‐type procathepsin S in macrophages, recombinant procathepsin S in human embryonic kidney cells (HEK 293 cells), recombinant nonglycosylated procathepsin S in HEK 293 cells, wild‐type procathepsin S in the established nonsmall cell lung carcinoma cell line 97TM1. The half‐lives of both wild‐type procathepsins S expressed in macrophages and in HEK 293 cells were 1 h, whereas that of procathepsin S in the tumor cell line was 2 h. Nonglycosylated procathepsin S was not processed. The degradation of mature cathepsin S proceeded with a half‐life of 16–18 h. All cell lines studied secreted substantial amounts of procathepsin S into the culture medium. No further maturation of secreted procathepsin S has been observed in the culture medium. We suggest a disturbed sorting mechanism in tumor cells.
Veratryl alcohol‐mediated oxidation of isoeugenyl acetate by lignin peroxidase
Authors: ten Have Rimko , de Thouars Robbert G. , Swarts Henk J. , Field Jim A. 
Published: March 2003

The mechanism of the veratryl alcohol (VA)‐mediated oxidation of isoeugenyl acetate (IEA) by lignin peroxidase, and the subsequent spontaneous Cα–Cβ cleavage of IEA to vanillyl acetate were studied. IEA oxidation only occurred in the presence of VA. It probably did not bind to lignin peroxidase as evidenced by an unaffected Km for VA in the presence of IEA, and by the fact that a 10‐fold molar excess of the unreactive IEA counterpart, eugenyl acetate, did not affect the IEA oxidation rate. IEA was very efficient in recycling VA. Up to 34 mol of IEA were oxidized per mol VA. Formation of the predominant VA oxidation product, veratraldehyde, was postponed until IEA was almost completely oxidized. Together these findings suggest that IEA was oxidized by VAz.rad;+ rather than directly by lignin peroxidase. Thus, VA functioned as a redox mediator during IEA oxidation which is remarkable considering the high calculated ionization potential of 8.81 eV. Regardless of the presence of O2, ≈ 2 mol of IEA were consumed per mol H2O2, which indicated that IEA was enzymatically oxidized by one electron to the putative radical cation (IEAz.rad;+). After formation of IEAz.rad;+, a series of O2‐dependent chemical reactions were responsible for Cα–Cβ cleavage to the major oxidation product vanillyl acetate, as evidenced by the observation that an N2 atmosphere did not inhibit IEA oxidation, but almost completely inhibited vanillyl acetate formation. GC‐MS analyses revealed that under an air atmosphere 1‐(4′‐acetoxy‐3′‐methoxyphenyl)‐2‐propanone, 1‐(4′‐acetoxy‐3′‐methoxyphenyl)‐1‐hydroxy‐2‐propanone, and 1‐(4′‐acetoxy‐3′‐methoxyphenyl)‐2‐hydroxy‐1‐propanone were also formed. Formation of the latter two was diminished under an N2 atmosphere.
Down‐regulation of mammalian 3‐hydroxy‐3‐methylglutaryl coenzyme A reductase activity with highly purified liposomal cholesterol
Authors: Plemenitas Ana , Watson John A. 
Published: March 2003

Chinese hamster ovary‐215 cells (CHO‐215) cannot synthesize C27 and C28 sterols because of a defect in the reaction that decarboxylates 4‐carboxysterols [Plemenitas, A., Havel, C.M. & Watson, J.A. (1990) J. Biol. Chem.265, 17012–17017]. Thus, CHO‐215 cell growth is dependent on an exogenous metabolically functional source of cholesterol. We used CHO‐215 cells to (a) determine whether highly purified (> 99.5%) cholesterol, in egg lecithin liposomes, could down‐regulate derepressed 3‐hydroxy‐3‐methylglutaryl coenzyme A (HMG‐CoA) reductase activity and if so (b) determine whether the loss in reductase catalytic activity correlated kinetically with the synthesis and accumulation of detectable oxycholesterol derivatives.Liposomal cholesterol (26–39 µm) supported maximum CHO‐215 growth and initiated suppression of HMG‐CoA reductase activity at concentrations greater than 50 µm. Maximum suppression (50–60%) of reductase activity was achieved with 181.3 µm liposomal cholesterol in 6 h. Also, regulatory concentrations of highly purified liposomal [3H]cholesterol were not converted (biologically or chemically) to detectable levels of oxy[3H]cholesterol derivatives during 3–6 h incubations. Lastly, a broad‐spectrum cytochrome P450 inhibitor (miconazole) had no effect on liposomal cholesterol‐mediated suppression of HMG‐CoA reductase activity.These observations established that (a) highly purified cholesterol, incorporated into egg lecithin liposomes, can signal the down‐regulation of derepressed mammalian cell HMG‐CoA reductase activity and (b) if oxycholesterol synthesis was required for liposomal cholesterol‐mediated down‐regulation, the products had to be more potent than 24‐, 25‐, or 26‐/27‐hydroxycholesterol.
The pneumococcal common antigen C‐polysaccharide occurs in different forms
Authors: Karlsson Camilla , Jansson Per‐Erik , Skov Sørensen Uffe B. 
Published: March 2003

The structure of the pneumococcal common antigen, C‐polysaccharide, from a noncapsulated pneumococcal strain, CSR SCS2, was studied using 1H‐NMR, 13C‐NMR and 31P‐NMR spectroscopy. The dependence of NMR chemical shifts on the variation in pD was also studied. It was established that the C‐polysaccharide is composed of a backbone of tetrasaccharide‐ribitol repeating units that are linked to each other by a phosphodiester linkage between position 5 of a d‐ribitol residue and position 6 of a β‐d‐glucopyranosyl residue. The polysaccharide is substituted with one residue of phosphocholine at position 6 of the 4‐substituted 2‐acetamido‐2‐deoxy‐α‐d‐galactopyranosyl residue. Both galactosamine residues in the polysaccharide are N‐acetylated.                    O)‐P‐Cho                      |                    66)‐β‐d‐Glcp‐(1→3)‐α‐AATp‐(1→4)‐α‐d‐GalpNAc‐(1→3)‐β‐d‐GalpNAc‐(1→1)‐d‐ribitol‐5‐P‐(O→where AAT is 2‐acetamido‐4‐amino‐2,4,6‐trideoxy‐d‐galactose and Cho is choline. This structure differs, concerning phosphocholine substituents and N‐acetylation, from those reported previously for pneumococcal C‐polysaccharide [Jennings, H.J., Lugowski, C. & Young, N.M. (1980) Biochemistry19, 4712–4719; 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]. The structures of the C‐polysaccharides present in three pneumococcal types were also examined. They contain one (in 18B) or two (in 32F and 32A) phosphocholine residues in the repeating unit. The degree of substitution was not determined. The backbone of all examined C‐polysaccharides was identical and in all cases both galactosamine residues appeared to be N‐acetylated.
Supramolecular organization of photosystem II and its light‐harvesting antenna in partially solubilized photosystem II membranes
Authors: Boekema Egbert J. , van Roon Henny , van Breemen Jan F. L. , Dekker Jan P. 
Published: March 2003

We present an extended analysis of the organization of green plant photosystem II and its associated light‐harvesting antenna using electron microscopy and image analysis. The analysis is based on a large dataset of 16 600 projections of negatively stained PSII–LHCII supercomplexes and megacomplexes prepared by means of three different pretreatments. In addition to our previous work on this system [Boekema, E.J., van Roon, H., Calkoen, F., Bassi, R. and Dekker, J.P. (1999) Biochemistry38, 2233–2239], the following results were obtained.The rotational orientation of trimeric LHCII at the S, M and L binding positions was determined. It was found that compared to the S trimer, the M and L trimers are rotationally shifted by about −20° and −50°, respectively.The number of projections with empty CP29, CP26 and CP24 binding sites was found to be about 0, 18 and 4%, respectively. We suggest that CP26 and CP24 are not required for the binding of trimeric LHCII at any of the three binding positions.A new type of megacomplex was observed with a characteristic windmill‐like shape. This type III megacomplex consists of two C2S2 supercomplexes connected at their CP26 tips.Structural variation in the region of the central dimeric photosystem II complex was found to occur at one specific position near the periphery of the complex. We attribute this variation to the partial absence of an extrinsic polypeptide or one or more small intrinsic membrane proteins.
Release of the cyano moiety in the crystal structure of N‐cyanomethyl‐N‐(2‐methoxyethyl)‐daunomycin complexed with d(CGATCG)
Authors: Saminadin Pascale , Dautant Alain , Mondon Martine , Langlois d’Estaintot Béatrice , Courseille Christian , Précigoux Gilles 
Published: March 2003

Doxorubicin is among the most widely used anthracycline in cancer chemotherapy. In an attempt to avoid the cardiotoxicity and drug resistance of doxorubicin therapy, several analogues were synthesized. The cyanomorpholinyl derivative is the most cytotoxic. They differ greatly from their parent compound in their biological and pharmacological properties, inducing cross‐links in drug DNA complexes.The present study concerns N‐cyanomethyl‐N‐(2‐methoxyethyl)‐daunomycin (CMDa), a synthetic analogue of cyanomorpholino‐daunomycin. Compared to doxorubicin, CMDa displays a cytotoxic activity on L1210 leukemia cells at higher concentration but is effective on doxorubicin resistant cells. The results of fluorescence quenching experiments as well as the melting temperature (ΔTm = 7.5 °C) studies are consistent with a drug molecule which intercalates between the DNA base pairs and stabilizes the DNA double helix. The crystal structure of CMDa complexed to the hexanucleotide d(CGATCG) has been determined at 1.5 Å resolution. The complex crystallizes in the space group P41212 and is similar to other anthracycline‐hexanucleotide complexes. In the crystal state, the observed densities indicate the formation of N‐hydroxymethyl‐N‐(2‐methoxyethyl)‐daunomycin (HMDa) with the release of the cyano moiety without DNA alkylation. The formation of this degradation compound is discussed in relation with other drug modifications when binding to DNA. Comparison with two other drug‐DNA crystal structures suggests a correlation between a slight change in DNA conformation and the nature of the amino sugar substituents at the N3′ position located in the minor groove.
Nucleotide binding of an ADP analog to cooperating sites of chloroplast F1‐ATPase (CF1)
Authors: Günther Stephan , Huchzermeyer Bernhard 
Published: March 2003

Pre‐steady state nucleotide binding to the chloroplast F1‐ATPase (CF1) was measured in a stopped‐flow apparatus by monitoring the change of fluorescence intensity of TNP‐ADP upon binding. The analysis of the time courses led to the proposal of a mechanism of nucleotide binding with the following characteristics. (a) It involves three sites binding nucleotides in a concerted manner. (b) Each binding site is able to undergo a conformational change from a loose binding state into a state refraining from any direct release of the bound nucleotide into the medium. Only the reverse reaction via the loose binding state enables release out of the tight binding state. (c) Due to very strong negative cooperativity, a maximum of two of the three sites can be found in the state of tight binding. (d) Cooperativity between the three sites leads to a slower nucleotide binding of the second nucleotide compared to the first nucleotide. Furthermore, the conformational change from the loose binding state to the tight binding state is slowed down if one of the other sites already is in the tight binding state.Three‐sites mechanisms in which rotation leads to an exchange of the properties of the binding sites failed to simulate the observed time courses of nucleotide binding. However, as the experimental set up was designed to prevent catalysis taking place, our results entirely agree with the current finding that rotation requires catalytic turnover of the enzyme.
Activity and stability of a neutral protease from Vibrio sp. (vimelysin) in a pressure‐temperature gradient
Authors: Ikeuchi Hidekazu , Kunugi Shigeru , Oda Kohei 
Published: March 2003

The apparent second‐order rate constant of hydrolysis of Fua‐Gly‐LeuNH2 by vimelysin, a neutral protease from Vibrio sp. T1800, was measured in a variable pressure–temperature gradient (0.1–400 MPa and 5–40 °C). The apparent maximum rate was observed at approximately 15 °C and 150–200 MPa; the pressure‐activation ratio (kcat/Km(max)/kcat/Km(0.1 MPa)) was reached about sevenfold. The pressure dependence of the kcat and Km parameters at constant temperature (25 °C) revealed that the pressure‐activation below 200 MPa was mainly caused by a change in the kcat parameter.The change in the intrinsic fluorescence intensity of vimelysin was also measured in a pressure‐temperature plane (0.1–400 MPa and −20 to +60 °C). The fluorescence intensity was found to decrease by increasing pressure and temperature, and the isointensity contours were more or less circular. The tangential lines to the contours at high temperatures and low to medium pressures seem to have slightly positive slopes, which was reflected by the higher residual activities left after incubations at higher temperatures and medium pressure (200 MPa and 50 °C) and by the almost intact secondary structure left after 1 h of incubation at 200 MPa and 40 °C, as studied by circular dichroism.These results were compared with the corresponding results for thermolysin, a moderately thermostable protease from Bacillus thermoproteolyticus. Apparent differences that might be related to the temperature adaptations of the respective source microbes are also discussed.
New functions of a long‐known molecule
Authors: Ziegler Mathias 
Published: March 2003

Over the past decades, the pyridine nucleotides have been established as important molecules in signaling pathways, besides their well known function in energy transduction. Similarly to another molecule carrying such dual functions, ATP, NAD(P)+ may serve as substrate for covalent protein modification or as precursor of biologically active compounds.Protein modification is catalyzed by ADP‐ribosyl transferases that attach the ADP‐ribose moiety of NAD+ to specific amino‐acid residues of the acceptor proteins. For a number of ADP ribosylation reactions the specific transferases and their target proteins have been identified. As a result of the modification, the biological activity of the acceptor proteins may be severely changed. The cell nucleus contains enzymes catalyzing the transfer of ADP‐ribose polymers (polyADP‐ribose) onto the acceptor proteins. The best known enzyme of this type is poly(ADP‐ribose) polymerase 1 (PARP1), which has been implicated in the regulation of several important processes including DNA repair, transcription, apoptosis, neoplastic transformation and others.The second group of reactions leads to the synthesis of an unusual cyclic nucleotide, cyclic ADP‐ribose (cADPR). Moreover, the enzymes catalyzing this reaction may also replace the nicotinamide of NADP+ by nicotinic acid resulting in the synthesis of nicotinic acid adenine dinucleotide phosphate (NAADP+). Both cADPR and NAADP+ have been reported to be potent intracellular calcium‐mobilizing agents. In concert with inositol 1,4,5‐trisphosphate, they participate in cytosolic calcium regulation by releasing calcium from intracellular stores.