bluuebunny/crossref_metadata_2025 huggingface dataset:
Functional analysis of O‐linked oligosaccharides in threonine/serine‐rich region of Aspergillus glucoamylase by expression in mannosyltransferase‐disruptants of yeast
Authors: Goto Masatoshi , Tsukamoto Masaki , Kwon Il , Ekino Keisuke , Furukawa Kensuke
Published: March 2003

The glaA gene encoding glucoamylase I (GAI) of Aspergillus awamori var. kawachi was heterologously expressed in mannosyltransferase mutants of Saccharomyces cerevisiae, in which the pmt1 gene and the kre2 gene were disrupted. The GAI enzymes expressed in these yeast mutant cells exhibited a lesser extent of O‐glycosylation. Secretion of GAI expressed in the pmt1‐disruptant and in the kre2‐disruptant, respectively, was almost the same as that of GAI expressed in wild type (wt) strains. The number of O‐linked mannose in GAI from wt yeast strain ranged in size from one (Man1) to five (Man5). On the other hand, the O‐linked oligosaccharides of GAI from the pmt1‐disruptant ranged in size from Man1 to Man4. Man5 was not detected and Man2–Man4 were reduced in proportion to the reduction of Man1. The O‐linked oligosaccharides of GAI from the kre2‐disruptant ranged from Man1 to Man4, and the molar amount of Man4 was reduced to 27.3%, compared to that of the wt strain. The hydrolyzing abilities for soluble starch and the adsorbing abilities on raw starch were comparable between both disruptants and wt strains. However, the digesting abilities for raw starch of the disruptants were decreased to 70% of those of the wt strains. Stabilities of GAI of the disruptants were reduced toward extreme pH and high temperature, compared to those of the wt strains. These results demonstrate that the O‐linked oligosaccharides of GAI are responsible for the enzyme stability and activity toward insoluble substrates but not for secretion.
NMR studies on the 46‐kDa dimeric protein, 3,4‐dihydroxy‐2‐butanone 4‐phosphate synthase, using 2H, 13C, and 15N‐labelling
Authors: Richter Gerald , Kelly Mark , Krieger Cornelia , Yu Yihua , Bermel Wolfgang , Karlsson Gøran , Bacher Adelbert , Oschkinat Hartmut
Published: March 2003

3,4‐Dihydroxy‐2‐butanone 4‐phosphate synthase catalyses the release of C‐4 from the substrate, ribulose phosphate, via a complex series of rearrangement reactions. The cognate ribB gene of Escherichia coli was hyperexpressed in a recombinant E. coli strain. The protein was shown to be a 46‐kDa homodimer by hydrodynamic analysis. A variety of protein samples labelled with different grades of 13C, 15N and 2H, i.e. one with 100% 2H and 15N, one with 75% 2H, 99% 13C,15N, and one with 100% 2H, 99% 13C,15N were prepared. Despite the large molecular size, 2‐ and 3‐dimensional NMR spectra of reasonable quality were obtained. Attempts at the assignment of individual 13C, 15N and 1H signals show, in principle, the feasibility of structure determination. The number of NMR signals shows unequivocally that the homodimeric protein obeys strict C2 symmetry.
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.
Uptake and fate of class B scavenger receptor ligands in HepG2 cells
Authors: Rhainds David , Falstrault Louise , Tremblay Caroline , Brissette Louise
Published: March 2003

Class B scavenger receptors (SR‐Bs) interact with native, acetylated and oxidized low‐density lipoprotein (LDL, AcLDL and OxLDL), high‐density lipoprotein (HDL3) and maleylated BSA (M‐BSA). The aim of this study was to analyze the catabolism of CD36‐ and LIMPII‐analogous‐1 (CLA‐1), the human orthologue for the scavenger receptor class B type I (SR‐BI), and CD36 ligands in HepG2 (human hepatoma) cells. Saturation binding experiments revealed moderate‐affinity binding sites for all the SR‐B ligands tested with dissociation constants ranging from 20 to 30 µg·mL−1. Competition binding studies at 4 °C showed that HDL and modified and native LDL share common binding site(s), as OxLDL competed for the binding of 125I‐LDL and 125I‐HDL3 and vice versa, and that only M‐BSA and LDL may have distinct binding sites. Degradation/association ratios for SR‐B ligands show that LDL is very efficiently degraded, while M‐BSA and HDL3 are poorly degraded. The modified LDL degradation/association ratio is equivalent to 60% of the LDL degradation ratio, but is three times higher than that of HDL3. All lipoproteins were good cholesteryl ester (CE) donors to HepG2 cells, as a 3.6–4.7‐fold CE‐selective uptake ([3H]CE association/125I‐protein association) was measured. M‐BSA efficiently competed for the CE‐selective uptake of LDL‐, OxLDL‐, AcLDL‐ and HDL3‐CE. All other lipoproteins tested were also good competitors with some minor variations. Hydrolysis of [3H]CE‐lipoproteins in the presence of chloroquine demonstrated that modified and native LDL‐CE were mainly hydrolyzed in lysosomes, whereas HDL3‐CE was hydrolyzed in both lysosomal and extralysosomal compartments. Inhibition of the selective uptake of CE from HDL and native modified LDL by SR‐B ligands clearly suggests that CLA‐1 and/or CD36 are involved at least partially in this process in HepG2 cells.
Solution properties of the free and DNA‐bound Runt domain of AML1
Authors: Wolf‐Watz Magnus , Xie Xiao‐Qi , Holm Magnus , Grundström Thomas , Härd Torleif
Published: March 2003

The Runt domain is responsible for specific DNA and protein‐protein interactions in a family of transcription factors which includes human AML1. Structural data on the Runt domain has not yet become available, possibly due to solubility and stability problems with expressed protein fragments. Here we describe the optimization and characterization of a 140‐residue fragment, containing the Runt domain of AML1, which is suitable for structural studies. The fragment of AML1 including amino acids 46–185 [AML1 Dm(46–185)] contains a double cysteine→serine mutation which does not affect Runt domain structure or DNA‐binding affinity. Purified AML1 Dm(46–185) is soluble and optimally stable in a buffer containing 200 mm MgSO4 and 20 mm sodium phosphate at pH 6.0. Nuclear magnetic resonance and circular dichroism spectroscopy indicate that the Runt domain contains β‐sheet, but little or no α‐helical secondary structure elements. The 45 N‐terminal residues of AML1 are unstructured and removal of the N‐terminal enhances sequence‐specific DNA binding. The NMR spectrum of AML1 Dm(46–185) displays a favorable chemical shift dispersion and resolved NOE connectivities are readily identified, suggesting that a structure determination of this Runt domain fragment is feasible. A titration of 15N‐labelled AML1 Dm(46–185) with a 14‐bp cognate DNA duplex results in changes in the 15N NMR heteronuclear single quantum coherence spectrum which indicate the formation of a specific complex and structural changes in the Runt domain upon DNA binding.
Subcellular localization of the BtpA protein in the cyanobacterium Synechocystis sp. PCC 6803
Authors: Zak Elena , Norling Birgitta , Andersson Bertil , Pakrasi Himadri B.
Published: March 2003

Photosystem I is a large pigment–protein complex embedded in the thylakoid membranes of chloroplasts and cyanobacteria. In the cyanobacterium Synechocystis sp. PCC 6803, the btpA gene encodes a 30‐kDa polypeptide. Mutations in this gene significantly affect accumulation of the reaction center proteins of photosystem I in Synechocystis 6803 [Bartsevich, V. V. & Pakrasi, H. B. (1997) J. Biol. Chem. 272, 6372–6378]. We describe here the intracellular localization of the BtpA protein. Immunolocalization in Synechocystis 6803 cells demonstrated that the BtpA protein is tightly associated with the thylakoid membranes. Phase fractionation in the detergent Triton X‐114 indicated that BtpA is a peripheral membrane protein. To determine which surface of the thylakoid membrane BtpA is exposed to, we used a two‐phase polymer partitioning technique to develop a novel method to isolate inside‐out and right‐side‐out thylakoid vesicles from Synechocystis 6803. Treatments of such vesicles with different salts and protease showed that the BtpA protein is an extrinsic membrane protein which is exposed to the cytoplasmic face of the thylakoid membrane.
Preferential degradation of polyadenylated and polyuridinylated RNAs by the bacterial exoribonuclease polynucleotide phosphorylase
Authors: Lisitsky Irena , Schuster Gadi
Published: March 2003

Polyadenylation of mRNA has been shown to target the RNA molecule for rapid exonucleolytic degradation in bacteria. To elucidate the molecular mechanism governing this effect, we determined whether the Escherichia coli exoribonuclease polynucleotide phosphorylase (PNPase) preferably degrades polyadenylated RNA. When separately incubated with each molecule, isolated PNPase degraded polyadenylated and non‐polyadenylated RNAs at similar rates. However, when the two molecules were mixed together, the polyadenylated RNA was degraded, whereas the non‐polyadenylated RNA was stabilized. The same phenomenon was observed with polyuridinylated RNA. The poly(A) tail has to be located at the 3′ end of the RNA, as the addition of several other nucleotides at the 3′ end prevented competition for polyadenylated RNA. In RNA‐binding experiments, E. coli PNPase bound to poly(A) and poly(U) sequences with much higher affinity than to poly(C) and poly(G). This high binding affinity defines poly(A) and poly(U) RNAs as preferential substrates for this enzyme. The high affinity of PNPase for polyadenylated RNA molecules may be part of the molecular mechanism by which polyadenylated RNA is preferentially degraded in bacterial cells.
Antibodies to yeast Sm motif 1 cross‐react with human Sm core polypeptides
Authors: Bahia Diana , Font Josep , Khaouja Amina , Carreras Narcís , Espuny Ruth , Cicarelli Regina Maria Barretto , Ingelmo Miguel , Bach‐Elias Montse
Published: March 2003

Two regions common to all UsnRNP core polypeptides have been described: Sm motif 1 and Sm motif 2. Rabbits were immunized with a 22 amino‐acid peptide containing one segment of Sm motif 1 (YRGTLVSTDNYFNLQLNEAEEF, corresponding to residues 11–32) from yeast F protein. After immunization, the rabbit sera contained antibodies that not only reacted specifically with the peptide from yeast F protein but also cross‐reacted with Sm polypeptides from mammals; that is, with purified human U1snRNPs. The results suggest that the peptide used and human Sm polypeptides contain a common feature recognized by the polyclonal antibodies. A large collection of human systemic lupus erythematosus sera was assayed using the yeast peptide as an antigen source. Seventy per cent of systemic lupus erythematosus sera contain an antibody specificity that cross‐reacts with the yeast peptide.
NMR studies of subunit c of the ATP synthase from Propionigenium modestum in dodecylsulphate micelles
Authors: Matthey Ulrich , Kaim Georg , Braun Daniel , Wüthrich Kurt , Dimroth Peter
Published: March 2003

The structure of the Na+, Li+ or H+‐binding c subunit of the ATP synthase from Propionigenium modestum was studied by NMR. Subunit c in dodecylsulphate micelles consists of four α‐helical segments, I–IV, that are connected by short linker peptides with non‐regular secondary structures. We propose that helices I (V4–I26) and IV (I69–V85) are membrane‐spanning structures, and that helices II and III and the intervening hydrophilic loop are located in the cytoplasm. The Na+‐binding residues Q32, E65 and S66 are located in the I→II and III→IV helix connections, probably near the membrane surface on the cytoplasmic side.
Comparison of expression and regulation of the high‐density lipoprotein receptor SR‐BI and the low‐density lipoprotein receptor in human adrenocortical carcinoma NCI‐H295 cells
Authors: Martin Geneviève , Pilon Antoine , Albert Caroline , Vallé Michel , Hum Dean W. , Fruchart Jean‐Charles , Najib Jamila , Clavey Véronique , Staels Bart
Published: March 2003

In rodents, cholesterol for adrenal steroidogenesis is derived mainly from high‐density lipoproteins (HDL) via the HDL receptor, scavenger receptor‐BI (SR‐BI). In humans cholesterol for steroidogenesis is considered to be derived from the low‐density lipoprotein (LDL) receptor pathway, and the contribution of SR‐BI to that is unknown. In the present study SR‐BI expression and regulation by steroidogenic stimuli was analysed in human adrenocortical cells and compared with LDL receptor expression. In addition, the functional contribution of both receptors for cholesteryl ester delivery to human adrenocortical cells was compared. Northern blot and reverse transcription–PCR amplification and sequence analysis demonstrated the presence of SR‐BI mRNA in foetal and adult human adrenal cortex. Furthermore, SR‐BI mRNA was expressed to similar levels in human primary adrenocortical and adrenocortical carcinoma NCI‐H295 cells, indicating its presence in the steroid‐producing cells. Treatment of NCI‐H295 cells with 8Br‐cAMP, a stimulator of glucocorticoid synthesis via the protein kinase A second messenger signal transduction pathway, resulted in an increase of both SR‐BI and LDL receptor mRNA levels in a time‐ and dose‐dependent manner. The induction of SR‐BI and LDL receptor by cAMP was independent of ongoing protein synthesis and occurred at the transcriptional level. Ligand blot experiments indicated that a protein of similar size to SR‐BI is the major HDL‐binding protein in NCI‐H295 cells. Western blot analysis demonstrated that cAMP treatment increased the levels of LDL receptor and, to a lesser extent, SR‐BI protein in NCI‐H295 cells. Binding and uptake of cholesterol was quantitatively smaller from HDL than from LDL, both in basal as well as in cAMP‐stimulated cells. Scatchard analysis under basal conditions indicated that NCI‐H295 cells express twice as many specific binding sites for LDL than for HDL. Dissociation constant values (Kd; in nm) were approximately five times higher for HDL than for LDL, indicating a lower affinity of HDL compared with LDL. The combined effects of these two parameters and the low cholesteryl ester content of HDL subfraction 3 (HDL3) contributes to a lower cholesteryl ester uptake from HDL than from LDL by the NCI‐H295 cells. In conclusion, both the SR‐BI and LDL receptor genes are expressed in the human adrenal cortex and coordinately regulated by activators of glucocorticoid synthesis. In contrast to rodents, in human adrenocortical cells the HDL pathway of cholesterol delivery appears to be of lesser importance than the LDL pathway. Nevertheless, the SR‐BI pathway may become of major importance in conditions of functional defects in the LDL receptor pathway.
Quantitative analysis of histone H1° protein synthesis in HTC cells
Authors: Cuisset Laurence , Tichonicky Lydie , Delpech Marc
Published: March 2003

H1°, a member of histone H1 family associated with cell growth arrest and differentiation, is barely expressed in most mammalian cells in culture. Depending on the cell type, serum deprivation or drugs, such as sodium butyrate, significantly increase H1° mRNA level and H1° protein accumulates. However, probably because of a lack of a simple quantitative procedure, little is known about the relationship between H1° mRNA content and its effective translation rate. Using a rat hepatoma cell line and sodium butyrate as a model system, we attempted to evaluate this in different cellular conditions by measuring H1° synthesis with a rapid quantitative procedure we described previously. We found that although the amount of H1° mRNA rapidly increased and then stabilized under sodium butyrate treatment, its transcription was delayed and H1° protein was synthesized in a progressive wave. Butyrate removal from cell culture confirmed that mRNA level and protein synthesis were independently regulated, and provided evidence that sodium butyrate would not directly target the translation apparatus. In contrast, during the S phase of the cell cycle, H1° gene transcription and protein synthesis were concomitantly activated. Taken together these data provide evidence that H1° accumulation results from an increase of its synthesis and that, depending on conditions, a cell exhibits a H1° translation efficiency which may or may not reflect the mRNA level.
Overexpression, purification and characterization of Mycobacterium bovis BCG alcohol dehydrogenase
Authors: Wilkin Jean‐Marc , Soetaert Karine , Stélandre Martine , Buyssens Paul , Castillo Gabriel , Demoulin Vincent , Bottu Guy , Laneelle Marie‐Antoinette , Daffe Mamadou , De Bruyn Jacqueline
Published: March 2003

A previous study of the effect of zinc deprivation on Mycobacterium bovis BCG pointed out the potential importance of an alcohol dehydrogenase for maintaining the hydrophobic character of the cell envelope. In this report, the effect of the overexpression of the M. bovis BCG alcohol dehydrogenase (ADH) in Mycobacterium smegmatis and M. bovis BCG is described. The purification of the enzyme was performed to apparent homogeneity from overexpressing M. bovis BCG cells and its kinetic parameters were determined. The enzyme showed a strong preference for both aromatic and aliphatic aldehydes while the corresponding alcohols were processed 100–1000‐fold less efficiently. The best kcat/Km values were found with benzaldehyde > 3‐methoxybenzaldehyde > octanal > coniferaldehyde. A phylogenetic analysis clearly revealed that the M. bovis BCG ADH together with the ADHs from Bacillus subtilis and Helicobacter pylori formed a sister group of the class C medium‐chain alcohol dehydrogenases, the plant cinnamyl alcohol dehydrogenases (CADs). Comparison of the kinetic properties of our ADH with some related class C enzymes indicated that the mycobacterial enzyme substrate profile resembled that of the CADs involved in plant defence rather than those implicated in lignification. A possible role for the M. bovis BCG ADH in the biosynthesis of the lipids composing the mycobacterial cell envelope is proposed.
Phospholipid bound to the flavohemoprotein from Alcaligenes eutrophus
Authors: Ollesch Gabriela , Kaunzinger Astrid , Juchelka Dieter , Schubert‐Zsilavecz Manfred , Ermler Ulrich
Published: March 2003

The structurally characterized flavohemoprotein from Alcaligenes eutrophus (FHP) contains a phospholipid‐binding site with 1‐16 : 0‐2‐cyclo‐17 : 0‐diacyl‐glycerophospho‐ethanolamine and 1‐16 : 0‐2‐cyclo‐17 : 0‐diacyl‐glycerophospho‐glycerol as the major occupying compounds. The structure of the phospholipid is characterized by its compact form, due to the ‐sc/β/‐sc conformation of the glycerol and the nonlinear arrangement of the sn‐1‐ and sn‐2‐fatty acid chains. The phospholipid‐binding site is located adjacent to the heme molecule at the bottom of a large cavity. The fatty acid chains form a large number of van der Waal’s contacts with nonpolar side chains, whereas the glycerophosphate moiety, which points towards the entrance of the channel, is linked to the protein matrix by polar interactions. The thermodynamically stable globin module of FHP, obtained after cleaving off the oxidoreductase module, also contains the phospholipid and can therefore be considered as a phospholipid‐binding protein. Single amino acid exchanges designed to decrease the lipid‐binding site revealed both the possibility of blocking incorporation of the phospholipid and its capability to evade steric barriers. Conformational changes in the phospholipid can also be induced by binding heme‐ligating compounds. Phospholipid binding is not a general feature of flavohemoproteins, because the Escherichia coli and the yeast protein exhibit less and no lipid affinity, respectively.
The interaction of thrombomodulin with Ca2+
Authors: Light David R. , Glaser Charles B. , Betts Melissa , Blasko Eric , Campbell Elizabeth , Clarke Jeffrey H. , McCaman Michael , McLean Kirk , Nagashima Mariko , Parkinson John F. , Rumennik Galina , Young Tish , Morser John
Published: March 2003

Thrombomodulin (TM) is a cofactor for protein C activation by thrombin and each residue of a consensus Ca2+ site in the sixth epidermal growth factor domain (EGF6) is essential for this cofactor activity [Nagashima, M., Lundh, E., Leonard, J.C., Morser, J. & Parkinson, J.F. (1993) J. Biol. Chem.268, 2888–2892]. Three soluble analogs of the extracellular domain of TM, solulin (Glu4–Pro490), TME1–6 (Cys227–Cys462) and TMEi4–6 (Val345–Cys462) were prepared for equilibrium dialysis experiments by exhaustive dialysis against Ca2+‐depleted buffer. However, all three analogs still contained one tightly bound Ca2+ (Kd≈ 2 µm), which could only be removed by EDTA. Epitope mapping with Ca2+‐dependent monoclonal antibodies to EGF6 provided further localization of this tight Ca2+ site. Equilibrium dialysis of the soluble TM analogs in [45Ca2+] between 10 and 200 µm revealed a second Ca2+ site (Kd = 30 ± 10 µm) in both solulin and TME1–6, but not in TMEi4–6. Ca2+ binding to this second site was unaffected by bound thrombin and we attribute it to the consensus Ca2+ site in EGF3. A 75‐fold decrease in the binding affinity of thrombin to TM was observed with immobilized solulin treated with EDTA to remove the high affinity Ca2+ by measuring kassoc and kdiss rates in a BIAcore™ instrument. Ca2+‐dependent conformational transitions detected by CD spectroscopy in the far UV indicate a more ordered structure upon Ca2+ binding. Bound Ca2+ stabilized soluble TM against protease digestion at a trypsin‐like protease‐sensitive site between Arg456 and His457 in EGF6 compared with protease treatment in EDTA. Finally, TM containing EGF domains 4–6, but lacking the interdomain loop between EGF3 and 4 (TME4–6), has an identical Ca2+ dependence for the activation of protein C as found for TMEi4–6, indicating this interdomain loop is not involved in Ca2+ binding.
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.
Interaction of CYP11B1 (cytochrome P‐45011β) with CYP11A1 (cytochrome P‐450scc) in COS‐1 cells
Authors: Cao Pei‐rang , Bernhardt Rita
Published: March 2003

The interactions of CYP11B1 (cytochrome P‐45011β), CYP11B2 (cytochrome P‐450aldo) and CYP11A1 (cytochrome P‐450scc) were investigated by cotransfection of their cDNA into COS‐1 cells. The effect of CYP11A1 on CYP11B isozymes was examined by studying the conversion of 11‐deoxycorticosterone to corticosterone, 18‐hydroxycorticosterone and aldosterone. It was shown that when human or bovine CYP11B1 and CYP11A1 were cotransfected they competed for the reducing equivalents from the limiting source contained in COS‐1 cells; this resulted in a decrease of the CYP11B activities without changes in the product formation patterns. The competition of human CYP11A1 with human CYP11B1 and CYP11B2 could be diminished with excess expression of bovine adrenodoxin. However, the coexpression of bovine CYP11B1 and CYP11A1 in the presence of adrenodoxin resulted in a stimulation of 11β‐hydroxylation activity of CYP11B1 and in a decrease of the 18‐hydroxycorticosterone and aldosterone formation. These results suggest that the interactions of CYP11A1 with CYP11B1 and CYP11B2 do not have an identical regulatory function in human and in bovine adrenal tissue.
Molecular cloning and functional properties of two early‐stage encapsulation‐relating proteins from the coleopteran insect, Tenebrio molitor larvae
Authors: Cho Mi Young , Lee Hyun Seong , Lee Kwang Moon , Homma Ko‐ichi , Natori Shunji , Lee Bok Luel
Published: March 2003

Encapsulation is a major defensive reaction against foreign materials that are too large to be phagocytosed by individual hemocytes; however, the biochemical process of encapsulation is still obscure. To isolate and characterize the early‐stage encapsulation‐relating protein (ERP), we used the coleopteran insect, Tenebrio molitor larvae, injecting three differing kinds of bead or inserting pieces of surgical suture into the abdomen of T. molitor larvae. The resulting proteins from the injected beads or the inserted pieces of surgical suture were recovered 10 min after injection or insertion, and were analyzed on SDS/PAGE under reducing conditions. Four different proteins (86, 78, 56 and 48 kDa) were enriched compared with the crude hemolymph. Among them, we purified 56‐kDa and 48‐kDa ERPs to homogeneity and raised polyclonal antibodies against each protein. Immunoblotting analysis showed that the affinity‐purified antibodies of the 56‐kDa and 48‐kDa ERPs cross‐reacted with the 48‐kDa and 56‐kDa ERPs, respectively. Analysis of the cDNA of 56‐kDa ERP consisted of 579 amino acid residues and showed a novel glutamine‐rich protein. Positive clones of the 48‐kDa ERP showed the same DNA sequence as 56‐kDa ERP. Interestingly, the chemically determined N‐terminal amino acid sequence and the three partial amino acid sequences of the 48‐kDa protein were found in the 56‐kDa ERP, suggesting that the 48 kDa ERP was produced by the cleavage of Arg101‐Gly102 of the 56‐kDa ERP by a limited proteolysis. Western blotting analysis showed that these ERPs were detected exclusively on membrane fractions of hemocytes. Also, when the early‐stage encapsulated beads were coated with both the 56‐kDa and 48‐kDa ERP antibodies and re‐injected into larvae, no further encapsulation reaction was observed. However, when the early‐stage encapsulated beads were incubated with 56‐kDa ERP antibody, 48‐kDa ERP antibody or nonimmunized rabbit IgG and re‐injected into larvae, further encapsulation did occur.
NMR investigation and secondary structure of domains I and II of rat brain calbindin D28k (1–93)
Authors: Klaus Werner , Grzesiek Stephan , Labhardt Alexander M. , Buchwald Petra , Hunziker Willi , Gross Myron D. , Kallick Deborah A.
Published: March 2003

Calbindin D28k, a member of the troponin C superfamily of calcium‐binding proteins, contains six putative EF hand domains but binds only four calcium‐atoms: one at a binding site of very high affinity and three calcium‐atoms at binding sites of lower affinity. The high‐affinity site could be located within domain I while domains III, IV, and V bind calcium less tightly. The recombinant protein construct calb I‐II (residues 1–93) comprising the first two EF hands affords a unique opportunity to study a pair of EF hands with one site binding calcium tightly and the second site empty. A series of heteronuclear 2D, 3D and 4D high‐resolution NMR experiments were applied to calb I‐II, and led to the complete assignment of the 1H, 13C and 15N resonances. The secondary structure of the protein was deduced from the size of the 3JHN‐Hα coupling constants, the chemical shift indices of 1Ηα, 13Cα, 13C′ and 13Cβ nuclei and from an analysis of backbone NOEs observed in 3D and 4D NOESY spectra. Four major α‐helices are identified: Ala13–Phe23, Gly33–Ala50, Leu54–Asp63, Val76–Leu90, while residues Ala2–Leu6 form a fifth, flexible helical segment. Two short β‐strands (Tyr30–Glu32, Lys72–Gly74) are found preceding helices B and D and are arranged in an anti‐parallel interaction. Based on these data a structural model of calb I‐II was constructed that shows that the construct adopts a tertiary structure related to other well‐described calcium‐binding proteins of the EF‐hand family. Surprisingly, the protein forms a homodimer in solution, as was shown by its NMR characterization, size‐exclusion chromatography and analytical ultra‐centrifugation studies.
Activation of the ubiquitin proteolytic system in murine acquired immunodeficiency syndrome affects IκBα turnover
Authors: Crinelli Rita , Bianchi Marzia , Gentilini Lucia , Magnani Mauro , Hiscott John
Published: March 2003

Murine acquired immunodeficiency syndrome (MAIDS) is a complex immunopathology caused by a defective murine leukemia virus (LP‐BM5) that mainly targets B‐lymphocytes. Lymphadenophathy, splenomegaly, hypergammaglobulinemia and progressive immunodeficiency are prominent features of MAIDS. Previously, we showed that the ubiquitin proteolytic system was upregulated in infected lymph nodes [Crinelli, R., Fraternale, A., Casabianca, A. & Magnani, M. (1997) Eur. J. Biochem.247, 91–97]. In this report, we demonstrate that increased 26S proteasome activity is responsible for accelerated turnover of the IκBα inhibitor in lymph node extracts derived from animals with MAIDS. The molecular mechanisms mediating IκBα proteolysis involved constitutive phosphorylation of IκBα at Ser32 and Ser36 and subsequent ubiquitination, suggesting persistent activation of an NF‐κB inducing pathway. Interestingly, enhanced IκBα degradation did not result in enhanced NF‐κB DNA binding activity, but rather in a different subunit composition. The modulation of NF‐κB/IκB system may affect multiple immunoregulatory pathways and may in part explain the mechanisms leading to the profound immune dysregulation involved in MAIDS pathogenesis.
Endogenous casein kinase I catalyzes the phosphorylation of the lens fiber cell connexin49
Authors: Cheng Hsueh‐Ling , Louis Charles F.
Published: March 2003

The lens fiber cell‐specific gap junction protein connexin49 is a substrate for a membrane‐associated Ser/Thr protein kinase that can be extracted from lens cell membranes by 0.6 m KCl. However, the identity of this protein kinase has not been defined. In this report, evidence is presented indicating that it is casein kinase I. Thus, connexin49 was shown to be a substrate for purified casein kinase I but not for casein kinase II; the endogenous connexin49 protein kinase activity extracted from lens membranes with KCl was inhibited by the casein kinase I‐specific inhibitor, N‐(2‐aminoethyl)‐5‐chloroisoquinoline‐8‐sulfonamide (CKI‐7); the connexin49 protein kinase activity in the lens membrane KCl extract, which could be partially purified by gel filtration and affinity purification with a casein–Sepharose 4B column, copurified with casein kinase activity; phosphopeptide analysis showed that casein kinase I and the connexin49 protein kinase activity in the lens membrane KCl extract probably share the same phosphorylation sites in connexin49. Reverse transcription‐PCR using total ovine lens RNA and casein kinase I isoform‐specific oligonucleotide primers resulted in the amplification of cDNAs encoding casein kinase I‐α and ‐γ, while an in‐gel casein kinase assay indicated casein kinase activity in the lens membrane KCl extract was associated with a major 39.2‐kDa species, which is consistent with the 36 to 40‐kDa size of casein kinase I‐α in other animal species. These results demonstrate that the protein kinase activity present in the lens membrane 0.6 m KCl extract that catalyzes the phosphorylation of connexin49 is casein kinase I, probably the α isoform.
Evidence that oleoyl‐CoA and ATP‐dependent elongations coexist in rapeseed (Brassica napus L.)
Authors: Domergue Frédéric , Chevalier Sylvette , Santarelli Xavier , Cassagne Claude , Lessire René
Published: March 2003

The elongation of different substrates was studied using several subcellular fractions from Brassica napus rapeseed. In the presence of malonyl‐CoA, NADH and NADPH, very‐long‐chain fatty acid (VLCFA) synthesis was observed from either oleoyl‐CoA (acyl‐CoA elongation) or endogenous primers (ATP‐dependent elongation). No activity was detected using oleic acid as precursor. Acyl‐CoA and ATP‐dependent elongation activities were mainly associated with the 15 000 g/25 min membrane fraction. Reverse‐phase TLC analysis showed that the proportions of fatty acids synthesized by these activities were different. Acyl‐CoA elongation increased up to 60 µm oleoyl‐CoA, and ATP‐dependent elongation was maximum at 1 mm ATP. Both activities increased with malonyl‐CoA concentration (up to 200 µm). Under all conditions tested, acyl‐CoA elongation was higher than ATP‐dependent elongation, and, in the presence of both ATP and oleoyl‐CoA, the elongation activity was always lower. ATP strongly inhibited acyl‐CoA elongation, whereas ATP‐dependent elongation was slightly stimulated by low oleoyl‐CoA concentrations (up to 15 µm) and decreased in the presence of higher concentrations. CoA (up to 150 µm) had no effect on the ATP‐dependent elongation, whereas it inhibited the acyl‐CoA elongation. These marked differences strongly support the presence in maturing rapeseed of two different elongating activities differently modulated by ATP and oleoyl‐CoA.
Entry of [(1,2‐13C2)acetyl]‐l‐carnitine in liver tricarboxylic acid cycle and lipogenesis
Authors: Aureli Tommaso , Puccetti Caterina , Di Cocco Maria Enrica , Arduini Arduino , Ricciolini Rita , Scalibastri Maurizio , Manetti Cesare , Conti Filippo
Published: March 2003

The biochemical pathways involved in acetyl‐l‐carnitine utilization were investigated in conscious, freely moving rats by 13C NMR spectroscopy. Following 4‐h [(1,2‐13C2)acetyl]‐l‐carnitine infusion in fasted animals, the free carnitine levels in serum were increased, and an efflux of unlabelled acetyl‐l‐carnitine from tissues was observed. [(1,2‐13C2)Acetyl]‐l‐carnitine was found to enter biosynthetic pathways in liver, and the acetyl moiety was incorporated into both cholesterol and 3‐hydroxybutyrate carbon skeleton. In accord with the entry of [(1,2‐13C2)acetyl]‐l‐carnitine in the mitochondrial acetylCoA pool associated with tricarboxylic acid cycle, the 13C label was also found in liver glutamate, glutamine, and glutathione. The analysis of the 13C‐labelling pattern in 3‐hydroxybutyrate and cholesterol carbon skeleton provided evidence that the acetyl‐l‐carnitine‐derived acetylCoA pool used for ketone bodies synthesis in mitochondria was homogeneous, whereas cholesterol was synthesized from two different acetylCoA pools located in the extra‐ and intramitochondrial compartment, respectively. Furthermore, cholesterol molecules were shown to be preferentially synthesized by the metabolic route involving the direct channelling of CoA‐activated mitochondria‐derived ketone bodies into 3‐hydroxy‐3‐methylglutarylCoA pathway, prior to equilibration of their acyl groups with extramitochondrial acetylCoA pool via acetoacetylCoA thiolase.
Molecular cloning and functional characterization of a snake venom metalloprotease
Authors: Jeon Ok‐Hee , Kim Doo‐Sik
Published: March 2003

A cDNA clone,MT‐d,encoding metalloprotease precursor was isolated from snake (Agkistrodon halys brevicaudus) venom gland cDNA library. MT‐d‐I protein containing both metalloprotease and disintegrin domains, and MT‐d‐II protein containing the metalloprotease domain only were expressed inEscherichia coliand refolded successfully into their functional forms. Each of the refolded enzyme species exhibited distinct substrate specificity. Proteolytic activity of the MT‐d‐1 was able to hydrolyse type I gelatin, type‐III and V collagens in contrast with the catalytic function of MT‐d‐II. MT‐d‐I protein having metalloprotease activity was also able to inhibit platelet aggregation. Functionally active MT‐d‐I protein underwent autoproteolytic processingin vitroto produce metalloprotease and disintegrin; this processing was accompanied by significant changes in the substrate specificity of the enzyme activity. Experimental evidence strongly suggests that the disintegrin domain in the metalloprotease precursor modulates the catalytic function of the enzyme in hydrolysing extracellular matrix proteins.
PepS from Streptococcus thermophilus
Authors: Fernandez‐Espla Maria Dolores , Rul Françoise
Published: March 2003

The proteolytic system of lactic acid bacteria is essential for bacterial growth in milk but also for the development of the organoleptic properties of dairy products. Streptococcus thermophilus is widely used in the dairy industry. In comparison with the model lactic acid bacteria Lactococcus lactis, S. thermophilus possesses two additional peptidases (an oligopeptidase and the aminopeptidase PepS). To understand how S. thermophilus grows in milk, we purified and characterized this aminopeptidase. PepS is a monomeric metallopeptidase of ≈ 45 kDa with optimal activity in the range pH 7.5–8.5 and at 55 °C on Arg‐paranitroanilide as substrate. PepS exhibits a high specificity towards peptides possessing arginine or aromatic amino acids at the N‐terminus. From the N‐terminal protein sequence of PepS, we deduced degenerate oligonucleotides and amplified the corresponding gene by successive PCR reactions. The deduced amino‐acid sequence of the PepS gene has high identity (40–50%) with the aminopeptidase T family from thermophilic and extremophilic bacteria; we thus propose the classification of PepS from S. thermophilus as a new member of this family. In view of its substrate specificity, PepS could be involved both in bacterial growth by supplying amino acids, and in the development of dairy products’ flavour, by hydrolysing bitter peptides and liberating aromatic amino acids which are important precursors of aroma compounds.
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.
Overexpression, purification and biochemical characterization of the wound‐induced leucine aminopeptidase of tomato
Authors: Gu Yong‐Qiang , Holzer Frances M. , Walling Linda L.
Published: March 2003

Wounding of tomato leaves results in the accumulation of an exoprotease called leucine aminopeptidase (LAP‐A). While the expression of LapA genes are well characterized, the specificity of the LAP‐A enzyme has not been studied. The LAP‐A preprotein and mature polypeptide were overexpressed in Escherichia coli. PreLAP‐A was not processed and was inactive accumulating in inclusion bodies. In contrast, 55‐kDa mature LAP‐A subunits assembled into an active, 357‐kDa enzyme in E. coli. LAP‐A from E. coli cultures was purified to apparent homogeneity and characterized relative to its animal (porcine LAP) and prokaryotic (E. coli PepA) homologues. Similar to the porcine and E. coli enzymes, the tomato LAP‐A had high temperature and pH optima. Mn2+ was a strong activator for all three enzymes, while chelators, zinc ion, and the slow‐binding aminopeptidase inhibitors (amastatin and bestatin) strongly inhibited activities of all three LAPs. The substrate specificities of porcine, E. coli and tomato LAPs were determined using amino‐acid‐p‐nitroanilide and ‐β‐naphthylamide substrates. The tomato LAP‐A preferentially hydrolyzed substrates with N‐terminal Leu, Met and Arg residues. LAP‐A had substantially lower levels of activity on other chromogenic substrates. Several differences in substrate specificities for the animal, plant and prokaryotic enzymes were noted.
Stabilization of DNA triple helices by a series of mono‐ and disubstituted amidoanthraquinones
Authors: Keppler Melanie D. , Read Martin A. , Perry Philip J. , Trent John O. , Jenkins Terence C. , Reszka Anthony P. , Neidle Stephen , Fox Keith R.
Published: March 2003

We have used quantitative DNase I footprinting to measure the relative affinities of four disubstituted and two monosubstituted amidoanthraquinone compounds for intermolecular DNA triplexes, and have examined how the position of the attached base‐functionalized substituents affects their ability to stabilize DNA triplexes. All four isomeric disubstituted derivatives examined stabilize DNA triplexes at micromolar or lower concentrations. Of the compounds studied the 2,7‐disubstituted amidoanthraquinone displayed the greatest triplex affinity. The order of triplex affinity for the other disubstituted ligands decreases in the order 2,7 > 1,8 = 1,5 > 2,6, with the equivalent monosubstituted compounds being at least an order of magnitude less efficient. The 1,5‐disubstituted derivative also shows some interaction with duplex DNA. These results have been confirmed by molecular modelling studies, which provide a rational basis for the structure–activity relationships. These suggest that, although all of the compounds bind through an intercalative mode, the 2,6, 2,7 and 1,5 disubstituted isomers bind with their two side groups occupying adjacent triplex grooves, in contrast with the 1,8 isomer which is positioned with both side groups in the same triplex groove.
Down‐regulation of endothelial cell growth inhibitors by enhanced MYCN oncogene expression in human neuroblastoma cells
Authors: Fotsis Theodore , Breit Stephen , Lutz Werner , Rössler Jochen , Hatzi Elissavet , Schwab Manfred , Schweigerer Lothar
Published: March 2003

Recent evidence indicates that the genetic alterations of the multistage process of malignant transformation appear to activate tumor neovascularization by altering the balance between stimulators and inhibitors of angiogenesis. In the present study, we have attempted to define the effect of enhanced MYCN oncogene expression on the profile of endothelial cell growth modulators in neuroblastoma cells. We report here that conditioned medium of human neuroblastoma cells with normal MYCN expression contains three inhibitors of endothelial cell proliferation, which appear to be novel proteins as judged by their physicochemical, immunological and biological properties. All three inhibitors are diminished or become undetectable upon experimental increase of MYCN expression. Our results suggest that enhanced MYCN expression in human neuroblastoma cells alters the angiogenic balance by down‐regulating endothelial cell growth inhibitors but leaving the expression of the stimulators unaffected. These data shed light on the molecular mechanisms linking the genetic changes of malignant transformation with initiation of tumor angiogenesis. Moreover, our observations might explain the poor prognosis of human neuroblastomas following MYCN oncogene amplification through initiation of angiogenesis and subsequent tumor growth and spread.