bluuebunny/crossref_metadata_2025 huggingface dataset:
Calorimetric measurements of thermal denaturation of stefins A and B
Authors: ŽEROVNIK Eva , LOHNER Karl , JERALA Roman , LAGGNER Peter , TURK Vito
Published: March 2005

Thermal denaturation of two homologous proteins, low‐Mr cysteine‐proteinase inhibitors stefins A and B, has been investigated by microcalorimetry. Calorimetric enthalpies, as well as the temperatures at maximum heat capacity, were determined as a function of pH for each protein. Transitions were found reversible at all pH values examined (5.0, 6.5, 8.1) for the thermally more stable stefin A, in contrast to stefin B. Stefin B shows a sharp irreversible transition around 65°C at pH 6.5 and 8.1, probably due to unfolding of a dimeric state followed by oligomerisation. At pH 5.0, both proteins exhibit a reversible transition with temperatures of half‐denaturation at 50.2°C and 90.8°C for stefins B and A, respectively. The calorimetric enthalpies, which equal the van't Hoff enthalpies to within 10%, are 293 kJ/mol and 490 kJ/mol for stefins B and A, respectively.

Using the predictive method of Ooi and Oobatake (1991) [Proc. Natl Acad. Sci. USA 88, 2859] the thermodynamic functions of unfolding were calculated for stefin B, whose three‐dimensional structure has been determined. The calculated enthalpy, heat‐capacity change on unfolding and the temperature of half denaturation compare well to the microcalorimetric data.
1H‐NMR study of neurotoxin B‐IV from the marine worm Cerebratulus lacteus
Authors: HANSEN Poul Erik , KEM William R. , BIEBER Allan L. , NORTON Raymond S.
Published: March 2005

Sequence‐specific resonance assignments are reported for the 500‐MHz 1H‐NMR spectrum of the 55‐residue neurotoxin B‐IV, isolated from the heteronemertine worm Cerebratulus lacteus. A range of two‐dimensional homonuclear correlated and NOE spectra was used in making these assignments, which include NH, CαH and CβH resonances, as well as most resonances from longer‐chain spin systems, with the exception of the ten Lys residues, where spectral overlap prevented complete, unambiguous assignments. The secondary structure of B‐IV was identified from the pattern of sequential (i, i+ 1) and medium range (i, i+ 2/3/4) NOE connectivities and the location of slowly exchanging backbone amide protons. Two helices are present, incorporating residues 13–26 and 33–49, and the C‐terminal five residues form a helix‐like structure. A type‐I reverse turn, involving residues 28–31 is present in a small loop linking the two major helices, and the N‐terminus appears to be unordered at 27°C, although it may adopt a more ordered conformation at lower temperatures. These elements of secondary structure, together with the four disulfide bonds in the protein, provide sufficient information to define the global fold of the molecule in solution. The pH and temperature dependence of the toxin have been investigated by 1H‐NMR and the pKa values of several ionisable sidechains determined.
The O18 antigens (lipopolysaccharides) of Escherichia coli
Authors: JANN Barbara , SHASHKOV Alexander S. , GUPTA Dhirendra S. , JANN Klaus
Published: March 2005

The O‐specific polysaccharide moieties (PS) of the O18A, O18A1, O18B, and O18B1 antigens (lipopolysaccharides, LPS) consist of L‐rhamnose (Rha), N‐acetyl‐d‐glucosamine, D‐galactose, and D‐glucose in different molar ratios. By using chemical fragmentation, methylation, as well as one‐and two‐dimensional NMR spectroscopy, the structures of these polysaccharides were found to be

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In O18A‐PS and O18A1‐PS x= 2, whereas in O18B‐PS and in O18B11‐PS x= 3. In all four polysaccharides α‐d‐Galp (residue D) is substituted at O‐3. This substituent L (residue E) is β‐d‐GlcpNAc‐(1 in O18A‐PS and O18A1‐PS and it is α‐d‐Glcp‐(1 in O18B‐PS and O18B1‐PS. Whereas there is no further substituent on the main chain of the O18A and O18B polysaccharides, in O18A1‐PS and O18B1‐PS the α‐d‐GlcpNAc residue A is substituted with α‐Glcp‐(1 (residue F), which is linked to O‐6 in O18A1‐PS and to O‐4 in O18B1‐PS. These results show that the O18 antigen comprises a group of four related LPS (O18A and O18B, with their glucosylated forms O18A1 and O18B1). The results are discussed with respect to epitope definition and biochemical implications.
The use of alternative substrates in the characterization of actin‐methylating and carnosine‐methylating enzymes
Authors: RAGHAVAN Malini , LINDBERG Uno , SCHUTT Clarence
Published: March 2005

Actin isolated from nearly every eukaryotic species contains approximately 1 mol 3‐methylhistidine/mol protein. His 73 in actin has been shown, by protein sequencing, to be the site of methylation. The methylation occurs enzymically and post‐translationally. A rabbit skeletal muscle myofibrillary fraction has previously been shown to contain a histidine methyltransferase activity that is actin specific. Detailed study of this enzyme has been hampered by lack of a suitable substrate assay. Naturally occurring actins are poor substrates for the enzyme, presumably due to prexistent methylation at His 73. In this study, two potential alternative substrates have been investigated. These are a chicken β‐actin expressed in Escherichia coli as a fusion protein with 80 amino acids of an influenza protein, NS1, and a synthetic peptide, Tyr‐Pro‐Ile‐Glu‐His‐Gly‐Ile‐Ile‐Thr, corresponding to residues 69–77 of actin. Both substrates were covalently methylated at histidine residues in the presence of S‐adenosylmethionine and partially purified enzyme fractions from rabbit muscle. In methylation experiments employing the fusion actin in the form of inclusion bodies, 3‐methylhistidine is the major product, as is the case when soluble muscle or non‐muscle actin is used. However, for the synthetic peptide, the methylated product primarily contained 1‐methylhistidine and only a small amount of the isomeric 3‐methylhistidine. Further investigations revealed that the peptide was recognized by carnosine N‐methyltransferase, another histidine methyltransferase found in muscle tissue. Carnosine N‐methyltransferase appears to copurify with the actin‐methylating enzyme in preliminary fraction‐ation experiments. Separation of the two methyltransferase activities is described.
Tyrosine phosphorylation and the mechanism of signal transduction by the B‐lymphocyte antigen receptor
Authors: DEFRANCO Anthony L.
Published: March 2005

Lymphocytes provide a powerful defense against infectious agents with their exquisite ability to distinguish between macromolecules of the host and macromolecules of foreign invaders. This ability derives from the antigen receptors, which are created from precursor minigenes by a series of genetic‐recombination reactions [1, 2] and from cellular mechanisms that inactivate lymphocytes expressing self‐reactive antigen receptors [3, 4]. Central to the problem of distinguishing self from non‐self is the means by which these antigen receptors recognize antigen and transmit the information of that recognition to the interior of the cell This information ultimately leads to lymphocyte activation or inactivation, depending upon the context. In this review, I shall summarize recent advances in understanding the structural elements of the antigen receptor complex of B lymphocytes and in understanding the signal‐transduction events initiated by this receptor.
Substitution of Arg214 at the substrate‐binding site of p, ‐hydroxybenzoate hydroxylase from Pseudomonas fluorescens
Authors: VAN BERKEL Willem , WESTPHAL Adrie , ESCHRICH Klaus , EPPINK Michel , DE KOK Arie
Published: March 2005

The gene encoding p‐hydroxybenzoate hydroxylase from Pseudomonas fluorescens was cloned in Escherichia coli to provide DNA for mutagenesis studies on the protein product. A plasmid containing a 1.65‐kbp insert of P. fluorescens chromosomal DNA was obtained and its nucleotide sequence determined. The DNA‐derived amino acid sequence agrees completely with the chemically determined amino acid sequence of the isolated protein. The enzyme is strongly expressed under influence of the vector‐encoded lac promotor and is purified to homogeneity in a simple three‐step procedure.

The relation between substrate binding, the effector role of substrate and hydroxylation efficiency was studied by use of site‐directed mutagenesis. Arg214, in ion‐pair interaction with the carboxy moiety of p‐hydroxybenzoate, was replaced with Lys, Gln and Ala, respectively. The affinity of the free enzymes for NADPH is unchanged, whereas the affinity for the aromatic substrate is strongly decreased. For enzymes Arg214→Ala and Arg214→Gln, the effector role of substrate is lost. For enzyme Arg214→Lys, binding of p‐hydroxybenzoate highly stimulates the rate of flavin reduction. In the presence of substrate or substrate analogues, the reduced enzyme Arg214→Lys fails to stabilize the 4a‐hydroperoxyflavin intermediate, essential for efficient hydroxylation. Like the wild‐type, enzyme Arg214→Lys is susceptible to substrate inhibition. From spectral and kinetic results it is suggested that secondary binding of the substrate occurs at the re side of the flavin, where the nicotinamide moiety of NADPH is supposed to bind.
Purification, properties and primary structure of thioredoxin from Aspergillus nidulans
Authors: LE MARECHAL Pierre , HOANG Bôi Minh Châu , SCHMITTER Jean‐Marie , VAN DORSSELAER Alain , DECOTTIGNIES Paulette
Published: March 2005

This paper reports the purification and the properties of a thioredoxin from the fungus Aspergillus nidulans. This thioredoxin is an acidic protein which exhibits an unusual fluorescence emission spectrum, characterized by a high contribution of tyrosine residues. Thioredoxin from A. nidulans cannot serve as a substrate for Escherichia coli thioredoxin reductase. Corn NADP‐malate dehydrogenase is activated by this thioredoxin in the presence of dithiothreitol, while fructose‐1,6‐bisphosphatase is not. The amino acid sequence of Aspergillus thioredoxin was determined by automated Edman degradation after cleavage with trypsin, SV8 protease, chymotrypsin and cyanogen bromide. The masses of tryptic peptides were verified by plasma‐desorption mass spectrometry. The mass of the protein was determined by electrospray mass spectrometry and shown to be in agreement with the calculated mass derived from the sequence (Mr= 11 564). Compared to thioredoxins from other sources, the protein from A. nidulans displays a maximal sequence similarity with that from yeast (45%).
Subtilisin removes the surface layer of the phage fd coat
Authors: SCHWIND Peter , KRAMER Hansgerd , KREMSER Andreas , RAMSBERGER Uwe , RASCHED Ihab
Published: March 2005

The major coat protein of native filamentous phage fd is vulnerable to digestion by subtilisin, but not by any of a number of other proteolytic enzymes. Degradation by the non‐specific protease subtilisin occurs at specific sites in the N‐terminal portion of g8p. The N‐terminal part of the protein is considered to be the outer layer of a two‐layered coat. Thus, subtilisin treatment results in a monolayered phage particle. These particles posses the morphology and stability of native pahge fd. Furthermore, subtilisin proteolysis proved to be an efficient instrument in detecting variations in the topology of the g8p of related filamentous phages.
Characterization of S1 nuclease
Authors: GITE Sadanand , SHANKAR Vepatu
Published: March 2005

Modification of the carboxylate groups of purified S1 nuclease resulted in a loss of its single stranded DNAase, RNAase and phosphomonoesterase activities. The inactivation was due to the removal of zinc atoms from the enzyme and this in turn was dependent on the degree of modification. While the removal of one zinc atom resulted in the partial inactivation of the enzyme, removal of the remaining zinc atoms resulted in the complete inactivation of the enzyme. Similar results were obtained when the purified enzyme was incubated with various concentrations of the metal chelator, EDTA. The EDTA‐(1 mM)‐treated enzyme, depleted of one zinc atom, showing 40–45% residual activity, when incubated with 1 mM Zn2+ or 1 mM Co2+, regained a significant amount of its initial activity towards all the substrates. However, Woodward's‐Reagent‐K‐modified enzyme depleted of one zinc atom and having the same level of activity (40–45%) could not regain its activity, indicating that the carboxylate groups are involved in the metal binding. Data obtained with carboxylate‐group modification, EDTA‐treatment, reconstitution with metal ions, zinc estimation and CD analysis of the enzyme suggests that, out of three zinc atoms present in S1 nuclease, zinc I is easily replaceable and is probably involved in the catalytic activity while zinc II and zinc III are involved in maintaining the enzyme structure.
Purification and characterization of a mitochondrial endonuclease from Drosophila melanogaster embryos
Authors: HAROSH Itzik , MEZZINA Mauro , HARRIS Paul V. , BOYD James B.
Published: March 2005

A mitochondrial endonuclease from Drosophila melanogaster embryos was purified to near homogeneity by successive fractionation with DEAE‐cellulose and heparin‐avidgel‐F, followed by FPLC chromatography on mono s, Superose 12 and a second mono S column. This enzyme digests double stranded DNA more efficiently than heat‐denatured DNA. The endonuclease activity has a molecular mass of 44 kDa, as determined under native conditions using a gel‐filtration Superose 12 column. The prominent peptide detected by SDS/polyacrylamide gel electrophoresis likewise has a molecular mass of 44 kDa, suggesting a monomeric protein. The enzyme has an absolute requirement for divalent cations, preferring Mg2+ over Mn2+. No activity could be detected when these cations were replaced by Ca2+ or Zn2+. The pH optimum for this enzyme activity is 6.5–7.4 and its isoelectric point is 4.9. Both single‐strand and double‐strand breaks are introduced simultaneously into a supercoiled substrate for DNA polymerase I from Escherichia coli, suggesting that 3′‐OH termini are generated during cleavage. The enzyme is free from any detectable DNA exonuclease activity but not from RNase activity. Partial inhibition by antibodies raised against mitochondrial endonucleases derived from bovine heart and Saccharomyces cerevisia have revealed a potential structural homology between these nucleases.
The structure of the O‐antigenic polysaccharide from lipopolysaccharide of Vibrio cholerae strain H11 (non‐O1)
Authors: VINOGRADOV Evgeny V. , HOLST Otto , THOMAS‐OATES Jane E. , BROADY Kevin W. , BRADE Helmut
Published: March 2005

After acid degradation of the lipopolysaccharide (LPS) of Vibrio cholerae strain H11 (non‐O1), a tetrasaccharide was obtained, the structure of which was determined by quantitative and methylation analyses, periodate oxidation, one‐ and two‐dimensional NMR spectroscopy, and fast‐atom‐bombardment and four‐sector tandem mass spectrometry as β‐d‐GalANGro‐(1–3)‐β‐d‐QuiNAc‐(1–4)‐α‐d‐GalANGro‐(1–4)‐NeuAc, in which GalANGro is N‐galacturonoyl‐2‐aminoglycerol and QuiN 2‐amino‐2,6‐dideoxy‐glucopyranose. In addition, the trisaccharide β‐d‐GalANGro‐(1–3)‐β‐d‐QuiNAc‐(1–4)‐d‐altro‐heptulose and the disaccharide α‐d‐GalANGro‐(1–4)‐NeuAc were isolated from acid‐degraded lipopolysaccharide; the occurrence of sedoheptulose in lipopolysaccharide has not been described before. Based on the result of methylation analysis showing that galacturonic acid was the terminal sugar of the polysaccharide chain, and on the assumption that the tri‐ and the disaccharide represented the reducing and the non‐reducing ends of the polysaccharide, respectively, the chemical structure of the O‐specific chain of V. cholerae H11 is proposed as α‐d‐GalANGro‐(1–4)‐α‐NeuAc‐(2–3)‐β‐d‐GalANGro‐(1–3)‐β‐d‐QuiNAc‐(1‐[4)‐α‐d‐GalANGro‐(1–4)‐α‐NeuAc‐(2–3)‐β‐d‐GalANGro‐(1–3)‐β‐d‐QuiNAc‐(1‐]n‐(1–4)‐d‐altro‐heptulose. However, other possible structures can not be ruled out since the tri‐ and the disaccharide could be localised at different positions.
The α subunit of the stimulatory guanine‐nucleotide‐binding regulatory protein forms high‐molecular‐mass aggregates, concomitant with iloprost‐induced desensitization of human platelet adenylyl cyclase
Authors: MOLLNER Stefan , DEPPISCH Heike , PFEUFFER Thomas
Published: March 2005

Prolonged treatment of human platelets with the prostacyclin analong iloprost led to desensitization of the response to various prostaglandin derivatives. However, basal adenylyl cyclase activity and stimulation by agents acting directly via Gs, the stimulatory guanine‐nucleotide‐binding regulatory protein of adenylyl cyclase, were likewise decreased. Reconstitution of desensitized membranes with purified Gs from turkey erythrocytes indicated no alteration in the catalyst itself. However, the function of Gs (in cholate extracts) appeared to be severely impaired when reconstituted with adenylyl cyclase catalyst. Modification of Gs was also indicated by its altered sedimentation in sucrose density gradients. From Western blots, the α subunit of Gs, αs, from control platelets sedimented as a 5.6S species, while that from desensitized cells appeared at higher S values (in a polydisperse distribution). Activation by guanosine 5′‐[γ‐thio]triphosphate of Gs from control platelets shifted αs to 3.5–3.7S, while activation of Gs from desensitized platelets induced such shift only for a minor portion of αs. This small fraction alone appeared to be susceptible to ADP‐ribosylation by cholera toxin/[32P]NAD. Furthermore, an antibody directed against the C‐terminal hexadecapeptide of αs precipitated much less αs from cholate extracts derived from desensitized platelets. Modification of αs during desensitization was also suggested from cross‐linking experiments using the homobifunctional agent bismaleimidohexane: αs from desensitized platelets formed a single product of 80 kDa, while that from untreated platelets yielded a doublet (100 kDa and 110 kDa).
Analysis of soluble human and mouse interferon‐γ receptors expressed in eukaryotic cells
Authors: GENTZ Reiner , HAYES Ashley , GRAU Nicole , FOUNTOULAKIS Michael , LAHM Hans‐Werner , OZMEN Laurence , GAROTTA Gianni
Published: March 2005

The extracellular domains of the human and mouse interferon‐γ receptors were produced in insect Spodoptera frugiperda cells infected with recombinant baculoviruses and in mammalian Chinesehamster‐ovary cells. The receptors expressed in both systems are secreted into the culture medium. Their signal peptides are cleaved off and the proteins show heterogeneity in glycosylation which, however, does not affect the capacity to bind interferon γ or specific antibodies. The soluble mouse receptors exhibit binding capacities similar to those of cell‐surface‐anchored receptors, whereas the human receptors exhibit a lower binding capacity. All soluble receptors inhibit the binding of interferon γ to cellular receptors and neutralize the activity exerted by interferon γ. These receptors could therefore be useful for structure/function analyses and in vivo studies.
Oleate stimulation of diacylglycerol formation from phosphatidylcholine through effects on phospholipase D and phosphatidate phosphohydrolase
Authors: SIDDIQUI Rafat A. , EXTON John H.
Published: March 2005

Hydrolysis of exogenous phosphatidylcholine (PtdCho) to 1,2‐diacylglycerol by rat liver plasma membranes was stimulated by oleate concentrations as low as 0.1 mM. In the presence of 75 mM ethanol, the fatty acid also enhanced phosphatidylethanol (PtdEtOH) formation from PtdCho. These effects were also observed with linoleate and arachidonate, but not with saturated fatty acids or detergents, and were minimal in microsomes or mitochondria. Release of [3H]choline form exogenous Ptd[3H]Cho was stimulated by oleate, whereas phosphory1[3H]choline formation was inhibited. Oleate and other unsaturated, but not saturated, fatty acids also stimulated the conversion of exogenous [14C]phosphatidic acid to [14C]diacylglycerol. These data are consistent with stimulatory effects of these fatty acids on both phospholipase D and phosphatidate phosphohydrolase in liver plasma membranes. The stimulatory effect of guanosine 5′‐O‐[3‐thio]triphosphate) (20 μM) on PtdEtOH and diacylglycerol formation from PtdCho was enhanced by low concentration of oleate. Phospholipase A2 also stimulated PtdEtOH and diacylglycerol formation from exogenous PtdCho. It is proposed that unsaturated fatty acids may play a physiological role in the regulation of diacylglycerol production through activation of phospholipase D and phosphatidate phosphohydrolase.
Chromatin structure and conserved sequence elements in genes encoding ribosomal proteins in Tetrahymena thermophila
Authors: NØRAARD Peter , DREISIG Hanne , KRISTIANSEN Karsten
Published: March 2005

The chromatin structure of the macronuclear genes encoding ribosomal proteins S25 and L1 in the ciliated protozoan Tetrahymena thermophila was analyzed. Using the indirect end‐labelling technique, DNase‐I‐hypersensitive regions were located in the promoter regions as well as in the 3′regions of the genes. The DNase‐I‐hypersensitive regions were present in chromatin of exponentially growing cells, where the rate of ribosomal‐protein gene transcription is high, and in chromatin from starved cells, where transcription of ribosomal‐protein genes is severely depressed. Micrococcal‐nuclease‐digestion experiments revealed that the promoter regions of the S25 gene and the L1 gene are devoid of nucleosomes in exponentially growing cells. In starved cells, no nucleosomal organisation of the promoter region of the L1 gene could be detected, whereas nucleosomal structures were discernible in the promoter region of the S25 gene. A conspicuous polypurine sequence motif, AARGGGAAA, is present within or adjacent to the DNase‐I‐hypersensitive regions in the promoter of the S25 and the L1 gene, and interestingly, the same motif is found also in the promoter regions of the genes encoding ribosomal proteins L21 and L37.
Ceruloplasmin in human plasma and its relationships with the copper‐albumin complex
Authors: MUSCI Giovanni , BONACCORSI DI PATTI Maria Carmela , CARLINI Patrizia , CALABRESE Lilia
Published: March 2005

The electron paramagentic spectrum of human plasma is dominated, in the g=2 region, by resonances from copper atoms bound to ceruloplasmin, and does not reveal the fraction of copper normally associated with albumin, except in a few cases, where a copper‐albumin signal increases with time after blood withdrawl. This copper‐albumin complex is responsible for a resonance at a g value below g=2 in the spectrum of human serum, which has been recently attributed to a modified form of type 2 copper bound to ceruloplasmin [Rylkov, V. V., Tarasiev, M. Y., & Moshkov, K. A. (1991) Eur. J. Biochem. 197, 185–189]. In the plasma, copper associated to albumin comes from cerulplasmin. Purified ceruloplasmin is unable to exchange copper with albumin, either purified or in plasma. It can not be ruled out that some serum components trigger the metal exchange, in a defence mechanism operating when ceruloplasmin leaks, by unknown processes, its copper content before discharging the metal into the various organs.
Fluorescence study of the ligand stereospecificity for binding to lumazine protein
Authors: LEE John , GIBSON Bruce G. , O'KANE Dennis J. , KOHNLE Angelika , BACHER Adelbert
Published: March 2005

6,7‐Dimethyllumazine derivatives, substituted at the 8‐position with aldityls or monohydroxyalkyl groups, have been examined for their binding ability to lumazine apo‐protein from two strains of Photobacterium phosphoreum using fluorescence dynamics techniques. On the protein the lumazine has a nearly monoexponential decay of fluorescence with lifetime 13.8 ns (20°C). In free solution the lifetime is 9.6 ns. The concentration of free and bound lumazine in an equilibrium mixture can be recovered readily by analysis of the fluorescence decay. Only the aldityl derivatives D‐xylityl and 3′‐deoxy‐d‐ribityl, having stereoconfigurations at the 2′ and 4′ positions identical to the natural ligand, 8‐(1′‐d‐ribityl), show comparable dissociation constants (0.3 μM, 20°C, pH 7.0). D‐Erythrityl and L‐arabityl have dissociation constants of 1–2 μ. All other ligands show no interaction at all or have dissociation constants in the range 6–80 μ, which can still be determined semi‐quantitatively using the fluorescence decay technique. In the case of these very weakly bound ligands, unambiguous detection of bound ligand can be shown by a long correlation time (23 ns, 2°C) for the fluorescence anisotropy decay. Examination of the bound D‐xylityl compound's fluorescence anisotropy decay at high time resolution (< 100 ps) shows rigid association, i.e. no mobility independent of the macromolecule. All bound ligands appear to be similarly positioned in the binding site. The influence of the stereoconfiguration at the 8‐position found for lumazine protein parallels that previously observed for the enzyme riboflavin synthase, where the lumazines are substrates or inhibitors. This is consistent with the finding of significant sequence similarity between these proteins. The binding rigidity may have implications for the mechanism of the enzyme.
Metabolic effects of short‐chain ceramide and glucosylceramide on sphingolipids and protein kinase C
Authors: ABE Akira , WU Dorothy , SHAYMAN James A. , RADIN Norman S.
Published: March 2005

Recent studies have identified a potential role for glucosylceramide (GlcCer) in growth promotion and hormonal signalling. In an effort to demonstrate a growth‐promoting activity of GlcCer, we prepared a GlcCer having a short‐chain acid (octanoyl), in the belief that this glycolipid could be absorbed more readily and more uniformly by cultured cells. By using a mixture of two specific lecithins, dioleoylglycerophosphocholine and 1‐stearoyl‐2‐palmitoylglycerophosphocholine, we were able to prepare dispersions containing a high molar proportion of the GlcCer and the related ceramide, octanoyl sphingosine. Unexpectedly, both sphingolipids inhibited protein and DNA synthesis in Madin‐Darby canine kidney cells and produced large increases in the levels of the natural lipids, GlcCer, ceramide, free sphingosine, and an amine that may be glucosylsphingosine (GlcSph). Decreases were seen in the level of sphingomyelin and the proportion of protein kinase C in the cell membranes. The level of lactosylceramide was diminished by octanoyl GlcCer but elevated considerably by octanoyl sphingosine. Diacylglycerols were increased by the lecithins in the liposomes, but the exogenous sphingolipids had no effect. Octanoyl sphingosine labeled in the sphingoid base yielded labeled GlcCer and sphingomyelin labeled in both long‐chain and very‐long‐chain fatty acid families, as well as the octanoyl version. The two families of ceramides, however, had relatively little radioactivity. Some of these changes are attributed to rapid hydrolysis of the added lipids with the formation, particularly from the ceramide, of sphingosine and its anabolic metabolite, GlcSph. Several obser‐vations support the idea that the octanoyl sphingosine inhibited the phosphocholinetransferase that synthesizes sphingomyelin while the octanoyl GlcCer inhibited GlcCer β‐glucosidase and GlcCer galactosyltransferase. The use of unnatural short‐chain lipids in the study of cell growth and other phenomena may result in unexpected changes in related metabolites and the findings from such experiments should therefore be interpreted cautiously.
A mutant Kex2 enzyme with a C‐terminal HDEL sequence releases correctly folded human insulin‐like growth factor‐1 from a precursor accumulated in the yeast endoplasmic reticulum
Authors: CHAUDHURI Bhabatosh , LATHAM Sarah E. , STEPHAN Christine
Published: March 2005

Mutations in the pro region of the yeast DNA hybrid of prepro‐α‐factor and human insulinlike growth factor‐1 (IGF‐1) cause the accumulation, in the yeast Saccharomyces cerevisiae, of an unglycosylated precursor protein where the pre sequence is missing. The prepro sequence of the prepro‐α‐factor consists of a pre or signal sequence and a proregion which possesses three sites for N‐glycosylation. Isolation of a precursor, where the pro region is still linked to IGF‐1 through a pair of dibasic amino acid residues, implies that the polypeptide may have translocated into the endoplasmic reticulum (ER) but has not been processed by the Golgi membrane‐bound Kex2 endoprotease. However, the lack of any N‐glycosylation in the translocated polypeptide is surprising. The mutated pro region, can be processed, in vitro, by treatment with a soluble form of the Kex2 enzyme. It is also possible to release the pro region, in vivo, by coexpressing a mutant Kex2 protease which is partially retained in the ER with the help of the C‐terminal tetrapeptide sequence, HDEL. The mature IGF‐1, which is secreted from the intracellular pool of precursor proteins, is predominantly an active, monomeric molecule, corroborating observations that early removal of the pro region before folding in the ER helps to prevent aberrant intermolecular disulfide‐bond formation in IGF‐1. These results have revealed the utility of the ER‐retained Kex2 enzyme as a novel in vivo biochemical tool.
Characterization of a biologically active extracellular domain of fibroblast growth factor receptor 1 expressed in Escherichia coli
Authors: BERGONZONI Laura , CACCIA Paolo , CLETINI Ornella , SARMIENTOS Paolo , ISACCHI Antonella
Published: March 2005

The functional features of a recombinant fibroblast growth factor (FGF) receptor (FGF‐R) were investigated by expressing at high level in Escherichia coli a soluble non‐glycosylated form of FGF‐R1. The extracellular domain of the mature protein (XC‐FGF‐R), comprising the first 356 amino acids, was purified from a large‐scale fermentation. After cell lysis, the protein was quantitatively found in the pellet. XC‐FGF‐R was solubilized using guanidine/HCl and allowed to refold using two dialysis steps. The refolded protein was obtained in a homogeneous form after ammonium sulphate precipitation and gel‐filtration chromatography. The soluble receptor had the ability to form a complex with recombinant human basic FGF (rhbFGF) in solution, as demonstrated by immunoprecipitation with anti‐(FGF‐R) serum. Formation of a rhbFGF/XC‐FGF‐R complex was visualized by cross‐linking experiments. Quantitative binding experiments with the XC‐FGF‐R immobilized on Affi‐Gel resin showed high binding affinity for 125I‐bFGF (Kd= 5–10 nM). Purified XC‐FGF‐R inhibited binding of 125I‐bFGF to its high‐affinity receptors on baby hamster kidney cells. These data suggest that glycosylation of the FGF‐R is not necessary for its ligand‐binding activity. The use of an E. coli expression system resulted in the efficient production of a soluble receptor in a form suitable for ligand/receptor structural studies and screening of new potential agonists and antagonists of angiogenesis. These results indicate that E. coli can be used for the production of complex molecules such as Ig‐like receptors.
Cardiolipin liposomes sequester a reactivatable partially folded rhodanese intermediate
Authors: ZARDENETA Gustavo , HOROWITZ Paul M.
Published: March 2005

The interaction was studied between the mitochondrial enzyme thiosulfate sulfurtransferase and liposomes, in the form of large unilamellar vesicles (LUV), prepared from either cardiolipin (CL), PtdCho or PtdSer. At equivalent concentrations of lipid, more partially folded thiosulfate sulfurtransferase bound to CL/LUV than to PtdSer/LUV, and only traces were bound to PtdCho/LUV. Native thiosulfate sulfurtransferase did not bind to any of these LUV. We show that CL/LUV‐sequestered thiosulfate sulfurtransferase is inactive but may be reactivated (approximately 56%) with the aid of detergents, thiosulfate, β‐mercaptoethanol and phosphate buffer. Reactivations in the presence of PtdSer/LUV or PtdCho/LUV was only 9% or 1%, respectively. Analysis of the complex by protease digestion and fluorescence spectroscopy indicated that thiosulfate sulfurtransferase was held by CL/LUV and PtdSer/LUV as a folding intermediate. Data presented here suggest that detergents may not interat directly with the protein, but, rather, their primary role in reactivation is to disrupt the LUV, allowing flexibility to the anchored thiosulfate sulfurtransferase molecule, thereby promoting folding. These studies complement other reports which imply a possible role for CL in protein translocation across the mitochondria, since we find that CL binds to thiosulfate sulfurtransferase and sequesters it in a translocation‐competent prefolded conformation, which may readily lead to a correctly folded enzyme.
Single‐stranded DNA binding protein from calf thymus
Authors: ATRAZHEV Alexey , ZHANG Suisheng , GROSSE Frank
Published: March 2005

A binding protein for single‐stranded DNA (ssDNA) was purified from calf thymus to near homogeneity by chromatography on DEAE‐cellulose, blue‐Sepharose, ssDNA‐cellulose and FPLC Mono Q. The most purified fraction consisted of four polypeptides with molecular masses of 70, 55, 30, and 11 kDa. The polypeptide with the molecular mass of 55 kDa is most likely a degraded form of the largest polypeptide. The complex migrated as a whole on both glycerol gradient ultracentrifugation (s= 5.1 S) and gel filtration (Stokes' radius ∼ 5.1 nm). Combining these data indicates a native molecular mass of about 110 kDa, which is in accord with a 1:1:1 stoichiometry for the 70 + 55/30/11‐kDa complex. The ssDNA binding protein (SSB) covered approximately 20–25 nucleotides on M13mp8 ssDNA, as revealed from both band shift experiments and DNase I digestion studies. The homologous DNA‐polymerase‐α‐primase complex was stimulated by the ssDNA binding protein 1.2‐fold on poly(dA) · (dT)14 and 10–13‐fold on singly primed M13mp8 DNA. Stimulation was mainly due to facilitated DNA synthesis through stable secondary structures, as demonstrated by the vanishing of many, but not all, pausing sites. Processivity of polymerase‐primase was not affected on poly(dA) · (dT)14; with poly(dT) · (rA)10 an approximately twofold increase in product lengths was observed when SSB was present. The increase was attributed to a facilitated rebinding of polymerase α to an already finished DNA fragment rather than to an enhancement of the intrinsic processivity of the polymerase. Similarly, products 300–600 nucleotides long were formed on singly primed M13 DNA in the presence of SSB, in contrast to 20–120 nucleotides when SSB was absent. DNA‐primase‐initiated DNA replication on M13 DNA was inhibited by SSB in a concentration‐dependent manner. However, with less sites available to begin with RNA priming, more homogeneous products were formed.
Determination of the three‐dimensional solution structure of the histidine‐containing phosphocarrier protein HPr from Escherichia coli using multidimensional NMR spectroscopy
Authors: van NULAND Nico A. J. , GRÖTZINGER Joachim , DIJKSTRA Klaas , SCHEEK Ruud M. , ROBILLARD George T.
Published: March 2005

We recorded several types ofheteronuclear three‐dimensional (3D) NMR spectra on 15N‐enriched and 13C/15N‐enriched histidine‐containing phosphocarrier protein, HPr, to extend the backbone assignments [van Nuland, N. A. J., van Dijk, A. A., Dijkstra, K., van Hoesel, F. H. J., Scheek, R. M. & Robillard, G. T. (1992) Eur. J. Biochem. 203, 483–491] to the side‐chain 1H,15N and 13C resonances. From both 3D heteronuclear 1H‐NOE 1H‐13C and 1H‐NOE 1H‐15N multiple‐quantum coherence (3D‐NOESY‐HMQC) and two‐dimensional (2D) homonuclear NOE spectra, more than 1200 NOE were identified and used in a step‐wise structure refinement process using distance geometry and restrained molecular dynamics involving a number of new features. A cluster of nine structures, each satisfying the set of NOE restraints, resulted from this procedure. The average root‐mean‐square positional difference for the Cα atoms is less than 0.12 nm. The secondary structure topology of the molecule is that of an open‐face β sandwich formed by four antiparallel β strands packed against three α helices, resembling the recently published structure of Bacillus subtilis HPr, determined by X‐ray crystallography [Herzberg, O., Reddy, P., Sutrina, S., Saier, M. H., Reizer, J. & Kapafia, G. (1992) Proc. Natl. Acad. Sci. USA 89, 2499–2503).
Structural studies of Desulfovibrio vulgaris ferrocytochrome c3 by two‐dimensional NMR
Authors: TURNER David L. , SALGUEIRO Carlos A. , LeGALL Jean , XAVIER António V.
Published: March 2005

Two‐dimensional NMR has been used to make specific assignments for the four haems in Desulfovibrio vulgaris (Hildenborough) ferrocytochrome c3 and to determine their haem core architecture. The NMR signals from the haem protons were assigned according to type using two‐dimensional NMR experiments which led to four sets of signals, one for each of the haems. Specific assignments were obtained by calculating the ring current shifts which arise from other haems and aromatic residues. Observation of interhaem NOEs confirmed the assignments and established that the relative orientation of the haems is identical to that found in the crystal structure of D. vulgaris (Miyazaki F.) ferricytochrome c3. Assignments were also made for all the aromatic residues except for the haem ligands and F20, which is shifted under the main envelope of signals. The NOEs observed between these aromatic protons and haem protons confirm the similarity between the structures in solution and in the crystal. The assignments reported here are the basis for the cross‐assignments of the four microscopic haem redex potentials to specific haems in the protein structure [Salgueiro, C. A., Turner, D. L., Santos, H., LeGall, J. and Xavier, A. V. (1992) FEBS Lett., in the press].
Differences in the proteinase inhibition mechanism of human α2‐macroglobulin and pregnancy zone protein
Authors: JENSEN Poul Erik Hyldgaard , STIGBRAND Torgny
Published: March 2005

Different conformational states of human α2‐macroglobulin (α2M) and pregnancy zone protein (PZP) were investigated following modifications of the functional sites, i. e. the ‘bait’ regions and the thiol esters, by use of chymotrypsin, methylamine and dinitrophenylthiocyanate. Gel electrophoresis, mAb (7H11D6 and α 1:1) and in vivo plasma clearance were used to describe different molecular states in the proteinase inhibitors. In α2M, in which the thiol ester is broken by binding of methylamine and the ‘trap’ is closed, cyanylation of the liberated thiol group from the thiol ester modulates reopening of the ‘trap’ and the ‘bait’ regions become available for cleavage again. The trapping of proteinases in the cyanylated derivative indicates that the trap functions as in native α2M. In contrast, cyanylation has no effect on proteinase‐treated α2M. As demonstrated by binding to mAb, the methylamine and dinitrophenylthiocyanate‐treated α2M exposes the receptor‐recognition site, but the derivative is not cleared from the circulation in mice.

The trap is not functional in PZP. In native PZP and PZP treated with methylamine, the conformational states seem similar. The receptor‐recognition sites are not exposed and removal from the circulation in vivo is not seen for these as for the PZP‐chymotrypsin complex. Tetramers are only formed when proteinases can be covalently bound to the PZP. Conformational changes are not detected in PZP derivatives in which the thiol ester is treated with methylamine and dinitrophenylthio‐cyanate.

The results suggest that the conformational changes in α2M are generated by mechanisms different to these in PZP. The key structure gearing the conformational changes in α2M is the thiol ester, by which the events ‘trapping’ and exposure of the receptor‐recognition site can be separated. In PZP, the crucial step for the conformational changes is the cleavage of the ‘bait’ region, since cleavage of the thiol ester does not lead to any detectable conformational changes by the methods used.
Enzyme function in organic solvents
Authors: GUPTA Munishwar N.
Published: March 2005

Enzyme catalysis in organic solvents is being increasingly used for a variety of applications. Of special interest are the cases in which the medium is predominantly non‐aqueous and contains little water. A display of enzyme activity, even in anhydrous solvents (water <0.02% by vol.), perhaps reflects that the minimum necessity for water is for forming bonds with polar amino acids on the enzyme surface.

The rigidity of enzyme structure at such low water content results in novel substrate specificities, pH memory and the possibility of techniques such as molecular imprinting. Limited data indicates that, while enhanced thermal stability invariably results, the optimum temperature for catalysis may not change. If true in general, this enhanced thermostability would have extremely limited benefits.

Medium engineering and biocatalyst engineering are relevant techniques to improve the efficiency and stability of enzymes in such low water systems. Most promising, as part of the latter, is the technique of protein engineering.

Finally, this review provides illustrations of applications of such systems in the diverse areas of organic synthesis, analysis and polymer chemistry.
Rat liver DNA ligases
Authors: ELDER Rhoderick H. , MONTECUCCO Alessandra , CIARROCCHI Giovanni , ROSSIGNOL Jean‐Michel
Published: March 2005

A novel form of rat liver DNA ligase (molecular mass 100 kDa) can be differentiated from DNA ligase I by several biochemical parameters. It is a more heat‐labile enzyme and unable to join bluntended DNA, even in the presence of poly(ethylene glycol) concentrations which stimulate such joining by DNA ligase I and T4 DNA ligase. It also lacks the AMP‐dependent nicking/closing reaction, which is a property of all other DNA ligases tested so far, including DNA ligase I from rat liver. Both rat liver DNA ligases were inhibited by deoxyadenosinetriphosphate, however this inhibition was competitive with respect to ATP, for DNA ligase I (Ki 22 μM) and non‐competitive for the 100‐kDa DNA ligase (Ki 170 μM). These results support the idea that, when compared with other DNA ligases, the novel form of DNA ligase has a unique AMP‐binding site, may have an absolute requirement for single‐strand breaks and, furthermore, may have an altered reaction mechanism to that which is conserved from bacteriophage to mammalian DNA ligase I.
Effect of dexamethasone on mRNA levels for 5‐aminolevulinate synthase in different rat tissues
Authors: SRIVASTAVA Gopesh , KWONG Simon K. Y. , LAM Karen S. L. , MAY Brian K.
Published: March 2005

5‐Aminolevulinate synthase mRNA levels from different tissues were quantitated by Northern blot hybridization analysis utilizing the rat liver 5‐aminolevulinate synthase cDNA clone as probe. A 5‐aminolevulinate synthase mRNA species of size 2.3 kb was seen in all the tissues examined. Densitometric scanning of the autoradiographs demonstrated that the adrenal gland contained the largest amount of 5‐aminolevulinate synthase mRNA. Levels corresponding to approximately 50% of this amount were found in the small intestine, lung, heart, muscle and testes. In the liver and kidney the level was approximately 25% of that found in the adrenal gland. These results demonstrate the housekeeping role of this gene. Dexamethasone treatment for 1 day or 5 days dramatically induced 5‐aminolevulinate synthase mRNA levels in the liver and small intestine, and to a lesser extent in lung, heart, kidney and muscle. Nuclear run‐off experiments suggest that a post‐transcriptional mechanism predominantly contributes to the dexamethasone‐induced increase in 5‐aminolevulinate synthase mRNA levels observed in the liver. Interestingly, in the steroidogenic tissues of the adrenal gland and testes, there was a substantial decrease in 5‐aminolevulinate synthase mRNA levels after dexamethasone administration but the mechanism of this control remains to be investigated.
Glycosylation of interleukin‐6 purified from normal human blood mononuclear cells
Authors: PAREKH Raj B. , DWEK Raymond A. , RADEMACHER Thomas W. , OPDENAKKER Ghislain , VAN DAMME Jo
Published: March 2005

Interleukin 6 (IL‐6) is a glycosylated cytokine which is important in exerting cell‐specific growth‐inducing, growth‐inhibiting and differentiation‐inducing effects. IL‐6 produced in mammalian cell lines is heterogeneous, reflecting specific cell‐type‐dependent post‐translational modifications. Native IL‐6 was purified from human blood mononuclear cells and the oligosaccharides released, radiolabelled and sequenced by a combination of sequential exoglycosidase digestion using Bio‐Gel P‐4 high‐resolution gel chromatography and acetolysis. N‐ and O‐linked glycans were found. The N‐linked glycans were sialylated di‐ and tri‐antennary complex‐type and oligomannose‐type structures. However, the most predominant N‐linked oligosaccharide was a small tetrasaccharide with the sequence Manα6Manβ4GlcNAcβ4GlcNAc. This is the first report of this structure on a circulating glycoprotein. This structure has only previously been reported to be present on the syncytiotrophoblast of human placenta. The presence of the oligomannose structures and the mannose‐terminating tetrasaccharide on IL‐6 may be important in maintaining a high local concentration of the cytokine while limiting its systemic serum level via interaction with soluble mannose‐binding serum lectins.
Incomplete functional differentiation of HL‐60 leukemic cells by synthetic lipopetides
Authors: SEIFERT Roland , SERKE Stefan , HUHN Dieter , BESSLER Wolfgang G. , HAUSCHILDT Sunna , METZGER Jörg , WIESMÜLLER Karl‐Heinz , JUNG Günther
Published: March 2005

In human neutrophils, the synthetic lipopeptide, N‐palmitoyl‐S‐[2,3‐bis(palmitoyoxy‐(2RS)‐propyl]‐ R)‐cysteinyl‐(S)‐seryl‐(S)‐lysyl‐(S)‐lysyl‐ S)‐lysyl‐(S)‐lysine [Pam3CysSer(Lys)4], activates NADPH‐oxidase catalyzed superoxide (O2−) formation through pertussis‐toxin‐sensitive and per‐tussis‐toxin‐insensitive mechanisms (Seifert, R., Schultz, G., Richter‐Freund, M., Metzger, J., Wiesmüller, K.‐H., Jung, G., Bessler, W. G. & Hauschildt, S. (1990) Biochem. J. 267, 795–802). We studied the effects of lipopeptides on differentiation of HL‐60 leukemic cells. Pam3CysSer(Lys)4 enhanced phorbol‐12‐myristate‐13‐acetate‐induced O2− formation (presumably through the expression of components of NADPH oxidase) in a concentration‐dependent manner with a half‐maximal effect at 100 ng/ml and a maximum at 1 μg/ml. The effect of the lipopeptide was evident after 24 h and reached a plateau after 48 h. (2S,6S)‐2‐Palmitoylamino‐6,7‐bis(palmitoyloxy)heptanoyl‐ (S)‐seryl‐(S)‐lysyl‐(S)‐lysyl‐(S)‐lysyl‐ (S)‐lysine enhanced O2− formation as well. The effects of Pam3CysSer(Lys)4 were potentiated by dibutyryl cAMP, dimethyl sulfoxide, retinoic acid, 1,25‐dihydroxyvitamin D3, interferon‐γ and tumor‐necrosis‐factor‐α. Pertussis toxin, but not its B‐oligomer, partially inhibited enhanced O2− formation induced by Pam3CysSer(Lys)4. O2− formation induced by arachidonic acid and γ‐hexachlorocyclohexane were more sensitive to inhibition by pertussis toxin than O2− formation induced by phorbol 12‐myristate 13‐acetate. Enhanced O2− formation induced by dibutyryl cAMP was not affected by pertussis toxin. Unlike ATP, histamine, prostaglandin E1 and the β‐adrenergic agonist, isoproterenol, Pam3CysSer(Lys)4 did not increase cytosolic Ca2+ ([Ca2+]i) in undifferentiated HL‐60 cells. Histamine but not lipopeptides stimulated high‐affinity GTPase of guanine‐nucleotide‐binding proteins in membranes of undifferentiated HL‐60 cells. In Pam3CysSer(Lys)4‐differentiated HL‐60 cells, the responsiveness to the [Ca2+]i‐increasing agonists, N‐formyl‐l‐methionyl‐l‐leucyl‐l‐phenylalanine, C5a and leukotriene B4, was increased, whilst the responsiveness to prostaglandin E1 and isoproterenol was decreased. Pam3CysSer(Lys)4 did not inhibit proliferation of HL‐60 cells but decreased transferrin receptor expression and increased C3bi receptor expression. Pertussis toxin did not affect proliferation and expression of transferrin and C3bi receptors. Dibutyryl cAMP was considerably more effective than Pam3CysSer(Lys)4 at inducing alterations in the above parameters. Our results suggest that (a) Pam3CysSer(Lys)4 induces incomplete functional differentiation of HL‐60 cells through a mechanism which does not depend on a rise in [Ca2+]i and is different from that of other differentiation‐inducing substances and (b) the mechanism by which Pam3CysSer(Lys)4 induces differentiation involves pertussis‐toxin‐sensitive and pertussis‐toxin‐insensitive mechanisms.