bluuebunny/crossref_metadata_2025 huggingface dataset:
The vacuolar protein‐targeting signal of yeast carboxypeptidase is functional in oocytes from Xenopus laevis
Authors: ROITSCH Thomas , LEHLE Ludwig
Published: March 2005

Carboxypeptidase Y, a yeast vacuolar glycoprotein was expresed in oocytes from Xenopus laevis and its biosynthesis and sorting were examined. In yeast, targeting to the vacuole, the functional equivalent of the lysosome, is not mannose‐6‐phosphate‐receptor dependent. It was found that carboxypeptidase enters the secretory pathway of the oocyte and is there glycosylated, phosphorylated in the carbohydrate part and delivered to the lysosome. Deletion of an amino acid sequence, previously shown to determine intracellular targeting of this enzyme in yeast, caused a loss of phosphorylation and mislocalization of carboxypeptidase Y into the oocyte medium. Inhibition of glycosylation of carboxypeptidase by tunicamycin did not lead to its secretion. In‐frame fusion of the targeting domain to a secretory yeast glycoprotein, invertase, did not prevent its secretion. However, a hybrid containing 80% carboxypeptidase abolished invertase secretion. The results indicate that the vacuolar protein‐targeting signal from yeast carboxypeptidase can, in principal, function in a higher eukaryote.
Evolutionary analysis of the nucleus‐encoded subunits of mammalian cytochrome c oxidase
Authors: SACCONE Cecilia , PESOLE Graziano , KADENBACH Bernard
Published: March 2005

The cytochrome c oxidase enzyme complex of eukaryotes is made up of three mitochondrial‐coded subunits and a variable number of nuclear‐coded subunits. Some nuclear‐coded subunits are present in multiple forms and probably perform a tissue‐ or development‐specific function. A detailed evolutionary analysis of the cytochrome c oxidase subunits that have been sequenced to date is reported here. We have found that gene duplication events from which the liver and heart isoforms of rat subunits VIa and subunit VIII originated can both be dated at about 240 ± 90 million years ago, long before the radiation of mammalian lineages. Sequence divergence between the processed‐type pseudogenes for the subunits IV, VIc and VIII have been estimated. Our results indicate that they arose fairly recently, thus suggesting that retroposition is a continuing process. We show that the rate of silent substitution in mitochondrial‐coded subunits is 5–10 times higher than in nuclear‐coded subunits; on the other hand replacement rates, although differing from gene to gene, are roughly of the same order of magnitude in both nuclear and mitochondrial genes. In the case of most of the nuclear‐coded proteins we observed a slightly greater similarity between rats and cow, which agrees with the data obtained for mitochondrial‐coded subunits.
Endopeptidase variations among different life‐cycle stages of African trypanosomes
Authors: MBAWA Zeres R. , GUMM Ian D. , FISH Wallace R. , LONSDALE‐ECCLES John D.
Published: March 2005

Lysates of different life‐cycle stages of Trypanosoma congolense, Trypanosoma vivax and Trypanosoma brucei were analysed for endopeptidase activity, using reaction conditions which permitted a distinction to be made between lysosomal and non‐lysosomal activity [Lonsdale‐Eccles, J. D. & Grab, D. J. (1987) Eur. J. Biochem. 169, 467–475]. Hydrolysis of Z‐Arg‐Arg‐NHMec (Z = benzyloxycarbonyl, NHMec = 7‐amino‐4‐methylcoumaryl) and Z‐Gly‐Gly‐Arg‐NHMec occurred predominantly at alkaline pH and was observed in lysates of both insect and mammalian infective forms of T. brucei and T. congolense. Compared to their other life‐cycle stages, procyclic forms of T. brucei and epimastigote forms of T. congolense exhibited enhanced hydrolysis of these substrates. Low levels of hydrolysis of Z‐Arg‐Arg‐NHMec were observed in the bloodstream and epimastigote forms of T. vivax. The hydrolysis of Z‐Gly‐Gly‐Arg‐NHMec in each of the life‐cycle stages of T. vivax was generally below detectable levels.

In lysates of T. congolense, proteolytic and Z‐Phe‐Arg‐NHMec‐hydrolytic activity in bloodstream forms > metacyclic > epimastigote > procyclic forms. In T. vivax Z‐Phe‐Arg‐NHMec‐hydrolytic activity differed slightly according to the origin of the parasite but, in general, followed the same pattern (i.e. bloodstream forms > epimastigote forms, with metacyclic forms usually intermediate between these two). In T. brucei, Z‐Phe‐Arg‐NHMec‐hydrolytic activity in bloodstream forms > procyclic forms. Upon differentiation of the long, slender bloodstream forms into short, stumpy forms the Z‐Phe‐Arg‐NHMec‐hydrolytic activity was elevated even further. Thus, during their life cycle, each of these African trypanosomes exhibits complex changes of endopeptidase activity, suggestive of an induction of lysosomal activity between the insect and mammalian forms.
Linker mutagenesis in the lacZ gene of Escherichia coli yields variants of active β‐galactosidase
Authors: BREUL Astrid , KUCHINKE Wolfgang , von WILCKEN‐BERGMANN Brigitte , MÜLLER‐HILL Benno
Published: March 2005

Synthetic octameric oligonucleotides that code for a unique restriction site were cloned into a randomly linearized plasmid that carries the lacZ gene. The insertions were mapped by digestion with appropriate restriction endonucleases. 12 mutants were identified which carry an insertion within the lacZ gene and still express active β‐galactosidase. Small deletions or duplications of the wild‐type sequence occurred at these positions which restore the correct reading frame. The insertions occurred in the first and the last third of the internal duplication of the lacZ gene and within the domain homologous to dihydrofolate reductase.
The major serum protein of Drosophila larvae, larval serum protein 1, is dispensable
Authors: ROBERTS David B. , JOWETT Trevor , HUGHES Jane , SMITH Deborah F. , GLOVER David M.
Published: March 2005

Null alleles of all three genes (α, β and γ) coding for the major serum protein of Drosophila larvae, larval serum protein 1, have been characterized at the protein level and by analysis of their RNA and DNA. Each allele completely lacks one polypeptide chain. β° shows an unaltered gene but reduced levels of RNA, α° shows restriction‐fragment‐length polymorphism and shows low levels of transcript, while γ° is a deletion. The three null alleles have been combined to give a strain without the gene for larval serum protein 1 which survives
Mechanism of the inhibition of α‐thrombin by hirudin‐derived fragments hirudin(1–47) and hirudin(45–65)
Authors: SCHMITZ Thomas , ROTHE Markus , DODT Johannes
Published: March 2005

The kinetic mechanism of the inhibition of α‐thrombin by hirudin was analyzed using the hirudin‐derived fragments hirudin(1–47) and hirudin(45–65). Previously, these fragments have been shown to interact with α‐thrombin at distinct sites inhibiting thrombin‐mediated clot formation. Binding to the active site the N‐terminal fragment hirudin (1–47) competitively inhibits hydrolysis of the substrates Tos‐Gly‐Pro‐Arg‐NH‐Mec (Tos, tosyl; NH‐Mec, 4‐methylcoumaryl‐7‐amide) and fibrinogen with Ki values of 420 ± 18 nM and 460 ± 25 nM, respectively. Interacting with the anion‐binding site of α‐thrombin the C‐terminal fragment competitively inhibits the hydrolysis of fibrinogen with a Ki of 760 ± 40 nM. It was found, however, that this fragment acts as a hyperbolic uncompetitive inhibitor with respect to the hydrolysis of the peptide‐NH‐Mec substrate. According to the Botts‐Morales scheme for enzyme inhibition, the parameters Ki= 710 ± 38 nM, K′i= 348 ± 22 nM, as well as α=β= 0.49 of thrombin inhibition by the C‐terminal fragment hirudin(45–65), were obtained. The results are discussed in terms of the interaction of hirudin and thrombin.
NMR investigations of the N‐linked oligosaccharides at individual glycosylation sites of human lutropin
Authors: WEISSHAAR Gerhard , HIYAMA Jun , RENWICK Alistair G. C. , NIMTZ Manfred
Published: March 2005

Human lutropin or luteinizing hormone (hLH) is a heterodimeric glycoprotein, composed of two subunits, hLHα (N‐glycosylated at Asn52 and Asn78) and hLHβ (N‐glycosylated at Asn30). The sugar chains were liberated by hydrazinolysis from intact hLHβ and from glycopeptides obtained after tryptic digestion of hLHα, subsequently reduced and fractionated as alditols by anion‐exchange and ion‐suppression amine‐adsorption HPLC and identified mainly by one‐dimensional (1D) and two‐dimensional (2D) 1H‐NMR spectroscopy. The results indicate predominantly diantennary, N‐acetyllactosamine‐type structures at all three glycosylation sites. The oligosaccharides attached to Asn52 (hLHα) and Asn30 (hLHβ) show a remarkably similar pattern, with mainly chain‐terminating 4‐sulphated 2‐deoxy‐2‐N‐acetylamino‐d‐galactose (GalNAc) and a sulphated/sialylated structure as the major single component. However, virtually all N‐glycans on the β subunit bear a fucose residue α1‐6‐linked to the proximal GlcNAc, whereas those at Asn52 (and Asn78) of the α subunit are predominantly non‐fucosylated. The oligosaccharides at Asn78 (hLHα) are sialylated rather than sulphated and contain the unique sequence NeuAcα2‐6GalNAcβ1‐4GlcNAcβ1‐2Manα1‐3 as part of the majority of mono‐ and disialylated compounds. The major single constituent at Asn78 has the following structure:

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Conformational change of turkey‐gizzard caldesmon induced by specific chemical modification with carbodiimide
Authors: MARTIN François , HARRICANE Marie‐Cécile , AUDEMARD Etienne , PONS Françoise , MORNET Dominique
Published: March 2005

Water soluble 1‐ethyl‐3‐(3‐dimethylaminopropyl)carbodiimide was used to internally cross‐link carboxyl and lysyl groups of caldesmon. The modification did not involve the two cysteines of the molecule which were previously labelled with N‐iodoacetyl‐N′‐(5‐sulfo‐1‐naphthyl)ethylenediamine. The modified caldesmon exhibited a smaller Stokes radius (4.0 nm instead of 6.3 nm) and its electrophoretic mobility corresponded to an apparent molecular mass of approximately 82 kDa, appreciably lower than that of the native molecule (120 kDa), but more similar to the reported true molecular mass of 86 974 Da of chicken‐gizzard caldesmon (Bryan, J., Imai, M., Lee, R., Moore, P., Cook, R. G. & Lin, W. (1989) J. Biol. Chem. 264, 13 873–13 879). Comparative circular dichroism analysis indicated a decrease of the α‐helix content from 43% to 36% resulting from the chemical modification. The 1H‐NMR spectra of the native and modified caldesmon showed that the covalent cross‐linking affected mainly the central and N‐terminal parts of the molecule. The C‐terminal part, rich in aromatic amino acids, was unmodified by the carbodiimide treatment. This was also corroborated by the continued ability of the modified caldesmon to bind to actin and calmodulin, and by the property of the 90‐kDa proteolytic N‐terminal fragment to give an internally cross‐linked species of 60 kDa. Using electron microscopy, the modified protein was shown to have a more compact shape and a reduced capacity to induce tight and long F‐actin bundles.

These conformational changes were obtained when the carbodiimide reaction was conducted at pH 6.0 and were not observed at pH 8.0. This suggests that local variation of the pH might affect the conformation of caldesmon which changes from an elongated to more compact shape, stabilized by electrostatic interactions. It is proposed that the flexibility of caldesmon might be involved in the regulatory function of this protein in the smooth muscle and might favour tightly packed F‐actin bundles or weaker interactions between actin filaments.
An unusual class I (Schiff base) fructose‐1,6‐bisphosphate aldolase from the halophilic archaebacterium Haloarcula vallismortis
Authors: KRISHNAN Gomathi , ALTEKAR Wijaya
Published: March 2005

An electrophoretically homogeneous class I (Schiff base) alsolase has been isolated for the first time from the archaebacterial halophile Haloarcula (Halobacterium) vallismortis. The aldolase was characterized with respect to its molecular mass, amino acid composition, salt dependency, immunological cross‐reactivity and kinetic properties. The subunit mass of aldolase is 27 kDa, which is much smaller than other class I aldolases. By the gel filtration method, the molecular mass of the halobacterial enzyme was estimated as 280 + 10 kDa, suggesting a decameric nature. In contrast to many halobacterial proteins, the H. vallismortis aldolase, though a halophilic enzyme, did not show an excess of acidic residues. Unlike the eukaryotic aldolases, the activity of the halobacterial enzyme was not affected by carboxypeptidase digestion. The general catalytic features of the enzyme were similar to its counterparts from other sources. No antigenic similarity could be detected between the H. vallismortis aldolase and class I aldolase from eubacteria and eukaryotes or class II halobacterial aldolases.
Fine tuning of the catalytic properties of human carbonic anhydrase II
Authors: BEHRAVAN Gity , JONSSON Bengt‐Harald , LINDSKOG Sven
Published: March 2005

The active‐site residue Thr200 in human carbonic anhydrase II has been replaced by several different amino acids by site‐directed mutagenesis. The CO2 hydration and 4‐nitrophenyl acetate hydrolase activities of these variants have been measured, as well as inhibition by the monovalent anion, SCN−. The results show that the replacement of Thr200 with Ser or Ala has no significant effect on the catalyzed rates of CO2 hydration. Also, variants with Asn200 and Gly200 have high activities, whereas the activities of variants with Val, Ile or Arg at position 200 are reduced by factors of 2–3 compared to the unmodified enzyme. The variant with Asp200 has a very low activity in both reactions studied, while most of the other variants have enhanced esterase activities, Thr200↔ Arg isoenzyme II as much as sevenfold. The Asp200 variant has a low affinity for SCN− as well as for a sulfonamide inhibitor, whereas all the other variants bind SCN− more strongly than unmodified enzyme. While His200 characterizes carbonic anhydrases I, the presence of Arg, Val or Ile as well as His at position 200 in human isoenzyme II seems to result in isoenzyme‐I‐like functional properties.
Secondary structure and orientation of the amphipathic peptide GALA in lipid structures
Authors: GOORMAGHTIGH Erik , DE MEUTTER Joëlle , SZOKA Francis , CABIAUX Véronique , PARENTE Roberta A. , RUYSSCHAERT Jean‐Marie
Published: March 2005

GALA, a synthetic, amphipathic 30‐amino‐acid peptide, based upon a Glu‐Ala‐Leu‐Ala motive, was designed to mimic the behavior of viral fusion proteins. GALA is a water‐soluble peptide with an aperiodic conformation at neutral pH, and becomes an amphipathic α helix as the pH is lowered to 5, where it interacts with phospholipid bilayers. Attenuated total‐reflection infrared spectroscopy, using polarized light, provides information on the structure and orientation of the peptide and the lipids, which is not subject to artifacts due to light scattering with large particles. H/2H‐exchange rate of the amide N‐H group and analysis of the shape of the amide I′ by Fourier self‐deconvolution and curve fitting indicate that the α‐helical content increases from 19% to 69% on lowering the pH. A further increase to 100%α helix is observed after interaction with palmitoyloleoylglycerophosphocholine (PamOleGroPCho) vesicles. Dichroism data obtained with oriented bilayers of the PamOleGroPCho‐GALA complex demonstrate that PamOleGroPCho hydrocarbon chains and the peptide α helical axis are essentially perpendicular (± 15°) to the membrane plane. At neutral pH, in the presence of dimyristoylglycerophosphocholine (Myr2GroPCho), GALA is known to form discoidal structures similar to those formed under the same conditions by apolipoproteins AI and AII. In these discoidal complexes, the α‐helical content was estimated to be 65%, with the rest of the structure being essentially unordered. No significant modification of the all‐trans conformation of the hydrocarbon chain of Myr2GroPCho was detected upon disc formation. Dichroism measurements show that the α‐helical axis is essentially parallel to the hydrocarbon chains. These data support a model in which a discoidal patch of the bilayer is surrounded by amphipathic helices which shield the hydrophobic region of the bilayer from the aqueous environment. The infrared spectrum of GALA in this complex was found to be very similar to those of apolipoproteins AI and AII which form discoidal complexes with Myr2GroPCho, but the spectrum is quite different from that of apolipoprotein B100 in low‐density lipoproteins, which does not form discoidal complexes.
Regulation of the proton/electron stoichiometry of mitochondrial ubiquinol:cytochromecreductase by the membrane potential
Authors: BECHMANN Georg , WEISS Hanns
Published: March 2005

The electron transfer reaction catalysed by mitochondrial ubiquinol:cytochromecreductase is linked to the outwards translocation of protons with an H+/e−stoichiometry of 1 under non‐membrane potential condition. The effect of the electrical membrane potential on the H+/e−stoichiometry was investigated. The enzyme was isolated fromNeurospora crassa, reconstituted into phospholipid vesicles and electrical membrane potentials of various values were generated across the membranes by means of the valinomycin‐induced potassium‐diffusion method. Using lithium ions as counterions for the intravesicular potassium, the induced membrane potential was stable for minutes and was not significantly changed by the protons ejected by the working enzyme. This allowed the assay of steady‐state reaction rates at pre‐given values of electrical membrane potential. The rate ratio between electron transfer and proton translocation declined from 1 to 0.6 with increase of the membrane potential from 0 to 100 mV. The activity of the quinol/cytochromecredox reaction followed a parabolic dependence, being activated by low (< 50 mV) potential and inhibited by high (> 100 mV) potential. This apparent non‐linear dependence was interpreted in terms of a linear flow/force relationship plus a membrane‐potential‐dependent slip. Evaluation of the parabolic course by means of a modified linear flow/force relation also indicated a decline of the H+/e−stoichiometry from 1 to 0.5 with increase of the membrane potential from 0 to 120 mV. These observations suggest that the membrane potential controls a change of ubiquinol:cytochromecreductase between two states that have different reaction routes.
Oxygen tension does not affect urea synthesis in perifused rat hepatocytes
Authors: BOON Louis , MEIJER Alfred J.
Published: March 2005

Perfusion of rat liver has led to the suggestion that oxygen tension, rather than the distribution of enzymes of urea synthesis, plays a key role in the regulation of urea synthesis in the periportal and pericentral areas of the liver lobule [F. W. Kari, H. Yoshihara and R. G. Thurman (1987)Eur. J. Biochem. 163, 1–7]. We have directly tested the effect of oxygen concentration on ureogenesis under steady‐state conditions in isolated hepatocytes perifused with physiological concentrations of ammonia. We found that ureogenesis is independent of the oxygen concentration. Only at oxygen concentrations below 25 μM (which is below the oxygen concentration in liver) was urea synthesis decreased. This was because insufficient production of ATP led to decreased flux through carbamoyl‐phosphate synthase. It is concluded that oxygen does not control urea synthesis.
A clonal rat pancreatic δ cell line (Rin 4B) expresses a high number of galanin receptors negatively coupled to a pertussis‐toxin‐sensitive cAMP‐production pathway
Authors: AMIRANOFF Brigitte , LORINET Anne Marie , LABURTHE Marc
Published: March 2005

Galanin, an ubiquitous neuropeptide, was recently shown to inhibit somatostatin release by the rat islet tumor cell line, Rin‐m. By using the clonal pancreatic delta cell line Rin14B, originating from Rin‐m cells, we were able to identify the presence of one type of specific galanin‐binding site of high affinity (Kd= 1.6 nM; maximal binding capacity = 270 fmol/mg protein) and high specificity for the peptide. Binding of 125I‐galanin to these receptors was time‐dependent and highly sensitive to guanine nucleotides. Using the cross‐linker disuccinimidyl tartarate, covalent linking of the galanin receptor to 125I‐galanin in membranes from Rin14B cells, followed by SDS/PAGE analysis of membrane proteins, indicated that the galanin receptor is a protein of 54 kDa. 0.1–100 nM galanin also exerted a marked inhibitory effect on the cAMP‐production system under basal conditions, as well as in the presence of the pancreatic peptide glucagon. At a maximal dose, galanin induces a 90–100% decrease of basal and glucagon‐stimulated cAMP production levels, with a median inhibition concentration (IC50) of 3 nM galanin. The direct inhibitory effect of galanin on the adenylate cyclase activity in Rin14B cell membranes was also demonstrated (IC50= 3 nM galanin). The inhibitory effect of galanin on the basal and glucagon‐stimulated cAMP production in Rin14B cells was reversed by pertussis toxin. The toxin was also shown to specifically ADP‐ribosylate a protein of 41kDa in membranes from Rin14B cells. Taken together, these data show that the pancreatic delta cell line Rin14B expresses high affinity galanin receptors negatively coupled to a pertussis‐toxin‐sensitive cAMP‐production system.
Structures and characterization of sex‐specific mouse cytochrome P‐450 genes as members within a large family
Authors: LAKSO Merja , MASAKI Ryuichi , NOSHIRO Mitsuhide , NEGISHI Masahiko
Published: March 2005

We characterized two female‐specific testosterone 16α‐hydroxylase mouse cytochrome P‐450 genes, 16αoh‐a and 16αoh‐b. Gene 16αoh‐a, consisting of nine exons, is approximately 38 kbp in size. The exon sequence of this P‐450 gene is identical to cDNA pf26 nucleotide sequence [Noshiro, M., Lakso, M., Kawajiri, K. & Negishi, M. (1988) Biochemistry 27, 6434–6443], which encodes female‐specific testosterone 16α‐hydroxylase regulated by the murine Rip locus. Gene 16αoh‐b, containing nine exons with the same junctions as the 16αoh‐a, spans at least 20 kbp, and encodes a cytochrome P‐450 whose deduced amino acid sequence is 90% similar to the hydroxylase. Nucleotide sequences revealed that duplication of the two genes occurred 4–22 million years ago, and that the 5′ duplication boundary is located 1336 bp upstream from the putative transcription‐start site. In the flanking regions of both genes, there is a long stretch (100 bp) of CA repeats in addition to other motifs, including TATA box, glucocorticoid‐response‐element‐core and Simian‐virus‐40‐enhancer sequences and IgG light‐chain gene promoter. We isolated many genomic DNA clones which contain exon 1 sequences, and compared their restriction maps, cross‐hybridization and nucleotide sequences. The results indicate that these genomic clones represent closely related genes in the 16αoh family with a minimum of 16 members, which is further divided into classes a, b and c. 16αoh‐a and 16αoh‐b belong to the first and second classes, respectively. Moreover, extensive segmental gene conversion and nonreciprocal recombination were noted among the genes, particularly among those in class b. All genes in that class contain the long ATTT repeat sequences in intron 1, which may have triggered a rapid gene conversion and/or stabilize the duplicated genes.
Regulation of the expression of the sex‐specific isoforms of cytochrome P‐450 in rat liver
Authors: KOBLIAKOV Valerji , POPOVA Natalia , ROSSI Lorenzo
Published: March 2005

The hepatic metabolism of steroid hormones and of xenobiotics frequently depends on the expression of the sex‐specific isoforms of cytochrome P‐450 and on differences in sex hormones.

Following biochemical, immunological and molecular biological investigations, it was shown that in adult rat liver there exist at least four male‐specific and one female‐specific isoforms of cytochrome P‐450. The designation of these sex‐specific genes is IIC11, IIIA2, IIC13 and IIA2 in males, and IIC12 in females. The irreversible programming of the expression of these isoforms of cytochrome P‐450 in adulthood occurs during the perinatal period of life, and is named enzyme imprinting. One of the main factors that regulates the expression of the sex‐specific isoforms of cytochrome P‐450 is the level of androgens in the blood. Castration of adult rats decreased the level of the male isoforms of cytochrome P‐450 and the activity of the monooxygenase enzyme system that remained higher than in intact females.

The mechanism of enzyme imprinting can be explained as follows: neonatal androgens program the secretion of hypothalamic hormones, somatostatin and growth‐hormone‐releasing factor. These factors determine the type of growth hormone secretion in adult rats, and this controls the type of sex‐specific isoforms of cytochrome P‐450 expressed in adulthood.

Metabolic regulation similar to that outlined above was shown to occur for several metabolism‐dependent chemical carcinogens. Such a pathway may explain the different sensitivity displayed by male and female rats to treatment with these carcinogenic agents. One possible way of modulating the expression of some isoforms of cytochrome P‐450 in adult rats is by treating neonates with specific xenobiotics that change the constitutive expression of neonatal androgens. It appears that this enzyme imprinting plays an important role in determining the individual sensitivity to the carcinogenic effects of chemicals.
Tryptophan phosphorescence of G‐actin and F‐actin
Authors: STRAMBINI Giovanni B. , LEHRER Sherwin S.
Published: March 2005

The tryptophan phosphorescence spectrum, intensity and decay kinetics of G‐actin and F‐actin were measured over a temperature range of 140–293 K. The fine structure in the phosphorescence spectra at low temperature, with O,O vibrational bands centered at 405 nm and 415.5 nm for both species, reveals a marked heterogeneity of the chromophore environment. The thermal quenching profile distinguishes these sites in terms of their flexibility, and shows that probably only one of the four tryptophan residues is still phosphorescent at ambient temperature due to its location in a relatively rigid buried core. Although some differences are demonstrated between G‐actin and F‐actin at low temperature, the identity of the triplet lifetime at ambient temperature strongly supports the notion that the conformation of the macromolecule is largely unaffected by polymerization. Preliminary phosphorescence anisotropy measurements demonstrate both the occurrence of singlet‐singlet energy transfer among tryptophan residues and a strong immobilization of actin in the polymerized state.
Activation of a pertussis‐toxin‐sensitive guanine‐nucleotide‐binding regulatory protein during desensitization of Dictyostelium discoideum cells to chemotactic signals
Authors: SNAAR‐JAGALSKA B. Ewa , VAN ES Saskia , KESBEKE Fanja , VAN HAASTERT Peter J. M.
Published: March 2005

The chemoattractant cAMP induces the activation of adenylate cyclase in Dictyostelium discoideum. Upon prolonged incubation with cAMP, cells become desensitized via two distinct processes: a decrease in the number of available cAMP‐binding sites (down regulation) and modification of the receptor (presumably via phosphorylation) correlated with adaptation. These processes occur simultaneously, but differ in the cAMP dose dependency and reversibility. In this study we investigated the mechanism of adaptation; cells were incubated with a cAMP analog to induce desensitization mediated by adaptation. The cells were then washed, lysed and the interaction between cAMP, receptor, guanine‐nucleotide‐binding regulatory proteins (G proteins) and GTP was investigated. (1) cAMP receptors that are phosphorylated in vivo remain phosphorylated for at least 45 min after lysis. (2) Desensitization did not alter basal cAMP binding to the receptor nor the inhibitory effect of guanosine 5′‐[γ‐thio]triphosphate (GTP[S]) on this binding. (3) The stimulatory effect of cAMP on GTP[S] binding was also unchanged, while basal GTP[S] binding and the kinetics of binding were only slightly different. (4) Basal high‐affinity GTPase activity was not altered but cAMP stimulation was reduced from 43 ± 7% in control lysates to 14 ± 4% in lysates from desensitized cells. (5) cAMP stimulation of GTPase was decreased by pretreatment of cells with pertussis toxin from 43 ± 7% to 17 ± 8% but this was not further altered in lysates from desensitized pertussis‐toxin‐treated cells. These observations indicate that during desensitization the phosphorylated receptor can still interact with G proteins. Furthermore, desensitization did not affect cAMP stimulation of GTP[S] binding but strongly reduced cAMP stimulation of GTPase, suggesting that a G protein becomes activated. This G protein is pertussis toxin sensitive and may be the inhibitor G protein (Gi). This would imply that adenylate cyclase desensitizes because Gi becomes activated.
Identification of a novel nuclear protein synthesized in growth‐arrested human hepatoblastoma HepG2 cells
Authors: DONADEL Giulia , GARZELLI Carlo , FRANK Rainer , GABRIELLI Franco
Published: March 2005

DNA synthesis of human hepatoblastoma HepG2 cells is reversibly inhibited by butyrate. When butyrate is removed from the culture medium, cells re‐enter the cell cycle, synthesizing DNA with a time lag of about 12 h. HepG2 cells, growth‐inhibited for 30 h with butyrate, synthesize and accumulate a nuclear protein, called D. Protein D synthesis is inhibited in cells which, released from the butyrate block, have resumed DNA synthesis as well as in growing cells never exposed to butyrate. Protein D has been purified from growth‐arrested cells and partially sequenced. The amino acid sequences of five internal trypsin peptides indicate that protein D is a novel nuclear protein.
Structural and functional characteristics of polypeptide subunits of the bovine heart ubiquinol—cytochrome‐c reductase complex
Authors: COCCO Tiziana , LORUSSO Michele , SARDANELLI Anna Maria , MINUTO Michele , RONCHI Severino , TEDESCHI Gabriella , PAPA Sergio
Published: March 2005

Structural and functional characteristics of subunits of bovine heart cytochrome‐c reductase have been investigated by controlled digestion of soluble and membrane‐reconstituted purified bc1 complex and direct amino acid sequencing of native and digested protein subunits.

The results obtained show that the N‐terminal segments of core protein II and the 14‐kDa protein extend at the periphery of the complex, protruding into the inner matrix space. The Fe‐S protein, located at the outer C‐periphery of the complex, is shown to be anchored to other subunits of the complex by the amphipathic N‐terminal region. Proteolytic cleavage of 7–11 residues from the N‐terminal segment of the 14‐kDa protein is apparently associated with decoupling of redox‐linked proton pumping. Partial digestion of core protein II, the 6.4‐kDa protein, and the C‐terminal region of the 9.2‐kDa protein, is without effect on the redox and proton‐motive activity of the complex.
Covalent binding of dolichyl phosphate to proteins in rat liver
Authors: THELIN Anders , LÖW Peter , CHOJNACKI Tadeusz , DALLNER Gustav
Published: March 2005

Rats were injected via the portal vein with (RS)‐[5‐3H]‐mevalonolactone and the lipids were extracted. From fractions of liver homogenate, all labeled dolichol, cholesterol and ubiquinone could be extracted, but about 40% of microsomal and lysosomal dolichyl phosphate was only released after alkaline hydrolysis. Only a small amount of the non‐extractable radioactivity was found to be associated with α‐unsaturated polyprenyl phosphate. There was no difference in the polyisoprenoid pattern when the two pools of dolichyl phosphate were compared. On the other hand, the specific activity of the bound lipid was only half that of the extractable form. After phenyl‐Sepharose chromatography, a peak of protein was isolated exhibiting a 25‐fold enrichment in bound radioactive dolichyl phosphate. Treatment with a non‐specific protease, followed by chromatography, gave polypeptide fragments associated with bound lipids. On SDS/PAGE a major protein band at 23 kDa and some minor bands with higher molecular masses were found to be associated with this lipid. The results indicate the presence of covalently bound dolichyl phosphate in rat liver.
An acyl‐carrier‐protein – thioesterase domain from the 6‐deoxyerythronolide B synthase of Saccharopolyspora erythraea
Authors: CAFFREY Patrick , GREEN Brian , PACKMAN Leonard C. , RAWLINGS Bernard J. , STAUNTON James , LEADLAY Peter F.
Published: March 2005

The C‐terminal region of a multifunctional polypeptide from the 6‐deoxyerythronolide B synthase of Saccharopolyspora erythraea is predicted to contain an acyl carrier protein and a thioesterase or acyltransferase activity [Cortes, J., Haydock, S. F., Roberts, G. A., Bevitt, D. J. & Leadlay, P. F. (1990) Nature 348, 176–178]. Site‐directed mutagenesis by means of the polymerase chain reaction was used to construct an efficient pT7‐based expression plasmid for this domain. The recently developed technique of electrospray mass spectrometry was used to demonstrate that the purified protein had not been post‐translationally modified by attachment of a 4′‐phosphopantetheine group. However, treatment with the serine proteinase inhibitor phenylmethylsulphonyl fluoride led to highly selective labelling of the predicted active site of the thioesterase or acyltransferase.
Structure/function relationship of proteins belonging to the family of receptors coupled to GTP‐binding proteins
Authors: STROSBERG Arthur Donny
Published: March 2005

The structural properties of a number of proteins belonging to the family of receptors coupled to GTP binding proteins are discussed in relation to their function.

The structure of the ligand binding site and of the regions involved in coupling to the G proteins are analyzed mainly for the adrenergic and muscarinic cholinergic receptors, for which site‐directed mutagenesis and chimaeric constructions have been studied. The structure of the genes are compared and the presence of various regulatory elements is discussed in relation to control of expression.

Mechanism of desensitization and internalization, while mostly studied for the β2‐adrenergic receptor, are proposed to be generally applicable to all G‐protein‐coupled receptors.
Three‐dimensional structure of natural charybdotoxin in aqueous solution by 1H‐NMR Charybdotoxin possesses a structural motif found in other scorpion toxins
Authors: BONTEMS François , ROUMESTAND Christian , BOYOT Philippe , GILQUIN Bernard , DOLJANSKY Yvon , MENEZ André , TOMA Flavio
Published: March 2005

A 600‐MHz proton NMR study of natural charybdotoxin, a toxin acting on K+ channels, is reported. The unambiguous sequential assignment of all the protons of the toxin was achieved. The analysis of NOEs and of backbone coupling constants showed the existence of an α‐helix (residues 10–19) and of an antiparallel β‐sheet in the 26–35 part. Three‐dimensional structures were generated by distance geometry, using a set of 114 inter‐residual calibrated constraints (63 sequential, 47 medium and long range, 4 hydrogen bonds) and 29 π angles. These structures show that charbdotoxin is composed of a β‐sheet linked to an α‐helix by two disulphide bridges and to an extended fragment by the third disulphide bridge. Comparison with the other known structures of long and short scorpion toxins shows that this structural motif is common to all these proteins.
Kinetics of inositol 1,4,5‐trisphosphate and inositol 1,3,4,5‐tetrakisphosphate generation in dog‐thyroid primary cultured cells stimulated by carbachol
Authors: VERJANS Benoît , ERNEUX Christophe , RASPE Eric , DUMONT Jacques E.
Published: March 2005

The action of carbachol on the generation of inositol trisphosphate and tetrakisphosphate isomers was investigated in dog‐thyroid primary cultured cells radiolabelled with [3H]inositol. The separation of the inositol phosphate isomers was performed by reverse‐phase high pressure liquid chromatography. The structure of inositol phosphates co‐eluting with inositol 1,4,5‐trisphosphate [Ins(1,4,5)P3] and inositol 1,3,4,5‐tetrakisphosphate [Ins(1,3,4,5)P4] standards was determined by enzymatic degradation using a purified Ins(1,4,5)P3/Ins(1,3,4,5)P4 5‐phosphatase. The data indicate that Ins(1,3,4,5)P4 was the only [3H]inositol phosphate which co‐eluted with a [32P]Ins(1,3,4,5)P4 standard, whereas 80% of the [3H]InsP3 co‐eluting with an Ins(1,4,5)P3 standard was actually this isomer. In the presence of Li+, carbachol led to rapid increases in [3H]Ins(1,4,5)P3 and [3H]Ins(1,3,4,5)P4. The level of Ins(1,4,5)P3 reached a peak at 200% of the control after 5–10 s of stimulation and fell to a plateau that remained slightly elevated for 2 min. The level of Ins(1,3,4,5)P4 reached its maximum at 20 s. The level of inositol 1,3,4‐trisphosphate [Ins(1,3,4)P3] increased continously for 2 min after the addition of carbachol. Inositol‐phosphate generation was also investigated under different pharmacological conditions. Li+ largely increased the level of Ins(1,3,4)P3 but had no effect on Ins(1,4,5)P3 and Ins(1,3,4,5)P4. Forskolin, which stimulates dog‐thyroid adenylate cyclase and cyclic‐AMP accumulation, had no effect on the generation of inositol phosphates. The absence of extracellular Ca2+ largely decreased the level of Ins(1,3,4,5)P4 as expected considering the Ca2+‐calmodulin sensitivity of the Ins(1,4,5)P3 3‐kinase. Staurosporine, an inhibitor of protein kinase C, increased the levels of Ins(1,4,5)P3, Ins(1,3,4,5)P4 and Ins(1,3,4)P3. This supports a negative feedback control of diacylglycerol on Ins(1,4,5)P3 generation.
Immunolocalization of cytochromes P‐450olf1 and P‐450olf2 in rat olfactory mucosa
Authors: ZUPKO Karin , PORIA Yitzhak , LANCET Doron
Published: March 2005

Previously, we described two olfactory‐specific cytochromes P‐450: rat cytochrome P‐450olf1 (IIG1), identified by cDNA cloning, and bovine cytochrome P‐450olf2 (IIA), identified by peptide microsequencing of a trans‐membranal polypeptide (p52). Here we describe the preparation of polyclonal antisera against peptide sequences of these proteins and their use in the immunolocalization of cytochromes P‐450olf1 and P‐450olf2 in rat olfactory mucosa. Immunoreactivities related to both enzymes are found in the subepithelial Bowman's glands of olfactory mucosa. Practically no immunoreactivity was found in other rat tissues, including liver, lung, kidney and respiratory mucosa. In addition, double‐labeling experiments demonstrated that cytochromes P‐450olf1 and P‐450olf2 are present in the same population of Bowman's glands. The olfactory‐specific localization of cytochromes P‐450olf1 and P‐450olf2 is consistent with a role for these enzymes in the modification or clearance of odorants from the chemosensory tissue.
Discrimination between rat thiostatin (T‐kininogen) and one of its cystatin‐like inhibitory fragments by a monoclonal antibody, and localization of the epitope
Authors: LALMANACH Gilles , ADAM Albert , MOREAU Thierry , GUTMAN Ninette , GAUTHIER Francis
Published: March 2005

A monoclonal antibody (mAb D3) raised against rat thiostain (T‐kininogen) strongly interacted with a fragment, identified as cystatin‐like domain 3, which inhibits cysteine proteinases but did not recognize intact, native thiostatin. The antigen‐antibody reaction requires cleavage of the single peptide chain of thiostatin in its inter‐domain 2–3 region. This mAb can also differentiate between the two molecular varieties of thiostatin, reacting only with immobilized domain 3 from T1 thiostatin, which differs from the T2 variety by only 10 out of 125 residues. mAb D3 did not react with an N‐terminally truncated domain 3 of T1 thiostatin prepared by submaxillary gland kallikrein k10 proteolysis. This suggests that the epitope, or an essential part of it, is located on a stretch of 12 residues at the N‐terminal of the T1 thiostatin domain 3. This sequence in T1 thiostatin differs from that in T2 thiostatin by four amino acids, two of which are arginyl residues in T1. Chemical modification of these residues located at positions 246 and 250 decreased the reactivity of T1 domain 3 towards the antibody, suggesting that at least one of them is a critical residue of the epitope. Arginine 246 is part of a small disulfide loop between cysteines 245 and 248 which is also necessary for antibody recognition. This antibody does not change the inhibitory properties of purified domain 3 towards papain or rat liver cathepsin L, indicating that the N‐terminal part of domain 3 is not involved in inhibition. mAb D3 was used to demonstrate the presence of inhibitory thiostatin fragments in ascites fluid but not in plasma from normal or turpentine‐injected rats.
Structure of the human brain calcium‐binding protein calretinin and its expression in bacteria
Authors: PARMENTIER Marc , LEFORT Anne
Published: March 2005

Calbindin D28k and calretinin are two closely related intracellular calcium‐binding proteins belonging to the troponin C superfamily. Calbindin is known to be involved in the vitamin‐D‐dependent calcium absorption through intestinal and renal epithelia, while the function of neuronal calbindin and calretinin is poorly understood. Using antibodies directed against chick intestinal calbindin D28k, human calretinin cDNA clones were isolated from brain cDNA libraries. The sequence of the calretinin cDNA revealed an open reading frame of 271 codons coding for a protein of 31 520 Da, and sharing 58% identical residues with human calbindin D28k. Calretinin contains five presumably active and one presumably inactive calcium‐binding domains. Comparison with the partial sequences available for chick and guinea pig calretinins revealed that the protein is highly conserved in evolution (evolutionary rate: 0.27 × 10−9 amino acid−1 year−1). The calretinin message was detected in the brain, while absent from heart muscle, kidney, liver, lung, spleen, stomach and thyroid gland. Recombinant calretinin was expressed in Escherichia coli, and the calcium‐binding properties were confirmed on both the natural and the recombinant proteins. Part of the human gene coding for calretinin was isolated and the region corresponding to the promoter and the first exon was sequenced.
Cellobiose oxidase from Phanerochaete chrysosporium can be cleaved by papain into two domains
Authors: HENRIKSSON Gunnar , PETTERSSON Göran , JOHANSSON Gunnar , RUIZ Alberto , UZCATEGUI Edgar
Published: March 2005

Cellobiose oxidase from the white rot fungus Phanerochaete chrysosporium has been purified to homogeneity by a new method. The enzyme has been cleaved by papain into two fragments: one containing the heme group and one containing the flavin group. The flavin fragment can oxidize cellobiose and is reoxidized by oxygen. Cellobiose oxidase binds to cellulose to approximately the same extent as cellobiohydrolase I. The cellulose‐binding site is located on the flavin domain. The enzyme cannot be totally displaced from cellulose by cellobiose, and it is still active when adsorbed to cellulose. The possible role of the enzyme in lignocellulose degradation is discussed.
Core region of Citrobacter O23 lipoplysaccharide
Authors: KATZENELLENBOGEN Ewa , GAMIAN Andrzej , ROMANOWSKA Elzbieta , DABROWSKI Ursula , DABROWSKI Janusz
Published: March 2005

A novel enterobacterial core region in Citrobacter O23 lipopolysaccharide is described. Its structure was determined by methylation analysis/mass spectrometry, chemical degradation and one‐ and two‐dimensional 1H‐NMR spectroscopy:

image

where PPEtN stands for diphosphorylethanolamine, and dOclA for 3‐deoxy‐D‐manno‐octulosonic acid.
Mobility of the spin‐labeled side chains of some novel antifolate inhibitors in their complexes with dihydrofolate reductase
Authors: BLAKLEY Raymond L. , PIPER James R. , MAHARAJ Geeta , APPLEMAN James R. , DELCAMP Tavner J. , FREISHEIM James H. , KULINSKI Robert F. , MONTGOMERY John A.
Published: March 2005

Four spin‐labeled inhibitors of dihydrofolate reductase (DHFR) have been synthesized, each of which has the 2,2,6,6‐tetramethyl‐1‐piperidinyloxy (TEMPO) reporting group at a different distance from the 2,4‐diaminopyrimidine moiety by which the inhibitors are anchored and oriented in the active site. Inhibitors in which the TEMPO group is attached by a short side chain are weakly bound to DHFR from bacteria (Streptococcus faecium and Lactobacillus casei), to the bovine enzyme and to recombinant human DHFR. However, binding is sufficiently tight, especially in the ternary complexes with NADPH, for recording of the EPR spectra of the bound ligands. The spectra indicate that when these inhibitors are bound to the enzyme the TEMPO group is highly immobilized with correlation times, τc, 4 – 20 ns. Inhibitors that have the reporter group attached to the glutamate moiety of methotrexate bind to all four DHFRs more tightly than the inhibitors with shorter side chains by factors of up to 106. However, in most complexes formed by the inhibitors with longer side chains immobilization of the TEMPO group is slight (τc 0.2–4 ns). These results are in general agreement with predictions from X‐ray crystallographic results including thermal factors but there are some unanticipated differences between some results for bacterial and eukaryotic enzymes. Three of the splin‐labeled inhibitors would provide good probes for distance measurements in and around the active site of mammalian DHFR.
Hydrolysis of intact and Cys‐Phe‐cleaved human atrial natriuretic peptide in vitro by human tissue kallikrein
Authors: VANNESTE Yves , MICHEL Alain , DESCHODT‐LANCKMAN Monique
Published: March 2005

Atrial natriuretic peptide (ANP) is a 28‐amino‐acid hormone involved in the regulation of fluid balance. In circulation, the proteolytic inactivation of ANP has been demonstrated to involve both membrane metallo‐endopeptidase and an aprotonin‐sensitive activity, probably corresponding to kallikrein [Vanneste, Y., Pauwels, S., Lambotte, L., Michel, A., Dimaline, R. & Deschodt‐Lanckman, M. (1990) Biochem. J. 269, 801–806]. In the present study, we focused on the aprotinin‐sensitive pathway of ANP metabolism. In order to identify the cleavage sites recognized by kallikrein within the sequence of the hormone, tissue kallikrein was purified to homogeneity from human urine and the degradation of human ANP by the enzyme preparation was studied.

Our results demonstrate that both intact and Cys7‐Phe8‐cleaved ANP, the initial metabolite produced in circulation by the metallo‐endopeptidase, are substrates in vitro for purified tissue kallikrein. However, the Cys‐Phe‐cleaved peptide was degraded approximately fourfold faster than the intact hormone by the purified enzyme. The first degradation step of ANP by tissue kallikrein involves two cleavages occurring at the bonds Arg3‐Arg4 and Gly16‐Ala17, generating an inactive, open‐ring metabolite. Incubation of ANP for a longer period with the enzyme led to the generation of several additional degradation fragments. Ten peaks were separated by HPLC and characterized by amino acid analysis. The results allowed the identification of a total of eight peptide bonds susceptible to hydrolysis by tissue kallikrein in the sequence of ANP: Arg3‐Arg4, Ser5‐Ser6, Cys7‐Phe8, Arg11‐Met12, Gly16‐Ala17, Gly20‐Leu21, Ser25‐Phe26 and Arg27‐Tyr28.

These results indicate that the aprotinin‐sensitive activity involved in the metabolism of ANP in circulation could correspond to tissue kallikrein. However, clear identification of ANP as a novel physiological substrate of the enzyme will need further investigation.