bluuebunny/crossref_metadata_2025 huggingface dataset:
Phenylalanine positively modulates the cAMP‐dependent phosphorylation and negatively modulates the vasopressin‐induced and okadaic‐acid‐induced phosphorylation of phenylalanine 4‐monooxygenase in intact rat hepatocytes
Authors: DØSKELAND Anne P. , VINTERMYR Olav K. , FLATMARK Torgeir , COTTON Richard G. H. , DØSKELAND Stein Ove
Published: March 2005

The state of phosphorylation of phenylalanine hydroxylase was determined in isolated intact rat hepatocytes. 32P‐labeled phenylalanine hydroxylase was immunoisolated from cells loaded with 32Pi or from cell extracts ‘back‐phosphorylated’ with [γ‐32P]ATP by cAMP‐dependent protein kinase.

The rate of phenylalanine hydroxylase phosphorylation in cells with elevated cAMP was similar to that observed for the isolated enzyme phosphorylated by homogeneous cAMP‐dependent protein kinase. The phosphorylation rate in cAMP‐stimulated cells was increased up to four times (reaching 0.018 s–1) by the presence of phenylalanine, the phosphate content (mol/mol hydroxylase) increasing to 0.5 from the basal level (0.17) in 50 s. The half maximal effect of phenylalanine was obtained at a physiologically relevant concentration (110 μM). The synthetic phenylalanine hydroxylase cofactor dimethyltetrahydropterin also enhanced the cAMP‐stimulated phosporylation of phenylalanine hydroxylase, presumably by displacing the endogenous cofactor, tetrahydrobiopterin.

Phenylalanine was a negative modulator of the phosphorylation of phenylalanine hydroxylase induced by incubating cells with vasopressin or with the phosphatase inhibitor okadaic acid. The same site on the phenylalanine hydroxylase was phosphorylated in response to these two agents as in response to elevated cAMP. The available evidence suggested that not only vasopressin, but also okadaic acid, acted by stimulating the multifunctional Ca2+/calmodulin‐dependent protein kinase II or a kinase with closely resembling properties.
Atrial natriuretic peptide is phosphorylated by intact cells through cAMP‐dependent ecto‐protein kinase
Authors: KÜBLER Dieter , REINHARDT Dieter , REED Jennifer , PYERIN Walter , KINZEL Volker
Published: March 2005

Recently we demonstrated the presence of cell‐surface‐located cAMP‐dependent protein kinase (ecto‐PK A) activity in a number of different cell types [Kübler, D., Pyerin, W., Bill, O., Hotz, A., Sonka, J. and Kinzel, V. (1989) J. Biol. Chem. 264, 14549–14555]. The question of the physiological role of externally directed kinase activity prompted a search for potential natural substrates present in the intercellular fluid. In the present study we have investigated the phosphorylation by ecto‐PK A of the human atrial natriuretic peptide ANP99–126, a hormone released by cardiac cells. This 28‐amino‐acid peptide carries the phosphorylation consensus sequence Arg‐Arg‐Ser‐Ser for the PK A. Incubation of various cell lines (including epithelial, epidermal, myoblast and lymphoma cells) or freshly isolated blood cells (macrophages, erythrocytes and platelets) with ANP in the presence of low micromolar concentrations of ATP resulted in the phosphorylation of ANP at Ser residues. The ANP phosphorylation reaction proved strictly dependent on cAMP; cAMP could not be replaced by cGMP. The phosphorylation was inhibited by the PK A‐specific inhibitory peptide and increased linearily for up to 15 min and with a Km value of 3–5 μM for ANP. At higher ATP concentrations (> 100 μM) the incorporation rates amounted to about 0.3 mmol P (mol ANP)−1 min−1. The rise of intracellular cAMP in HEL30 (an epidermal cell line) after application of the β‐adrenergic receptor agonist isoproterenol led to an approximately three‐fold stimulation of ANP phosphorylation which appears to be brought about by an efflux of intracellular cAMP. Employing cell supernatant fluids and cell sonicates, it could be shown that the phosphorylation of ANP results from the ecto‐PK A. Comparison of ANP with ANP phosphorylated in vitro using purified catalytic subunit of PK A showed that phosphorylation is accompanied by certain changes in the average solution conformation of the peptide, consistent with the changes known to occur in its biological activity. Our results demonstrate cAMP‐dependent phosphorylation of the peptide hormone analogue ANP99–126 by intact cells through ecto‐PK A, an intriguing mechanism for post‐translational processing of ANP.
Specfic protein kinase from Saccharomyces cerevisiae cells phosphorylating 60S ribosomal proteins
Authors: PILECKI Marek , GRANKOWSKI Nikodem , JACOBS Jolanta , GASIOR Eugeniusz
Published: March 2005

A protein kinase, specific for 60S ribosomal proteins, has been isolated from Saccharomyces cerevisiae cells, purified to almost homogeneity and characterized. The isolated enzyme is not related to other known protein kinases. Enzyme purification comprised three chromatography steps; DEAE–cellulose, phosphocellulose and heparin – Sepharose. SDS/PAGE analysis of the purified enzyme, indicated a molecular mass of around 71 kDa for the stained single protein band. The specific activity of the protein kinase was directed towards the 60S ribosomal proteins L44, L44′, L45 and a 38 kDa protein. All the proteins are phosphorylated only at the serine residues. None of the 40S ribosomal proteins were phosphorylated in the presence of the kinase. For that reason we have named the enzyme the 60S kinase. An analysis of the phosphopeptide maps of acidic ribosomal proteins, phosphorylated at either the 60S kinase or casein II, showed almost identical patterns. Using the immunoblotting technique, the presence of the kinase has been detected in extracts obtained from intensively growing cells. These findings suggest an important role played by the 60S kinase in the regulation of ribosomal activity during protein synthesis.
Molecular cloning of a mouse 47‐kDa heat‐shock protein (HSP47), a collagen‐binding stress protein, and its expression during the differentiation of F9 teratocarcinoma cells
Authors: TAKECHI Hajime , HIRAYOSHI Kazunori , NAKAI Akira , KUDO Hitoshi , SAGA Shinsuke , NAGATA Kazuhiro
Published: March 2005

A 47‐kDa heat‐shock protein (HSP47) is a major collagen‐binding stress protein residing in the endoplasmic reticulum, and is assumed to be a molecular chaperone specific to collagen. Two‐dimensional gel electrophoresis and immunoprecipitation studies showed that the expression of HSP47 was significantly induced during the differentiation of mouse teratocarcinoma F9 cells by treatment with retinoic acid alone or with retinoic acid and dibutyryladenosine 3′,5′‐phosphate. The induction of type‐IV collagen was also observed during F9‐cell differentiation. For further analysis, we cloned cDNA encoding mouse HSP47 from a cDNA library of BALB/c 3T3 cells and performed Northern‐blot analysis. The cDNA contained a signal sequence at the N‐terminus and an endoplasmic‐reticulum‐retention signal, RDEL, at the C‐terminus. An homology search revealed that mouse HSP47, as well as chick HSP47, belonged to the serine protease inhibitor superfamily. While chick HSP47 mRNA was 4.5 kb with a long (2‐kb) 3′ untranslated region, mouse and human HSP47 mRNA were 2.5 kb, with a 0.8‐kb 3′ untranslated region. Northern‐blot analysis revealed that the concurrent induction of HSP47 and type‐IV collagen during F9‐cell differentiation, and the transient induction of HSP47 after heat shock was regulated at the level of mRNA accumulation. These results suggested that HSP47 was closely related to collagens in terms of its expression as well as in its functional relevance.
Conformational and functional properties of peptides covering the intersubunit region of influenza virus hemagglutinin
Authors: HOLLÓSI Miklos , ISMAIL Ashraf A. , MANTSCH Henry H. , PENKE Botond , VARADI Istvan G. , TOTH Gabor K. , LACZKÓ Ilona , KURUCZ Istvan , NAGY Zoltan , FASMAN Gerald D. , RAJNAVÖLGYI Eva
Published: March 2005

The functionally active part of influenza virus hemagglutinin was investigated through the synthesis of a series of peptides representing different parts of the intersubunit region. Secondary structure prediction, circular dichroism and Fourier transform infrared spectroscopic studies were undertaken to investigate the secondary structure of these peptides. The peptide fragments were found to adopt multiple conformations, depending on their concentration in solution, the presence of the non‐ionic detergent octyl‐β‐d‐glucoside and the polarity of the solvent. The results of biological studies with these peptide fragments are discussed in relation to their conformation, as inferred from the spectroscopic analysis.
Formatin of heterodimers of human‐immunodeficiency‐virus‐type‐1 reverse transcriptase by recombination of separately purified subunits
Authors: STAMMERS David K. , ROSS Carl K. , IDRISS Haitham , LOWE Denise M.
Published: March 2005

Human‐immunodeficiency‐virus‐type‐1 reverse transcriptase exists in virions as a heterodimer of a Mr 66000 subunit and its C‐terminally truncated form of Mr 66000 protein, a mixture of heterodimers and homodimers results which co‐purify by most conventional techniques. We describe a method of hydrophobic hromatography which co‐purify by most conventional techniques. We describe a method of hydrophobic chromatography which gives baseline separation of these two forms of the protein. This method has been applied to purify heterodimers formed by recombination of separately expressed and purified Mr 66000 and 51000 subunits, resulting in significantly more homogeneous heterodimer preparations. The recombined heterodimer showed similar kinetic properties and RNase H activity to the standard heterodimer and a specific activity significantly higher than the original homodimer of the Mr 66000 protein. Heterodimers having greater asymmetry have also been prepared by recombining Mr 66000 subunits containing single‐point or deletion mutations, with wild‐type Mr 51000 subunits, and the resulting heterodimers analysed.
Two pathways of pyrophosphate hydrolysis and synthesis by yeast inorganic pyrophosphatase
Authors: BAYKOV Alexander A. , SHESTAKOV Alexander S.
Published: March 2005

Initial rates of pyrophosphate hydrolysis and synthesis by baker's yeast inorganic pyrophosphatase and equilibrium amounts of enzyme‐bound and free pyrophosphate were measured over wide ranges of Mg2+ and respective substrate concentrations. Computer analysis of these data, in conjunction with those on phosphate/water oxygen exchange [Kasho, V. N. & Baykov, A. A. (1989) Biochem. Biophys. Res. Comm. 161, 475–480], yielded values of the equilibrium constants for Mg2+ binding to free enzyme and central complexes and values of the forward and reverse rate constants for the four reaction steps, namely, PPi binding/release, PPi hydrolysis/synthesis and two Pi binding/release steps. All catalytic steps were found to proceed through two parallel pathways, involving 3 or 4 Mg2+/PPi or 2 Pi bound. Product release is the slowest catalytic event in both hydrolysis and synthesis of pyrophosphate, at least, for the four‐metal pathway. In the hydrolytic reaction, magnesium pyrophosphate binding is faster for the four‐metal pathway, dissociation of the second Pi is faster for the three‐metal pathway, while PPi hydrolysis and the release of the first Pi may proceed with similar rates. Release of pyrophosphate formed on the enzyme is faster for the three‐metal pathway. Both pathways are expected to operate in vivo, and their relative contributions will vary with changes in the Mg2+ concentration, thus providing a means for pyrophosphatase‐activity regulation.
Purification and properties of prostaglandin 9‐ketoreductase from pig and human kidney
Authors: SCHIEBER Ardrea , FRANK Rainer W. , GHISLA Sandro
Published: March 2005

Prostaglandin 9‐ketoreductase (PG‐9‐KR) was purified from pig kidney to homogeneity, as judged by SDS/PAGE using an improved procedure. The enzyme is pro‐S stereoselective with regard to hydrogen transfer from NADPH with prostaglandin E2 as substrate and reduces its 9‐keto group with approximately 90% stereoselectivity to form prostaglandin F2α. Approximately 8% of the prostaglandin F formed has the β‐configuration. In addition to catalyzing the interconversion of prostaglandin E2 to F2α, PG‐9‐KR also oxidizes prostaglandin E2, F2α and D2 to their corresponding, biologically inactive, 15‐keto metabolites. Incubation of PG‐9‐KR with prostaglandin F2α and NAD+ leads to the preferential formation of 15‐keto prostaglandin F2α rather than prostaglandin E2. This suggests that the prostaglandin E2/prostaglandin F2α ratio is not determined by the NADP+/NADPH redox couple. The enzyme also reduces various other carbonyl compounds (e.g. 9,10‐phen‐anthrenequinone) with high efficiency. The catalytic properties measured for PG‐9‐KR suggest that its in vivo function is unlikely to be to catalyze formation of prostaglandin F2α. The monomeric enzyme has a molecular mass of 32 kDa and exists as four isoforms, as judged by isoelectric focusing. PG‐9‐KR contains 1.9 mol Zn2+/mol enzyme and no other cofactors. Human kidney PG‐9‐KR was also purified to homogeneity. The human enzyme has a molecular mass of 34 kDa and also exists as four isoforms. Polyclonal antibodies raised against pig kidney PG‐9‐KR cross‐react with human kidney PG‐9‐KR and also with human brain carbonyl reductase, as demonstrated by Western blot analysis. Sequence data of tryptic peptides from pig kidney PG‐9‐KR show >90% identity with human placenta carbonyl reductase. From comparison of several properties (catalytical, structural and immunological properties), it is concluded that PG‐9‐KR and carbonyl reductase are identical enzymes.
Rac1, a low‐molecular‐mass GTP‐binding‐protein with high intrinsic GTPase activity and distinct biochemical properties
Authors: MÉNARD Luc , TOMHAVE Eric , CASEY Patric J. , UHING Ronald J. , SNYDERMAN Ralph , DIDSBURY John R.
Published: March 2005

Rac1, a member of the family of low‐molecular‐mass GTP‐binding proteins, functions in phagocytic leukocytes as a component necessary for activation of the respiratory burst. To characterize the biochemical properties of rac1, the protein was expressed as a fusion protein in Escherichia coli and purified to greater than 99% homogeneity by affinity chromatography. Rac1 protein bound maximally bound and hydrolyzed GTP under low free‐Mg2+ concetrations. Under those conditions, (45 nm free Mg2+), purified rac1 exhibited a steady‐state GTPase activity of 18 nmol · min−1· mg protein−1 (turnover number ∼ 0.39 min−1 at 37°C), which is 40‐fold higher than H‐ras. The high intrinsic GTPase activity of rac1 under low free Mg2+ was mainly due to an increased Kcat, the rate constant for hydrolysis of bound GTP, which was 0.29 min−1 for rac1 vs 0.007 min−1 for H‐ras (at 20°C). Rac1 also released bound GDP faster than H‐ras (Koff·sGDP= 1.02 min−1 for rac1 vs0.33 min−1 for H‐ras at 20°C). In contrast, rac1 released bound guanosine 5′‐[γ‐thio]triphosphate (GTP[S] at a slower rate than H‐ras (koff · GTP[S]∼ 0.04 min−1 for rac1 vs 0.31 min−1 for H‐ras at 20°C). Rac1 was a very good substrate for in vitro geranylgernylation (C20) but not for farnesylation (C15, whereas the converse is true for H‐ras. Surprisingly, rac1 was a very poor substrate for in vitro ADP‐ribosylation by the C3 component of Clostridium botulinum toxin compared to rhoA. As a further characterization of rac1, a mutant was made in which the Thr115 was replaced by asparagine. This protein (referred to as [Thr115 → Asn]rac1) contains the consensus amino acids of all four GTP‐binding domains of H‐ras. The koff·GDP of [Thr115 → Asn]rac1 was reduced to that of H‐ras, but [Thr115 → Asn]rac1 exhibited essentially identical kcat (0.13 min−1 at 20°C) and koff·GTP[s] (0.03 min−1 at 20°C) values as the Wild‐type protein. thus, the region(s) in rac1 which control the dissociation of GTP[S] (and presumably GTP) do not entirely coincide with those controlling GDP dissociation. Biochemical analysis of [Thr115 → Asn]rac1 also suggests that the region responsible for the increased kcat of rac1 is not within the consensus amino acids of the four guanine‐nucleotide‐binding domains.
The binding of triazine herbicides to the photosynthetic reaction center of Rhodopseudomonas viridis
Authors: EGNER Ursula , HOYER Georg‐Alexander , SAENGER Wolfram
Published: March 2005

The binding of six herbicides of the triazine family to the photosynthetic reaction center of Rhodopseudomonas viridis was investigated with energy‐minimization techniques, in order to correlate experimental with calculated data. The inhibitors were modeled in the active site according to the X‐ray structure analysis of the complex formed between the triazine terbutryn (2‐ethylamino‐4‐t‐butylamino‐6‐methylthio‐s‐triazine) and the reaction center of R. viridis [Michel, H., Epp, O. & Deisenhofer, J. (1986) EMBO J. 5, 2445–2451].

40 different energy minimizations were carried out with varying cutoff radii, partial charges on inhibitor atoms and dielectric constants, i.e. 10 different combinations of these were tested. The impact of these parameters on the calculated binding and interaction energy was either examined for all protein/triazine complexes or, in the case of the dielectric constant, a smaller sample was used. The calculated energies are dominated by van der Waals interactions, which change by up to 20% when extending the cutoff radius from 0.8 nm to 1.5 nm. The use of uniform or distance‐dependent dielectric constant or partial charges on the inhibitor atoms does not severely influence the resulting structures, but shows a great impact on the calculated energies.

In the two groups of triazines, each containing three inhibitors with methoxy or methylthio substituents, correlations of biological and calculated data were found quite often, but only once with all six triazines. The energy‐minimized structures were compared and analysed. A third hydrogen bond, not seen in the X‐ray analysis of the reaction center/terbutryn complex, was found between the t‐butylamino moiety of terbutryn (and equivalent moieties in the other triazines) and the carbonyl oxygen of TyrL222.
Reversed micelles of polymeric surfactants in nonpolar organic solvents
Authors: KHMELNITSKY Yuri L. , GLADILIN Alexandr K. , ROUBAILO Valentin L. , MARTINEK Karel , LEVASHOV Andrey V.
Published: March 2005

A new microheterogeneous non‐aqueous medium for enzymatic reactions, based on reversed micelles of a polymeric surfactant, was suggested. The surfactant termed CEPEI, was synthesized by successive alkylation of poly(ethyleneimine) with cetyl bromide and ethyl bromide and was found to be able to solubilize considerable amounts of water in benzene/n‐butanol mixtures. The hydrodynamic radius of polymeric‐reversed micelles was estimated to be in the range 22–51 nm, depending on the water content of the system, as determined by means of the quasi‐elastic laser‐light scattering. Polymeric reversed micelles were capable of solubilizing enzymes (α‐chymotrypsin and laccase) in nonpolar solvents with retention of catalytic activity. Due to the strong buffering properties of CEPEI over a wide pH range, it could maintain any adjusted pH inside hydrated reversed micelles. It was found that catalytic behavior of enzymes entrapped in polymeric reversed micelles was rather insensitive to the pH of the buffer solution introduced into the system as an aqueous component, but determined mostly by acid‐base properties of the polymeric surfactant itself. Both catalytic activity and stability of entrapped α‐chymotrypsin and laccase were found to increase with increasing water content of the system. Under certain conditions, the entrapment of α‐chymotrypsin into CEPEI reversed micelles resulted in a considerable increase in catalytic activity and stability as compared to aqueous solution. CEPEI reversed micelles were demonstrated to be promising enzyme carriers for use in membrane reactors. Owing to the large dimensions of CEPEI reversed micelles, they are effectively kept back by a semipermeable membrane, thus allowing an easy separation of the reaction product and convenient recovery of the enzyme.
The mechanism of the increase in mitochondrial proton permeability induced by thyroid hormones
Authors: BRAND Martin D. , STEVERDING Dietmar , KADENBACH Bernhard , STEVENSON Patricia M. , HAFNER Roderick P.
Published: March 2005

Three possible mechanisms by which different levels of thyroid hormones in rats might cause the observed sevenfold change in the apparent proton permeability of the inner membrane of isolated liver mitochondria were investigated. (a) Cytochrome c oxidase was isolated from the livers of hypothyroid, euthyroid and hyperthyroid rats and incorporated into liposomes made with soya phospholipids. There was no difference between the proton current/voltage curves of the three types of vesicles. The hormonal effects, therefore, were not an inherent property of the enzymes, and were not due to different coupling of electron flow through the enzyme to proton transport. (b) The surface area of the mitochondrial inner membrane was shown by three different assays to be greater by a factor of between two and three in mitochondria from hyperthyroid animals than in mitochondria from hypothyroid animals; euthyroid controls were intermediate. This difference in surface area of the inner membrane explains less than half of the difference in apparent proton permeability. (c) The proton permeability of liposomes prepared from phospholipids extracted from mitochondrial inner membranes of hyperthyroid rats was three times greater than the proton permeability of those from hypothyroid rats; euthyroid controls were intermediate. This suggests, first, that the proton permeability of the phospholipid bilayer is an important component of the proton permeability in intact mitochondria and, second, thyroid hormone‐induced changes in the bilayer are a major part of the mechanism of increased proton permeability. Such changes may be due to the known differences in fatty acid composition of mitochondrial phospholipids in different thyroid states. Thus we have identified two mechanisms by which thyroid hormone levels in rats change proton flux/mass protein in isolated liver mitochondria: a change in the area of the inner membrane/mass protein and a change in the intrinsic permeability of the phospholipid bilayer.
The pro‐region of the yeast prepro‐α‐factor is essential for membrane translocation of human insulin‐like growth factor 1 in vivo
Authors: CHAUDHURI Bhabatosh , STEUBE Klaus , STEPHAN Christine
Published: March 2005

Four yeast secretion signals, the 19‐amino‐acid invertase signal sequence, the 17‐amino‐acid acid‐phosphatase signal sequence, and the pre‐sequence and prepro‐sequence of prepro‐α‐factor have been used to look for the secretion of recombinant human insulin‐like growth factor 1 (IGF1) from Saccharomyces cerevisiae. Only the prepro‐sequence, often referred to as the α‐factor leader and consisting of an N‐terminal 19‐amino‐acid pre‐sequence or signal sequence attached to a 66‐amino‐acid pro‐region, permits secretion of IGF1. The signal sequences alone do not allow the translocation of IGF1 into the endoplasmic reticulum. This is evident from the fact that IGF1‐like molecules, to which the signal sequences are still attached, accumulate intracellularly in the cytosol. Fusion of the pro‐region of the α‐factor leader to the C‐terminus of the acid‐phosphatase and invertase signal sequences allows IGF1 to be secreted once again. These results reveal the essential role of the pro‐region of the α‐factor leader in the secretion of IGF1 and indicate that it may have a function in guiding a nascent IGF1 polypeptide to a state in which translocation can occur.
Isolation and characterization of three chitinases from Trichoderma harzianum
Authors: DE, LA CRUZ Jesús , HIDALGO‐GALLEGO Antonio , LORA José M. , BENITEZ Tahia , PINTOR‐TORO José A. , LLOBELL Antonio
Published: March 2005

Three proteins which display chitinase activity were purified from the supernatants of Trichoderma harzianum CECT 2413 grown in minimal medium supplemented with chitin as the sole carbon source. Purification was carried out after protein precipitation with ammonium sulphate, adsorption to colloidal chitin and digestion, and, finally, chromatofocusing. By this procedure, two chitinases of 42 kDa (CHIT42) and 37 kDa (CHIT37) were purified to homogeneity, as judged by SDS/PAGE and gel filtration, whereas a third, of 33 kDa (CHIT33), was highly purified. The isoelectric points for CHIT42, CHIT37 and CHIT33 were 6.2, 4.6 and 7.8, respectively. The three enzymes displayed endochitinase activities and showed different kinetic properties. CHIT33 was able to hydrolyze chitin oligomers of a polymerization degree higher than n= 4, its Km for colloidal chitin being 0.3 mg/ml. CHIT42 and CHIT37 were able to hydrolyze chitin oligomers with a minimal polymerization degree of n= 3, their Km values for colloidal chitin being 1.0 mg/ml and 0.5 mg/ml respectively. With regard to their lytic activity with purified cell walls of the phytopathogenic fungus Botrytis cinerea, a hydrolytic action was observed only when CHIT42 was present. Antibodies against CHIT42 and CHIT37 specifically recognized the proteins and did not display cross‐reaction, suggesting that each protein is encoded by a different gene.
Differential tissue‐specific expression and induction of cytochrome P450IVA1 and acyl‐CoA oxidase
Authors: BELL David R. , BARS Remi G. , ELCOMBE Clifford R.
Published: March 2005

We have examined the tissue‐specific expression and inducibility of acyl‐CoA oxidase and cytochrome P450IVA1 (P450IVA1) RNA in rats. Groups of three rats were dosed daily by gavage with methylclofenapate at 25 mg/kg in 5 ml/kg corn oil for nine weeks, or were administered a vehicle control. P450IVA1 and acyl‐CoA oxidase RNA were detected using an RNase protection assay. Similar levels of acyl‐CoA oxidase RNA were present in control liver and kidney, but the level of this RNA in lung, muscle and testis was 6–11%, and in pancreas was 0.13%, of that in liver. Treatment of rats with methylclofenapate led to an 11‐fold induction of acyl‐CoA oxidase RNA in liver and also produced a significant induction of this RNA in kidney, lung, muscle and testis of 1.7‐fold, 1.3‐fold, 2‐fold and 1.7‐fold, respectively. Acyl‐CoA oxidase RNA was not induced in pancreas.

P450IVA1 RNA was present in control liver and also in kidney of control rats at 28% of the level in liver. In contrast to acyl‐CoA oxidase RNA, P450IVA1 RNA was not detected in lung, pancreas or testis. Methylclofenapate treatment of rats led to an 18‐fold induction of P450IVA1 RNA in liver, and a sevenfold induction in kidney. Induction of P450IVA1 was not detected in any of the other tissues examined. Quantification of the relative amounts of acyl‐CoA oxidase and P450IVA1 RNA in control liver revealed that acyl‐CoA oxidase RNA was present in a 17.5‐fold molar excess over P450IVA1 RNA.

Western blotting with an anti‐P450IVA IgG revealed two bands of similar apparent molecular mass in liver and kidney microsomes, but not in microsomes from the testis of control rats. Methylclofenapate treatment of rats caused an increase in the intensity of these bands in microsomes from liver, but no induction was obvious in kidney. Immunocytochemical staining for both the microsomal P450IVA and peroxisomal acyl‐CoA oxidase proteins was restricted to the proximal convoluted tubule in the kidney cortex, with staining being most intense in the S3 region.
Periodic properties of proton conformational shifts in isolated protein helices
Authors: JIMÉNEZ M. Angeles , BLANCO Francisco J. , RICO Manuel , SANTORO Jorge , HERRANZ José , NIETO José L.
Published: March 2005

In this work, the helix‐forming residues in fragments of several proteins (ribonuclease, thermolysin, tendamistat and angiogenin) were identified by NOE and the helix proton shifts were measured as δ changes associated with helix‐population increments driven by trifluoroethanol addition. When estimated in this way, a regular pattern of helix conformational shifts was clearly seen in the Δδ versus sequence profiles of all the peptides studied. The helix periodicity of the Hα and Hβ resonances was especially clear, an observation that earlier statistical studies of protein δ values failed to predict. Amide protons showed the largest helix shifts, but with a less‐sharply defined periodic character. Aromatic residues considerably distorted the periodicity of the helix amide shifts in some peptides, as evidenced by the δ shifts of a RNase A fragment 1–15 analog in which the two aromatic residues were replaced by Ala. The relationship between helix periodicity and peptide amphiphatic character is discussed.
Synthesis and spectroscopy of membrane receptor proteins
Authors: ANDERSON Graeme J. , HARIS Parvez I. , CHAPMAN Dennis , ROMER Justyna T. , TOTH Gabor K. , TOTH Istvan , GIBBONS William A.
Published: March 2005

The high‐affinity receptor for IgE is a tetrameric complex of subunits of the type αβγ2. We report here conformational studies of the intact γ subunit in trifluoroethanol and water/liposomes by circular dichroism and Fourier‐transform infrared (FTIR) spectroscopy. In trifluoroethanol, the FTIR amide I′ frequencies were consistent with two predominant conformational components, the β‐turn and α‐helix, whilst in liposomes consisting of D2O and dimyristoylglycerophosphocholine (Myr2GroPCho), three components were observed. The third component present may contain some left‐handed extended helix. Spectral simulation was carried out to demonstrate that the CD spectra were consistent with the component conformations identified from FTIR spectroscopy. The simulated CD spectra were in excellent agreement with the experimental spectra. The intact γ subunit conformation in trifluoroethanol was shown to possess 72%α‐helical and 28%β‐turn conformations. In water/Myr2GroPCho liposomes the percentage of each conformational component present is 37%, 38% and 25% for the α‐helix, β‐turn and extended structures, respectively. Assuming that the transmembrane fragment was α‐helical, an excellent correlation was found between this derived α‐helical content in water/liposomes (37%) and from hydrophobicity plots where the percentage of amino acids in the transmembrane domain is predicted by others to be 34%. It is suggested that the β‐turn detected by CD and FTIR was attributable to a 310 helix rather than a type I or type III reverse turn.
Biochemical characterization of Epstein‐Barr virus nuclear antigen 2A and an associated ATPase activity
Authors: RANDAHL* Håkan , FÅHRAEUS Robin , KLEIN George
Published: March 2005

A partial purification of the Epstein‐Barr‐virus nuclear antigen 2A (EBNA 2A) protein from the Epstein‐Barr‐virus‐infected lymphoblastoid cell line, Cherry, has been designed. The main purification step was immunoaffinity chromatography, based on the mAb, 115E, directed towards the carboxy terminus of EBNA 2A. This was followed by chromatography over a Blue Sepharose column. According to silver‐stained SDS/PAGE, EBNA 2A was estimated to be 20% pure. The purified fractions contained an ATPase activity that was inhibited by the mAb 115E. Immunopurification of six EBNA‐2A‐positive cell lines and their negative counterpart showed that only fractions from EBNA‐2A‐positive lines contained ATPase activity. In gel‐filtration experiments EBNA 2A eluted as a 75‐kDa protein in conjunction with an ATPase activity. The EBNA 2A protein was covalently labeled by the ATP analog [14C]5′‐[p‐(fluorosulfonyl)benzoyl]adenosine. The ATPase activity was found to be optimal in the presence of 0.25 mM MgCl2 or CaCl2, whereas, in the presence of MnCl2 and ZnCl2, the activity was only about 50% of the control. High concentrations of Na2 VO3 and heparin do not interfere with the activity, while 2.5 mM NaF or 0.5 M NaCl give a 50% reduction of the activity. The Km for ATP and for GTP was 13 μM and 11 μM, respectively, and the Vmax for ATP was about six‐times higher than with GTP as substrate. Other low‐molecular‐mass non‐protein phosphate esters, such as phosphoserine or phosphothreonine inhibited the ATPase activity with a Ki of 18 and 32 μM, respectively. Phosphotyrosine had a Ki of 480 μM. Serine, threonine and tyrosine had no inhibitory effect on the ATPase activity.
EPR and redox characterization of iron‐sulfur centers in nitrate reductases A and Z from Escherichia coli
Authors: GUIGLIARELLI Bruno , ASSO Marcel , MORE Claude , AUGIER Valérie , BLASCO Francis , POMMIER Jeanine , GIORDANO Gérard , BERTRAND Patrick
Published: March 2005

The redox properties of the iron‐sulfur centers of the two nitrate reductases from Escherichia coli have been investigated by EPR spectroscopy. A detailed study of nitrate reductase A performed in the range + 200 mV to −500 mV shows that the four iron‐sulfur centers of the enzyme belong to two classes with markedly different redox potentials. The high‐potential group comprises a [3Fe–4S] and a [4Fe–4S] cluster whose midpoint potentials are +60 mV and +80 mV, respectively. Although these centers are magnetically isolated, they are coupled by a significant anticooperative redox interaction of about 50 mV. The [4Fe–4S]1+ center occurs in two different conformations as shown by its composite EPR spectrum. The low‐potential group contains two [4Fe–4S] clusters with more typical redox potentials (−200 mV and −400 mV). In the fully reduced state, the three [4Fe–4S]1+ centers are magnetically coupled, leading to a broad featureless spectrum. The redox behaviour of the high‐pH EPR signal given by the molybdenum cofactor was also studied. The iron‐sulfur centers of the second nitrate reductase of E. coli, nitrate reductase Z, exhibit essentially the same characteristics than those of nitrate reductase A, except that the midpoint potentials of the high‐potential centers appear negatively shifted by about 100 mV. From the comparison between the redox centers of nitrate reductase and of dimethylsulfoxide reductase, a correspondence between the high‐potential iron‐sulfur clusters of the two enzymes can be proposed.
Isolation and characterization of a novel type of growth factor derived from serum‐free conditioned medium of chicken embryo fibroblasts
Authors: GEISTLICH Andreas , GEHRING Heinz
Published: March 2005

A strong mitogenic activity for fibroblastic cells has been found in serum‐free medium of growtharrested primary cultures of chicken embryo fibroblasts (CEF). This serum‐free conditioned medium promoted growth of NIH/3T3 cells and primary as well as secondary cultures of CEF. The mitogenic activity was as potent as 5% serum. Half‐maximum stimulation was obtained with 20% of the initial concentration of the conditioned medium. The activity eluted at high Mr (1–2 × 105) from a gelfiltration column under nondenaturing conditions and was trypsin insensitive and thiol insensitive. Treatment with acid or urea converted the mitogen to a low‐molecular‐mass form, which showed a delayed induction of DNA synthesis. Purification of this factor (10 000‐fold) to apparent homogeneity was achieved by preparative isoelectric focusing, gel filtration, reverse‐phase HPLC and nonreducing SDS/PAGE. The factor, termed CEF‐derived growth factor (CDGF) was a 32‐kDa, disulfide‐linked heterodimer of a 15‐kDa and a 17‐kDa subunit as judged by SDS/PAGE, with a pI of approximately 7 in 8 M urea. It exhibited partial stability towards heat treatment and was trypsin sensitive. CDGF was active only in its dimeric form and half‐maximum stimulation of NIH/3T3 cells was obtained at approximately 10 pM. The mitogenic activity was not suppressible by an antibody neutralizing the activity of transforming growth factor β 1, 2 and 3 (TGF‐β). The physico‐chemical properties suggest that CDGF is not identical with one of the common growth factors like fibroblast growth factor, platelet‐derived growth factor, epidermal growth factor, insulin‐like growth factor, or TGF‐β but rather represents a novel type of growth factor.
Futile cycling of glycogen in Fibrobacter succinogenes as shown by in situ1H‐NMR and 13C‐NMR investigation
Authors: GAUDET Geneviève , FORANO Evelyne , DAUPHIN Gérard , DELORT Anne‐Marie
Published: March 2005

Glycogen was synthesized during all the growth phases in the rumen anaerobic cellulolytic bacterium Fibrobacter succinogenes. Glycogen synthesis and degradation were monitored using in situ13C and 1H‐NMR spectroscopy in resting cells of F. succinogenes. The cells were incubated at 37°C under anaerobic conditions with [1‐13C]glucose and [2‐13C]glucose. 1H‐NMR spectra were used to quantify enrichment by 13C of metabolism products. Glucose was utilized for energy requirements of the bacterium essentially via the Embden‐Meyerhof pathway, leading to the synthesis of succinate and acetate, while glycogen was stored. From [1‐13C]glucose, labeling occurred on C2 of succinate and acetate, and on both C1 and C6 of glycogen, the labeling on C1 being predominant. The C6‐labeling of glycogen may be explained by scrambling and reversal of the glycolytic pathway at the triose‐phosphate and fructose 1,6‐bisphosphate level. When the bacteria were incubated first with [1‐13C]glucose, then washed and incubated with [2‐13C]glucose, the pattern of 13C labeling in the products of the metabolism, as shown by 13C and 1H‐NMR spectra, indicated that glycogen was degraded at the same time as it was being stored, suggesting futile cycling of glycogen. The hydrolysis of previously stored glycogen can provide, in the presence of glucose, up to 30% of the carbon source for the bacteria.
Distinct forms of human CDC2 identified by novel monoclonal antibodies
Authors: LUKÁŠ Jiří , DRAETTA Giulio , BARTEK Jiří
Published: March 2005

Studies on the functional and structural properties of the cdc2 kinase, a key cell‐cycle regulator, have been possible thanks to the availability of cdc2‐specific immunoreagents. In an attempt to elucidate the biochemical regulation of the cdc2 kinase in more detail, we have raised a series of novel mouse monoclonal antibodies against human recombinant cdc2 protein. The five Mab reported here can be subclassified into two groups according to their interspecies cross‐reactivity and distinct immunoprecipitation patterns. Thus, the target epitopes of Mab POH‐1, POH‐2 and POH‐7 (group 1) appear to be limited to a few mammalian species and the fraction of cdc2 immunoprecipitable by these Mab from cellular extracts is considerably enhanced by denaturation. In contrast, the POH‐3 and POH‐8 (group 2) Mab recognize a denaturation‐sensitive epitope on cdc2 which is present in all tested mammalian species. More importantly, each of the two groups of Mab immunoprecipitate forms of cdc2 associated with a characteristic set of cellular proteins, none of which appears to be cyclin A or cyclin B. None of the antibodies precipitated a histone‐H1 or casein‐kinase activity, although an activity which phosphorylated some of the coprecipitated proteins was coprecipitated with the group 2 Mab. These novel Mab did not interfere with the association of cdc2 with cyclin A in vitro and efficient immunoprecipitation of a panel of cdc2 mutant proteins suggests that the target epitopes may not involve amino acid residues essential for currently known cdc2 functions. The results of the present study provide evidence for the existence of additional forms of the cdc2 protein in exponentially growing human cells, distinct from both the monomeric and the cyclin‐bound cdc2 identified so far.
A hybrid protein of urokinase growth‐factor domain and plasminogen‐activator inhibitor type 2 inhibits urokinase activity and binds to the urokinase receptor
Authors: BALLANCE D. James , MARSHALL Julian M. , COTTINGHAM Ian R. , STEVEN John , BERRY Sarah J. , CEDERHOLM‐WILLIAMS Stewart A. , GOODEY Andrew R. , COURTNEY Michael
Published: March 2005

The binding of urokinase‐type plasminogen activator (uPA) to its specific cell‐surface receptor (uPAR) localises the proteolytic cascade initiated by uPA to the pericellular environment. Inhibition of uPA activity or prevention of uPA binding to uPAR might have a beneficial effect on disease states wherein this activity is deregulated, e.g. cancer and some inflammatory diseases. To this end, a bifunctional hybrid molecule consisting of the uPAR‐binding growth‐factor domain of uPA (amino acids 1–47; GFuPA) at the N‐terminus of plasminogen‐activator inhibitor type 2 (PAI‐2) was produced in Saccharomyces cerevisiae. The purified protein inhibited uPA with kinetics similar to placental or recombinant PAI‐2 and was also found to bind to U937 cells and to FL amnion cells. GFuPA–PAI‐2 competed with uPA, the N‐terminal fragment of uPA and a proteolytic fragment of uPA (amino acids 4–43) in cell binding experiments, indicating that the molecule bound to the cells via uPAR. Hence, both the uPA‐inhibitory and uPAR‐binding domains of the hybrid molecule were functional, demonstrating the feasibility of the novel concept of introducing an unrelated, functional domain onto a member of the serine‐protease‐inhibitor superfamily.
Generation of specific antibodies against the rap1A, rap1B and rap2 small GTP‐binding proteins
Authors: KLINZ Franz‐Josef , SEIFERT Roland , SCHWANER Ingo , GAUSEPOHL Heinrich , FRANK Rainer , SCHULTZ Günter
Published: March 2005

Specific antibodies against rap1A and rap1B small GTP‐binding proteins were generated by immunization of rabbits with peptides derived from the C‐terminus of the processed proteins. Immunoblot analysis of membranes from several mammalian cell lines and human thrombocytes with affinity‐purified antibodies against rap1A or rap1B demonstrated the presence of multiple immunoreactive proteins in the 22–23 kDa range, although at strongly varying levels. Whereas both proteins were present in substantial amounts in membranes from myelocytic HL‐60, K‐562 and HEL cells, they were hardly detectable in membranes from lymphoma U‐937 and S49.1 cyc− cells. Membranes from human thrombocytes and 3T3‐Swiss Albino fibroblasts showed strong rap1B immunoreactivity, whereas rap1A protein was present in much lower amounts. In the cytosol of HL‐60 cells, only small amounts of rap1A and rap1B proteins were detected, unless the cells were treated with lovastatin, an inhibitor of hydroxymethylglutaryl‐coenzyme A reductase, suggesting that both proteins are isoprenylated. By comparison with recombinant proteins, the ratio of rap1A/ras proteins in membranes from HL‐60 cells was estimated to be about 4:1. An antiserum directed against the C‐terminus of rap2 reacted strongly with recombinant rap2, but not with membranes from tested mammalian cells. In conclusion, rap1A and rap1B proteins are distributed differentially among membranes from various mammalian cell types and are isoprenylated in HL‐60 cells.
The multimeric structure and disulfide‐bonding pattern of bovine κ‐casein
Authors: RASMUSSEN Lone K. , HØJRUP Peter , PETERSEN Torben E.
Published: March 2005

Bovine κ‐casein was analyzed by SDS/PAGE, MS and amino acid sequence analysis in order to determine its multimeric composition and disulfide‐bonding pattern. SDS/PAGE revealed that κ‐casein in the native state can range in size from a monomer to a multimeric structure larger than a decamer. Three types of interchain disulfide linkage, Cys11–Cys11, Cys11–Cys88 and Cys88–Cys88, were all assigned in multimers purified from [14C]carboxymethylated and untreated bulk milk, as well as a milk sample from a κ‐casein‐variant‐B homozygote Co20. These results indicate that multimerization occurs in a random or at present unpredictable disulfide‐bonding pattern regardless of the size of the multimer or the genotype.
ADP receptor‐induced activation of guanine‐nucleotide‐binding proteins in human platelet membranes
Authors: GACHET Christian , CAZENAVE Jean‐Pierre , OHLMANN Philippe , HILF Gerhard , WIELAND Thomas , JAKOBS Karl H.
Published: March 2005

ADP receptor‐regulated binding of the labeled GTP analog, guanosine 5′‐O‐(3‐[35S]thiotriphosphate) ([35S]GTP[γS]), to guanine‐nucleotide‐binding proteins (G proteins) was studied in human platelet membranes. The potent ADP receptor agonist, 2‐methyl‐thio‐adenosine 5′‐diphosphate (2MeSADP), a non‐hydrolyzable analog of ADP, increased the binding of [35S]GTP[γS] without apparent lag phase. Under optimal conditions, i.e. in the presence of GDP (1–10 μM), 2MeSADP increased the binding up to about threefold, with half‐maximal and maximal increase observed at 10 nM and 1 μM 2MeSADP, respectively. ADP itself increased the binding of [35S]GTP[γS] by maximally about twofold, with half‐maximal increase occurring at 0.1 μM ADP. The agonist‐induced stimulation was competitively antagonized by the ADP receptor(s) antagonist, (1S)‐adenosine 5′‐O‐(1‐thiotriphosphate) {(Sp)‐ATP[αS]}. Other platelet receptor agonists known to act through receptors coupled to G proteins also increased binding of [35S]GTP[γS] in human platelet membranes, but without being inhibited by (Sp)‐ATP[αS]. The data presented indicate that the platelet ADP receptor(s) can interact with and efficiently activate G proteins, the nature of which remains to be identified.
Large‐scale purification and characterisation of a recombinant epidermal growth‐factor receptor protein‐tyrosine kinase
Authors: McGLYNN Elaine , BECKER Michael , METT Helmut , REUTENER Sarah , COZENS Robert , LYDON Nicholas B.
Published: March 2005

The human epidermal‐growth‐factor receptor (EGF‐R) is a 170‐kDa transmembrane glycoprotein that mediates the mitogenic response of cells to EGF and transforming growth factor α. Culture conditions have been developed for the large‐scale expression of the cytoplasmic domain of the EGF‐R in insect cells using a recombinant baculovirus. From 6 l Sf9 cells, grown to high density using a bioreactor, 20 mg of the EGF‐R kinase was purified to greater than 95% purity. Purification, which was carried out in the absence of detergents using classical purification methods, yielded an EGF‐R protein that was not phosphorylated on tyrosine. This procedure has enabled us to produce high quality enzyme for both structural and biochemical studies on the EGF‐R kinase. The in vitro activity of the cytoplasmic domain of the EGF‐R kinase was modulated by multiple assay factors which include substrates, divalent cations and conformational modulators. Kinetic analysis in the presence of Mn2+ gave an apparent Vmax value of 20 nmol min−1 mg−1 and Km values of 4.5 μM for ATP and 1.43 mM for angiotensin II. This corresponds to a turnover number of 1.4 mol min−1 mol−1. Ammonium sulfate (1 M) resulted in an eightfold stimulation of kinase activity when assayed using angiotensin II as substrate. The specific activity of the intracellular domain of the EGF‐R, when assayed at 20°C in the presence of 1M ammonium sulfate, was 160 nmol min−1 mg−1. Activation of the EGF‐R kinase by ammonium sulfate was found to be substrate‐specific. No activation was found when assayed using polymeric substrates. Addition of Me2+‐ATP to the purified enzyme resulted in autophosphorylation and was accompanied by retardation of SDS/PAGE migration. Kinetic constants and metal ion preferences of a number of co‐polymers and peptide substrates have been compared. Dramatic differences in kinetic constants were found which were dependent on both the substrate and metal ion used. Activation of EGF‐R autophosphorylation was found to be influenced by the use of charged polymers. The random polymer of Glu, Lys, Ala, Tyr (2:5:6:1), which was not phosphorylated by the EGF‐R kinase, dramatically activates autophosphorylation of the EGF‐R. Thus the intracellular domain of the EGF‐R appears to be in a low‐activity conformation which, under appropriate assay conditions, can be activated to a similar specific activity to that reported for the purified EGF‐R holoenzyme.
Characterization of yeast‐expressed β‐actins, site‐specifically mutated at the tumor‐related residue Gly245
Authors: ASPENSTRÖM Pontus , ENGKVIST Hans , LINDBERG Uno , KARLSSON Roger
Published: March 2005

The tumorigenic cell line HUT14 expresses a β‐actin carrying a mutation at position 245. In this study, two mutant β‐actins with amino acid changes at position 245 replacing the wild‐type glycine by an aspartic acid and a lysine residue, respectively, were produced in the yeast Saccharomyces cerevisiae, purified to homogeneity and characterized with respect to polymerization behaviour and interaction with myosin. The major functional effect of these mutations appears to be an impaired polymerization, while the interaction with myosin seems less influenced. In addition, the results also suggest the presence of a Ca2+‐binding site in the region of residue 245 in actin.