bluuebunny/crossref_metadata_2025_split huggingface dataset:
Esterified cholesterol and triglyceride are present in plasma membranes of Chinese hamster ovary cells
Authors: MACKINNON Wanda B. , MAY George L. , MOUNTFORD Carolyn E. 
Published: March 2005

The chemical composition of highly purified plasma membrane preparations from a series of malignant Chinese hamster ovary (CHO) cell lines were undertaken to ascertain if neutral lipid, including cholesteryl ester and triacylglycerol, were present. Triacylglycerols (33–41 nmol/mg total lipid) and cholesteryl ester (226–271 nmol/mg) were measured in the plasma membranes and differences in the chemical composition of these membranes recorded. The most significant difference was a gradual decrease in the level of free cholesterol from wild type (312 ± 7 nmol/mg total plasma membrane lipid), Pod RII‐6 (268 ± 64 nmol/mg total plasma membrane lipid), Col R‐22 (243 ± 39 nmol/mg total plasma membrane lipid) to EOT (204 ± 20 nmol/mg total plasma membrane lipid), with a concomitant increase in the degree of saturation of the cholesteryl ester fatty acids, particularly palmitic acid. No statistically significant differences were apparent in the chemical composition of the whole cells in this series.The one‐dimensional (1D) 1H‐NMR spectra of the four malignant cell lines showed a gradation in intensity of lipid resonances, in the order of wild type, Pod RII‐6, Col R‐22 and EOT, with EOT having the strongest lipid spectrum. Interestingly, the increase in acyl‐chain signal intensities in the 1H‐NMR spectra of this series of CHO cells and emergence of signals from cholesterol and/or cholesteryl ester, coincide with alterations in the amount of free choelsterol and the degree of saturation of the fatty‐acyl chain of the esterified cholesterol in the plasma membranes.It is our hypothesis that, together, cholesteryl ester and triacylglycerol form domains in the plasma membrane and that when the cholesteryl ester has a largely saturated fatty acid content, the lipids are in isotropic liquid phase and hence visible by NMR.
The effect of polylysine on casein‐kinase‐2 activity is influenced by both the structure of the protein/peptide substrates and the subunit composition of the enzyme
Authors: MEGGIO Flavio , BOLDYREFF Brigitte , MARIN Oriano , MARCHIORI Fernando , PERICH John W. , ISSINGER Olaf‐G. , PINNA Lorenzo A. 
Published: March 2005

The mechanism by which polybasic peptides stimulate the activity of casein kinase 2 (CK2) has been studied by comparing the effect of polylysine on the phosphorylation of a variety of protein and peptide substrates by the native CK2 holoenzyme and by its recombinant catalytic α subunit, either alone or in combination with the recombinant non‐catalytic β subunit.Calmodulin is not phosphorylated by the CK2 holoenzyme, in either the native or the reconstituted form, unless polylysine is added. In the presence of polylysine, it becames a good substrate for CK2 (Km, 14.2 μM, Kcat 4.6 mol · min−1· mol CK2−1). The recombinant α subunit, however, spontaneously phosphorylates calmodulin, this phosphorylation being actually inhibited rather than stimulated by polylysine. The calmodulin tridecapeptide, RKMKDTDSEEEIR, reproducing the phosphorylation site for CK2, is spontaneously phosphorylated by either CK2 holoenzyme or the recombinant α subunit with 5.8‐fold and 2.8‐fold stimulation by polylysine, respectively.The recombinant β subunit of CK2 is itself a good exogenous substrate for the enzyme, its phosphorylation, however, is inhibited rather than enhanced by polylysine. On the contrary, the phosphorylation of the nonapeptide, MSSSEEVSW, reproducing the β‐subunit phosphoacceptor site, is dramatically stimulated by polylysine.Using a variety of small peptide substrates, it was shown that phosphorylation rate is diversely stimulated by polylysine. The observed stimulation, moreover, is variably accounted for by changes in Vm and/or Km depending on the structure of the peptide substrate. Maximum stimulation with all protein/peptide substrates tested requires the presence of the β subunit, since the recombinant a subunit is much less responsive than CK2 holoenzyme, either native or reconstituted. While the phosphorylation of the peptide RRRDDDSDDD by CK2 is stimulated 2.8‐fold, with 15 nM polylysine being required for half‐maximal stimulation, a stimulation of only 1 Sfold, with 80 nM polylysine required for half‐maximal stimulation, is attained with recombinant α subunit. The concentration of polylysine required for half‐maximal stimulation is comparable to CK2 concentration and increases by increasing CK2 concentration, suggesting that polylysine primarily interacts with the enzyme, rather than with the peptide substrate.
Structural and redox relationships between Paracoccus denitrificans, porcine and human electron‐transferring flavoproteins
Authors: WATMOUGH Nicholas J. , KISS Julie , FRERMAN Frank E. 
Published: March 2005

Electron‐transferring flavoprotein (ETF) was purified from the bacterium Paracoccus denitrificans and the structural and redox relationships to the porcine and human ETFs were investigated. The three proteins have essentially identical subunit masses and the α‐helix content of the bacterial and porcine ETFs are very similar, indicating global structural similarity. An anti‐(porcine ETF) polyclonal antibody that crossreacts with the human large and small subunits also crossreacts strongly with the large subunit of Paracoccus ETF. However, crossreactivity with the small subunit is very weak. Nonetheless, an amino‐terminal peptide and four internal peptides of the small bacterial subunit show extensive sequence identity with the human small subunit. Local similarities in environment are also indicated by the intrinsic tryptophan fluorescence emission spectra of porcine and Parucoccus ETFs.Although the visible spectra of porcine and Paracoccus ETFs are virtually identical, flavin fluorescence in the bacterial protein is only 15% that of the mammalian protein. Further, the circular dichroic spectrum of the flavin in the bacterial protein is significantly more intense, suggesting that the microenvironment of the isoalloxazine ring is different in the two proteins.Enzymatic or photochemical reduction of Paracoccus ETF rapidly yields an anionic semiquinone; formation of the fully reduced flavin in the bacterial ETF is very slow. The spacing of the oxidation‐reduction potentials of the flavin couples in the bacterial ETF is essentially identical to that in porcine ETF as judged from the disproportionation equilibrium of the bacterial ETF flavin semiquinone. Together, the enzymatic reduction and disproportionation equilibria suggest that the flavin potentials of the two ETFs must be very close.The data indicate that the structural properties of the bacterial and mammalian proteins and the thermodynamic properties of the flavin prosthetic group of the proteins are very similar.
Accuracy of wheat‐germ RNA polymerase II
Authors: de MERCOYROL Laure , CORDA Yves , JOB Claudette , JOB Dominique 
Published: March 2005

We investigated the accuracy of the insertion process in RNA chain elongation catalyzed by wheat germ RNA polymerase II. Error frequencies varied from 1 misinserted nucleotide per 250 polymerized correct substrates to less than 1 in 2 × 105, depending on template sequence and nature of the divalent metal cofactor. Higher error ratios were observed in the presence of Mn2+ compared to Mg2+, and with alternating poly[d(G‐C)] · poly[d(G‐C)] compared to poly[d(A‐T)] · poly[d(A‐T)]. In this latter case the eukaryotic RNA polymerase was as accurate as Escherichia coli RNA polymerase holoenzyme.The fidelity of wheat germ RNA polymerase II was also examined in transcription of polynucleotide templates in the poly[d(G‐C)] family adopting either the right‐handed B or left‐handed Z conformations. Error ratios for noncomplementary ATP increased markedly under experimental conditions favoring the B‐to‐Z conformational transition of the alternating copolymers. In accordance with the results of previous studies, the rate of productive elongation, i.e. the synthesis of poly[r(G‐C)], was depressed, suggesting that the decreased accuracy of the enzyme derived from an altered competence of the enzyme to form elongation complexes on the left‐handed DNA. As judged by the large difference in apparent Km values of the enzyme for complementary and noncomplementary nucleoside triphosphates, part of the discrimination between substrates seemed to take place at the initial binding step. Furthermore, the results indicate that wheat germ RNA polymerase II was able to elongate a primer with a 3′‐terminal mismatch, and thus to incorporate the mismatched nucleotide stably in the nascent RNA. However, the probability of productive RNA chain elongation was much lower with noncognate than with the complementary substrates.
Mouse liver cytidine‐5′‐monophosphate‐N‐acetylneuraminic acid hydroxylase
Authors: SHAW Lee , SCHNECKENBURGER Petra , CARLSEN Jens , CHRISTIANSEN Kirsten , SCHAUER Roland 
Published: March 2005

In this paper, we present the results of an investigation into the catalytic properties of CMP‐Neu5Ac hydroxylase (Neu5Ac: N‐acetylneuraminic acid) in high‐speed supernatants of mouse liver. The enzyme was most active in Hepes/NaOH pH 7.4 and was markedly inhibited by relatively small increases in ionic strength, though the inhibition was abolished by desalting procedures. Several nonionic detergents could activate the hydroxylase to various degrees in a concentration‐dependent manner. Ionic detergents and a number of phospholipids were, however, generally inert or inhibitory.The lack of inhibitory influence of a wide range of nucleotides revealed that CMP‐Neu5Ac hydroxylase binds its sugar‐nucleotide substrate with a high degree of specificity. Thus, even millimolar concentrations of several cytidine nucleotides elicited virtually negligible inhibition, though the reaction product, CMP‐Neu5Gc (Neu5Gc: N‐glycoloylneuraminic acid), was a weak inhibitor. The results also indicate that the enzyme is not regulated by any nuceotides or sugar‐nucleotides.Dilution of high‐speed supernatants with buffer gave rise to a decrease in the specific activity of the hydroxylase, implicating the involvement of more than one component in catalysis. Activity could be restored by the addition of a heat extract of the supernatant. The active principle in this extract was found to be a heat‐stable protein with a molecular mass of about 17 kDa. Immunochemical studies allowed this protein to be identified as cytochrome b5 and it was shown that this electron carrier is essential for the activity of CMP‐Neu5Ac hydroxylase.Inhibition studies using iron ligands and activation by exogenous iron salts suggest the involvement of a non‐haem iron cofactor in the catalytic cycle of this hydroxylase. Cytochrome b5 may thus serve as an electron donor for this postulated cofactor.
Sequence of horse pancreatic lipase as determined by protein and cDNA sequencing
Authors: KERFELEC Brigitte , FOGLIZZO Edith , BONICEL Jacques , BOUGIS Pierre E. , CHAPUS Catherine 
Published: March 2005

The complete sequence of the horse pancreatic lipase was elucidated by combining polypeptide chain and cDNA sequencing. Among the structural features of horse lipase, it is worth mentioning that Lys373 is not conserved. This residue, which is present in human, porcine and canine lipases, has been assumed to be involved in p‐nitrophenyl acetate hydrolysis by pancreatic lipases. Kinetic investigation of the p‐nitrophenyl acetate hydrolysis by the various pancreatic lipases and by the C‐terminal domain (336–449) of human lipase reveals that this hydrolysis is the result of the superimposition of independant events; a specific linear hydrolysis occurring at the active site of lipase, a fast acylation depending on the presence of Lys373 and a non‐specific hydrolysis most likely occurring in the C‐terminal domain of the enzyme. This finding definitely proves that pancreatic lipase bears only one active site and raises the question of a covalent catalysis by pancreatic lipases.Moreover, based on sequence comparison with the above‐mentioned pancreatic lipases, three residues located in the C‐terminal domain, Lys349, Lys398 and Lys419, are proposed as possible candidates for lipase/colipase binding.
YmL9, a nucleus‐encoded mitochondrial ribosomal protein of yeast, is homologous to L3 ribosomal proteins from all natural kingdoms and photosynthetic organelles
Authors: GRAACK Hanns‐Rüdiger , GROHMANN Lutz , KITAKAWA Madoka , SCHÄFER Karl‐Ludwig , KRUFT Volker 
Published: March 2005

The nuclear gene for mitochondrial ribosomal protein YmL9 (MRP‐L9) of yeast has been cloned and sequenced. The deduced amino acid sequence characterizes YmL9 as a basic (net charge + 30) protein of 27.5 kDa with a putative signal peptide for mitochondrial import of 19 amino acid residues. The intact MRP‐L9 gene is essential for mitochondrial function and is located on chromosome XV or VII.YmL9 shows significant sequence similarities to Escherichia coli ribosomal protein L3 and related proteins from various organisms of all three natural kingdoms as well as photosynthetic organelles (cyanelles). The observed structural conservation is located mostly in the C‐terminal half and is independent of the intracellular location of the corresponding genes [Graack, H.‐R., Grohmann, L. & Kitakawa, M. (1990) Biol. Chem. Hoppe Seyler 371, 787–788]. YmL9 shows the highest degree of sequence similarity to its eubacterial and cyanelle homologues and is less related to the archaebacterial or eukaryotic cytoplasmic ribosomal proteins. Due to their high sequence similarity to the YmL9 protein two mammalian cytoplasmic ribosomal proteins [MRL3 human and rat; Ou, J.‐H., Yen, T. S. B., Wang, Y.‐F., Kam, W. K. & Rutter, W. J. (1987) Nucleic Acids Res. 15, 8919–8934] are postulated to be true nucleus‐encoded mitochondrial ribosomal proteins.
(R)‐Lactyl‐CoA dehydratase from Clostridium propionicum
Authors: HOFMEISTER Antje E. M. , BUCKEL Wolfgang 
Published: March 2005

A new two‐step method for purifying component EII of lactyl‐CoA dehydratase was developed. The source fo the enzyme was Clostridium propionicum grown on either d,l‐alanine or l‐threonine. No difference in these preparations was observed whether during purification or by SDS/PAGE of the pure enzymes. Bothe preparations exhibited similar activities towards (R)‐lactyl‐CoA as well as towards (R)‐2‐hydroxbutyryl‐CoA, the latter being the superior substrate.
Three species of (2R)‐2‐hydroxybutyrate labelled with 3H at C3 were prepared containing 96%, 37% and 63% of the 3H in the 3S‐position. By incubation of these species with acetyl‐CoA, propionate CoA‐transferse and lactyl‐CoA dehydratase 104%, 32% and 70% of the 3H, respectively, was released as 3HOH. The data indicate the stereospecific abstraction of the 3Si hydrogin of (2R)‐2‐hydroxybutyryl‐CoA during the dehydration.
The identity of the product of the dehydration as crotonyl‐CoA was established by the combined action of the enzymes crotonase and (S)‐3‐hydroxybutyryl‐CoA dehydrogenase. The results indicate that the elimination of water from (R)‐2‐hydroxybutyryl‐CoA occurs in a syn mode.
All enzyme activities necessary for the conversion of l‐threonine via (R)‐2‐hydroxybutyryl‐CoA to butyrate were detected in cell‐free extracts of C.propionicum.
A new mechanism for the dehydration of lactyl‐CoA is proposed.
The plasma membrane of yeast acquires a novel heat‐shock protein (hsp30) and displays a decline in proton‐pumping ATPase levels in response to both heat shock and the entry to stationary phase
Authors: PANARETOU Barry , PIPER Peter W. 
Published: March 2005

Recent studies have revealed that the action of the proton‐translocating ATPase of the plasma membrane of yeast is an important determinant of several stress tolerances and affects the capacity of cells to synthesise heat shock proteins in response to heat shock [Panaretou, B. & Piper, P. W. (1990) J. Gen. Microbiol. 136, 1763–1770; Coote, P. J., Cole, M. B. & Jones, M. V. (1991) J. Gen. Microbiol. 137, 1701–1708]. This study investigated the changes to the protein composition of the Saccharomyces cerevisiae plasma membrane that result from a heat shock to dividing cultures and the entry to stationary growth caused by carbon source limitation. Plasma membranes were prepared from exponential, heat‐shocked and stationary yeast cultures. The proteins of these membrane preparations were then analysed by polyacrylamide gel electrophoresis and immunoblot measurement of ATPase levels.The protein composition of plasma membranes displayed two prominent changes in response to both heat shock and the entry to stationary phase: (a) a reduction in the level of the plasma membrane ATPase; and (b) the acquisition of a previously uncharacterised 30 kDa heat‐shock protein (hsp30). The ATPase decline with heat shock probably exerts an important influence over the ability of the cell to maintain ATPase activity, and therefore intracellular pH, during extended periods of stress. Through in vivo pulse‐labelling of plasma membrane proteins synthesised before and during heat shock, followed by subcellular fractionation, it was shown that hsp30 is the only protein induced by the yeast heat‐shock response that substantially copurifies with plasma membranes. It might therefore exert a stress‐protective function specifically at this membrane.
Mitochondrial dolichyl‐phosphate mannose synthase
Authors: GASNIER Françoise , ROUSSON Robert , LERME Fabienne , VAGANAY Elisabeth , LOUISOT Pierre , GATEAU‐ROESCH Odile 
Published: March 2005

Mitochondrial dolichyl‐phosphate mannose synthase has been purified to homogeneity using an original procedure, reconstitution into specific phospholipid vesicles and sedimentation on a sucrose gradient as final step.The enzyme has an apparent molecular mass of 30 kDa on an SDS/polyacrylamide gel. Increased enzyme activity could be correlated with this polypeptide band.A specific antibody was raised in rabbits against this transferase. Specific IgG obtained from the immune serum removed enzymatic activity from a detergent extract of mitochondrial outer membrane and reacted specifically with the 30‐kDa band on immunoblots. Furthermore, an immunocytochemical experiment proved the localization of dolichyl‐phosphate mannose synthase on the cytosolic face of the outer membrane of mitochondria.
A novel cyclic analog of neuropeptide Y specific for the Y2 receptor
Authors: BECK‐SICKINGER Annette G. , GROUZMANN Eric , HOFFMANN Eike , GAIDA Wolfram , VAN MEIR Erwin G. , WAEBER Bernard , JUNG Günther 
Published: March 2005

The low‐molecular‐mass, cyclic analog of neuropeptide Y, [Ahx5–24, γ‐Glu2‐ɛ‐Lys30] NPY (; Ahx, 6‐aminohexanoic acid; NPY, neuropeptide Y), was synthesized and investigated for receptor binding, inhibition of forskolin‐stimulated cAMP accumulation, inhibition of electrically stimulated rat vas deferens contractions and ability to increase blood pressure. Like the linear peptide [Ahx5–24] NPY (YPSK‐Ahx‐RHYINLITRQRY), the more rigid, cyclic analog showed good correlation between receptor binding to rabbit kidney membranes and biological activity in the vas deferens assay. Binding of this peptide to a new Y2‐receptor‐expressing cell line was slightly reduced, compared to the linear peptide [Ahx5–24] NPY, however inhibition of cAMP accumulation was even more efficient.Unlike the linear peptide [Ahx5–24] NPY, the cyclic analog did not induce a blood pressure increase in rats. Reduced binding to Y1 receptor‐expressing SK‐N‐MC cells, as well as the loss of capability of signal transduction, suggest that only Y2‐mediated activity is preserved after cyclization. The selectivity of the cyclic compound for Y2 subtypes of NPY receptors with respect to inhibition of cAMP accumulation is more than fortyfold increased, as compared to the linear NPY‐(13–36) peptide, which has been used to determine Y2 selectivity so far.
DNase‐I hypersensitivity analysis of the L‐type pyruvate kinase gene in rats and transgenic mice
Authors: BOQUET Didier , VAULONT Sophie , TREMP Günter , RIPOCHE Marie‐Anne , DAEGELEN Dominique , JAMI Jacques , KAHN Axel , RAYMONDJEAN Michel 
Published: March 2005

The rat L‐type pyruvate kinase gene possesses two promoters located 500 bp apart. The L′ promoter is specific to erythroid cells. The L promoter is specific to liver and is regulated by diet and hormones; positively by glucose and insulin and negatively by glucagon via cAMP. The DNA elements involved in this tissue‐specific and hormone‐regulated gene expression are located within 3.2 kbp of 5′ flanking region as previously demonstrated by transgenic mice analysis [Tremp, G. L., Boquet, D., Ripoche, M. A., Cognet, M., Yu‐Chun, L., Jami, J., Kahn, A. and Daegelen, D. (1989) J. Biol. Chem. 264, 19 904–19 910]. Moreover, we have observed in these mice that gene expression was dependent on the transgene copy number and independent of the integration site. We present here DNase‐I‐hypersensitivity analysis of the endogenous rat L‐type pyruvate kinase gene and of two transgene constructs in relation to development, tissue differentiation, nutritional and hormonal status. In rats, two groups of proximal sites were detected on the endogenous gene; hypersensitive site (HSS) HSS‐1 in adult liver and HSS‐A in fetal liver (a major erythropoietic tissue). Both groups are probably related to the transcriptional initiation complexes at either the L or L′ promoter. Two other distal groups were detected; HSS‐2 at −3 kbp (with respect to the liver‐specific cap site) in adult liver and HSS‐B around −4 kbp in fetal liver. These sites are thought to correspond to activating sequences; in adult liver, deletion of a fragment encompassing HSS‐2 provokes a dramatic reduction of transcription starting at the L promoter of the transgene. In adult liver, HSS‐1 appears to be a transcription‐associated site, being greatly weakened in fasted rats, while HSS‐2 is transcription independent.The pattern of DNase‐I hypersensitivity is similar for the rat endogenous gene and for the complete rat transgene; the liver‐specific HSS‐1 and HSS‐2 are present and the intensity of the sites is correlated to the number of integrated copies. Interestingly, HSS‐1 is still detectable and its intensity remains proportional to the number of integrated copies in a truncated transgene with HSS‐2 deletion, while this transgene is very weakly (but nevertheless tissue‐specifically) expressed. These results strongly suggest that each transgene copy possesses a complete set of specific nucleoprotein complexes and that, with or without HSS‐2, the DNA is in a potentially active configuration.
The cholesterol‐side‐chain‐cleaving cytochrome P450 spin‐state equilibrium
Authors: LANGE Reinhard , LARROQUE Christian , ANZENBACHER Pavel 
Published: March 2005

We have investigated the spin‐state equilibrium of adrenal mitochondrial P450scc (cholesterol‐side‐chain‐cleaving, CYP11A1) by absorption spectroscopy in the Soret band as a function of pH and temperature. The van't Hoff plot of the high‐spin/low‐spin equilibrium is not linear and is shifted towards high spin by lowering the pH. This non‐linearity resolves clearly into two phases when the temperature range is extended from 37°C to −20°C using ethylene glycol as anti‐freeze cosolvent. This enabled us to measure the enthalpy and entropy changes which are ΔHA= 0.7 kJ · mol−1 and ΔSA= 5 J · K−1· mol−1 at low temperatures and ΔHB=−42 kJ · mol−1 and ΔSB=−152 J · K−1· mol−1 at high temperatures. The transition temperature, Tbreak, between both phases decreases as a function of pH.The experimental data can be fitted by a minimal reactional model comprising a temperature dependent conformational transition and two ionisation steps (one for each conformation), the pK of which is 1.5 ± 0.5 higher in the low‐temperature conformation. The deduced conformational equilibrium is affected by physiological effectors: Tbreak depends on the nature of the substrate intermediate and on the presence of the physiological electron donor, adrenodoxin.