bluuebunny/crossref_metadata_2025 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.
Characteristics of the α2/β‐adrenoceptor‐coupled adenylate cyclase system and their relationship with adrenergic responsiveness in hamster fat cells from different anatomical sites
Authors: DIEUDONNE Marie‐Noelle , PECQUERY Rene , GIUDICELLI Yves
Published: March 2005

Various studies have shown that the lipolytic response of white adipocytes to catecholamines was dependent on the anatomical origin of these cells. To provide a biological explanation for this phenomenon, we compared hamster white adipocytes, from femoral subcutaneous and epididymal fat, for their lipolytic activities, cAMP responses and adrenoceptor‐coupled adenylate cyclase system. Basal and maximal lipolytic responses to theβ‐adrenergic (isoproterenol) and the mixed α2/β‐adrenergic (epinephrine) agonists were lower in femoral subcutaneous cells than in epididymal cells, but the α2‐adrenergic antilipolytic response to 5‐bromo‐6‐(2‐imidazolin‐2‐ylamino)quinoxaline bitartate (UK 14304) was slightly greater in femoral subcutaneous fat cells than in epididymal fat cells. Identical results were observed for cAMP responses, except for the α2‐adrenergic inhibitory response which was identical in both fat deposits. Adrenoceptors studies revealed higher density of inhibitory α2‐adrenoceptors 2‐(2‐methoxy‐1,4‐benzodioxan‐2‐yl)‐2‐imidazoline ([3H]RX821002‐binding sites) in femoral subcutaneous fat cells than in epididymal fat cells, but identical density of stimulatoryβ‐adrenoceptors (125I‐cyanopindolol‐binding sites) and similar subdivision intoβ‐adrenoceptor subtypes in both adipose deposits. Finally, the level of the α‐subunits of the stimulatory and inhibitors guanine‐nucleotide‐binding regulatory proteins, as well as the adenylate cyclase catalytic activity were 40–50% lower in femoral subcutaneous fat cell membranes than in epididymal fat cell membranes. These results suggest that the differences in cAMP and lipolytic responses to catecholamines between epididymal and femoral subcutaneous adipocytes result at least in part from site‐related differences in the adenylate cyclase system rather than in the adrenoceptor status.
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.
Purification and characterization of a vimentin‐specific protease in mouse myeloid leukemia cells
Authors: NAKAMURA Nobuhiro , TSURU Akio , HIRAYOSHI Kazunori , NAGATA Kazuhiro
Published: March 2005

Strong vimentin‐degrading activity was found in a mouse myelomonocytic leukemic cell line, M1. When M1 cells were induced to differentiate into macrophage‐like cells, this degrading activity decreased, while expression of the vimentin gene increased as reported previously [Tsuru, A., Nakamura, N., Takayama, E., Suzuki, Y., Hirayoshi, K. and Nagata, K. (1990) J. Cell Biol. 110, 1655–1664]. This activity was not due to calpain, which was reported to degrade vimentin, because it was independent of the presence or absence of Ca2+. This activity was revealed to be strongly associated with membranes by differential‐centrifugation experiments. To identify this protease, purification of the degradation enzyme was performed. A membrane fraction was prepared and extracted with a buffer containing Triton X‐100, then subjected to column chromatography using carboxymethyl‐Sepharose and heparin‐Sepharose. Quantitative analysis using the purified protease revealed that the specificity of this protease was more than 1000‐fold higher for vimentin than for bovine serum albumin, ovalbumin and actin. Four protein bands expressing the activity were finally identified by SDS/PAGE. Amino‐terminal sequences of these four proteins were identical, suggesting lower‐molecular‐mass proteins were degradative products. Furthermore, it was revealed that the sequence had the highest similarity with that of human cathepsin G. This result was consistent with the cathepsin‐G‐like properties of the purified protease, such as the optimum pH and the specificities for inhibitors. The purified protease degraded a synthetic substrate for cathepsin G, succinyl‐alanyl‐alanyl‐prolyl‐phenylalanyl‐p‐nitroanilide, with a comparable specific activity to human cathepsin G and was specifically detected with anti‐(human cathepsin G) serum in immunoblot analysis. The purified protease thus belongs to the ‘cathepsin G family’, and perhaps is a mouse homologue of human cathepsin G.
Effect of removal and reconstitution of troponins C and I on the Ca2+‐activated tension development of single glycerinated rabbit skeletal muscle fibers
Authors: HATAKENAKA Masamitsu , OHTSUKI Iwao
Published: March 2005

The effect of troponin T treatment on the Ca2+‐activated tension of single glycerinated rabbit skeletal muscle fibers was examined. The tension of the fiber was completely desensitized to Ca2+ by incubation in a solution containing an excessive amount of troponin T and reached a level of about 70% of the maximum tension of the control fiber. SDS/PAGE showed that most of troponins C and I was removed from the fiber by troponin T treatment. During the course of troponin T treatment, the cooperativity of Ca2+ activation (Hill coefficient) was decreased while pCa at half‐maximal Ca2+‐sensitive tension @K) increased. Using the 26‐K fragment of troponin T, the study indicated that the removal of troponins C and I was due to the replacement of the troponin C · I · T complex in the myofibrils of the fiber with the added troponin T. The troponin‐T‐treated fiber was again sensitized to Ca2+ by the addition of troponin C · I. The removal of troponin C by treatment with trans‐1,2‐cyclohexanediamine‐N,N,N′,N′‐tetraacetic acid did not change the minimum tension of the fiber, from which troponin C · I was partially removed by troponin T treatment, but it decreased the height of maximum tension with a concomitant decrease in the Hill coefficient as well as a decrease in pK. The above findings suggested that pK is determined by the balance between two opposite actions through troponins C and I, while the extent of cooperativity of Ca2+ activation seemed to be related mainly to the content of troponin C.
An infrared study of 2H‐bond variation in myoglobin revealed by high pressure
Authors: LE TILLY Veronique , SIRE Olivier , ALPERT Bernard , WONG Patrick T. T.
Published: March 2005

A high‐pressure Fourier‐transform infrared technique was used to probe the evolution of 2H bonds inside the helical segments of myoglobin in relation to p2H, Tris concentration in the medium and iron‐ligand nature. The analysis was focused on changes in the conformation‐sensitive amide‐I′ band, reflecting the peptide C = O group stretching vibrations coupled to the in‐plane N‐2H bending and C=N stretching modes. From data obtained under high pressure, the strength of 2H bonds, inside the α‐helical segments of the protein at atmospheric pressure, is not simply a function of p2H and salt concentration. At low Tris concentration (50 mM), the strength of these 2H bonds increases with p2H, whereas for a higher Tris concentration (100 mM) this strength is lower at p2H 7 than at p2H 6.0 or 8.5. It is also observed that the azidometmyoglobin molecule exhibits tighter intrahelical interactions and lower sensitivity to pressure than aquametmyoglobin. Information is also presented regarding interhelical interactions in relation to the solvent.
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.
Expression, purification and biochemical characterisation of the human immunodificiency virus 1 nef gene product
Authors: WOLBER Vera , RENSLAND Hans , BRANDMEIER Birgit , SAGEMANN Martin , HOFFMANN Ralf , KALBITZER Hans Robert , WITTINGHOFER Alfred
Published: March 2005

The human immunodeficiency virus 1 (HIV‐1) nef gene encoded by the HIV‐1 isolate lymphadenopathy‐associated virus type 1 was expressed in Escherichia coli under the control of the tac promoter. The protein is found mainly in the soluble part of the bacterial lysate; a simple twocolumn purification scheme has been developed allowing isolation of the recombinant protein without using denaturing agents. Analysis of the circular dichroism spectra reveals that the purified protein is folded and has a helix content of 16% and a β‐pleated sheet content of 31%. GTPase activity and binding of guanine nucleotides were measured for Nef and compared with the results obtained under identical experimental conditions for p21rasC, which represents a typical, well‐characterized guaninenucleotide‐binding (GNB) protein. Within the limits of error, native Nef does not show GTPase activity and does not bind guanine nucleotides strongly (association constant, Kass <5 × 103 M−1). An upper limit for the association constant of Nef for ATP was determined by equilibrium dialysis as 5 × 103 M−1. Nef can be autophosphorylated by ATP; under the experimental conditions used, 1–2% of the protein become phosphorylated. Correspondingly, our Nef preparation shows a low, but significant, ATPase activity. In conclusion, Nef is not a member of the GNB protein family, but a possible role as a protein kinase cannot be excluded.
Purification and characterization of a binding protein related to the Z class of cytosolic proteins in guinea‐pig liver cytosol (guinea‐pig Z protein)
Authors: NICOLLIER Monique , ROBLIN Sylvie , CYPRIANI Benoit , REMY‐MARTIN Jean P. , ADESSI Gérard L.
Published: March 2005

The purification and characterization of a low‐molecular‐mass binding protein from female guinea‐pig liver cytosol is reported. Its molecular mass (14.4 kDa), amino acid composition, abundance and biological properties identify it as belonging to the Z class of liver cytosolic proteins [Levi, A. J., Gatmaitan, Z. & Arias, I. M. (1969) J. Clin. Invest. 48, 2956–2167]. Among the most important members of this class of proteins are the fatty‐acid‐binding proteins (FABPs) and the sterol carrier protein2 (SCP2). The guinea‐pig Z protein (G‐ZP) has some similarities in its amino acid composition and NH2‐terminal sequence with those of the rat liver FABP, but its isoelectric point is basic (pI 8.85), like that of SCP2. We also examined its binding affinities for a number of ligands bound by these two proteins. The results show that the purified G‐ZP binds dehydroepiandrosterone sulfate, estrone sulfate, oleic acid and cholesterol, but shows no affinity for free steroids such as estrone and DHEA. Thus it can be said that G‐ZP has some characteristics of FABPs and some of SCP2 but seems, however, to be different from both these proteins. The purified G‐ZP inhibits microsomal DHEA sulfate sulfatase activity in a mixed noncompetitive way. This protein could be involved in the transport and/or metabolism of sulfated steroids.
Specific in vitro O‐glycosylation of human granulocyte‐macrophage colony‐stimulating‐factor‐derived peptides by O‐glycosyltransferases of yeast and rat liver cells
Authors: LORENZ Claudia , STRAHL‐BOLSINGER Sabine , ERNST Joachim F.
Published: March 2005

Human granulocyte‐macrophage colony‐stimulating factor (hGM‐CSF) is O‐glycosylated at residues Ser9 and Thr10 during secretion by yeast and COS‐1 cells [Ernst, J. F., Mermod, J.‐J. and Richman, L. I. (1992) Eur. J. Biochem. 203, 663–667]. Two types of octapeptides encompassing residues 4–11 (peptide 4–11) and variants thereof, or residues 8–15 (peptide 8–15) of hGM‐CSF were tested as substrates for in vitro O‐glycosylation using dolichyl‐phosphate‐d‐mannose:protein O‐d‐mannosyltransferase (Man‐transferase) of the yeast Saccharomyces cerevisiae, or UDP‐N‐acetyl‐α‐d‐galactosamine:polypeptide N‐acetylgalactosaminyltransferase (GalNAc‐transferase) of rat liver cells. Peptide 8–15 was found to be O‐glycosylated at residues Ser9 and Thr10 by GalNAc‐transferase and, with reduced efficiency, also by Man‐transferase. Peptide 4–11 was a good substrate for yeast Man‐transferase, leading to mannosylation of only Thr10, whereas it was very poorly O‐glycosylated at positions Ser5 and Ser7 by GalNAc‐transferase. The observed differences in peptide‐acceptor activities indicate that the site of O‐glycosylation depends on similar, but not identical protein structural features in yeast and mammalian cells.
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.
Substrate preferences of glutamic‐acid‐specific endopeptidases assessed by synthetic Peptide Substrates based on intramolecular fluorescence quenching
Authors: BREDDAM Klaus , MELDAL Morten
Published: March 2005

The substrate preferences of the easily available Glu/Asp‐specific enzymes from Staphyllococcus aureus (V8), Bacillus licheniformis and Streptomyces griseus have been extensively investigated using a series of synthetic peptide substrates, containing an N‐terminal anthraniloyl group and a 3‐nitrotyrosine close to the C‐terminus, allowing the fluorimetric monitoring of substrate hydrolysis by the decrease in intramolecular quenching. All three enzymes hydrolysed Glu‐Xaa peptide bonds approximately 1000‐fold faster than Asp‐Xaa bonds and they are consequently more appropriately termed Glu‐specific enzymes. The difference in kcat/Km for the hydrolysis of substrates with Glu and Asp is primarily due to a difference in kcat. The enzymes appear to hydrolyse all types of Glu‐Xaa bonds, although those with Xaa as Asp and, in particular, Xaa as Pro, are hydrolysed with very low rates. The influence of the nature of the amino acid residues at the substrate positions P2, P3, P4, P′1 and P′2 has been determined and it is shown that the enzyme from S. griseus exhibits the most narrow substrate preference. The results are useful in connection with fragmentation of proteins for sequencing purposes as well as for cleavage of fusion proteins.
Highly efficient transfection of mammalian cells by electric field pulses
Authors: ROLS Marie‐Pierre , COULET Denis , TEISSIÉ Justin
Published: March 2005

We have transfected mammalian cells with plasmid DNA by application of electric pulses. Chinese hamster ovary cells were chosen as a model in order to study and to optimize the transfection protocol. A plasmid carrying the gene coding for β‐galactosidase activity was used to determine transient expression of the electrotransferred acitivity at the cell level. Optimum transient expression for cells in suspension was obtained by application of 10 square wave pulses of 5‐ms duration and 0.6‐kV/cm intensity. Under the best conditions, transfection frequencies as high as 50–60% could be obtained and appeared to be highly dependent on the age of the cell culture. The method was applicable to plated cells growing in a petri dish or on microcarriers. The possibility of extension of the technique to large volumes of cells is presented. A flow system, composed of a peristaltic pump connected to the electropulser chamber, allowed large volumes of cells to be treated with flow rates in the order of several milliliters/minute. Transfection frequencies for the large volumes were 25% for cells in suspension and 35% for cells on microcarriers. These results open new perspectives in large‐scale transfection technology of cells however they are grown.
Regulation of cyclooxygenase and thromboxane synthase in human monocytes
Authors: NÜSING Rolf , ULLRICH Volker
Published: March 2005

Stimulation of human monocytes by lipopolysaccharide or phorbol ester resulted in an increase in thromboxane‐B2 and prostaglandin‐E2 production, wherease interleukin1, tumour necrosis factor α and leukotriene C4 exerted no effects. Inhibitors of protein kinase C suppressed these increases. The activity of cyclooxygenase was induced 3.2‐fold by an 8‐h stimulation, whereas thromboxane‐synthase and prostaglandin‐E‐isomerase activities remained unchanged. A glucocorticoid, dexamethasone, blocked both basal and induced prostanoid release, as well as cyclooxygenase activity. By immunoprecipitation, we were able to demonstrate an enhanced de novo synthesis of cyclooxygenase protein induced by lipopolysaccharide and phorbol ester. Dexamethasone suppressed cyclooxygenase synthesis, whereas thromboxane synthase was induced. For cyclooxygenase, we calculated a half‐life of 3.2 h in human monocytes, and for thromboxane synthase, a half‐life of 28 h. These results suggest that the regulation of differential prostanoid production mainly occurs by up and down regulation of cyclooxygenase.
Biochemical and immunological characterization of A1 adenosine receptors purified from human brain membranes
Authors: NAKATA Hiroyasu
Published: March 2005

The A1 adenosine receptor was purified approximately 13000‐fold to apparent homogeneity from human cerebral cortex membranes using a novel affinity‐chromatography system developed for the purification of rat brain and rat testis A1 adenosine receptors [Nakata, H. (1989) J. Biol. Chem. 264, 16545–16551; Nakata, H. (1990) J. Biol. Chem. 265, 671–677]. The purified human brain receptor showed the ligand‐binding specificity expected of the A1 adenosine receptor. The Bmax and Kd for the purified receptor with a specific A1 adenosine receptor antagonist, 8‐cyclopentyl‐1,3‐[3H]dipropylxanthine, were approximately 16 nmol/mg protein and 2 nM, respectively. SDS/PAGE of the purified receptor preparation showed one broad protein band of molecular mass of approximately 35 kDa, which is very similar to that of purified A1 adenosine receptor from rat brain membranes. Endoglycosidase F treatment of the purified receptor reduced the molecular mass to approximately 30 kDa, suggesting that the human brain A1 adenosine receptor is a glycoprotein. Comparison of the purified human and rat brain A1 adenosine receptors by peptide mapping after the proteolytic digestion showed minor differences between these receptors. Immunological comparisons of the human brain A1 adenosine receptor with rat brain A1 adenosine receptor using polyclonal antibodies against the purified rat brain A1 adenosine receptor showed that the antibodies react preferentially with the rat brain receptor and weakly with human brain receptor.
Cloning of the human and mouse type X collagen genes and mapping of the mouse type X collagen gene to chromosome 10
Authors: APTE Suneel S. , SELDIN Michael F. , HAYASHI Masando , OLSEN Bjorn R.
Published: March 2005

Type X collagen, a homotrimer of α1(X) polypeptide chains, is specifically expressed by hypertrophic chondrocytes in regions of cartilage undergoing endochondral ossification. We have previously described the isolation of a small fragment of the human type X collagen gene (COL10A1) and its localization to the q21–q22 region of human chromosome 6 [Apte, S., Mattei, M.‐G. & Olsen, B. R. (1991) FEBS Lett. 282, 393–396]. Using this fragment as a probe to screen genomic libraries, we report here the isolation of human and mouse genomic clones which contain the major part of the human and mouse type X collagen genes. In both species, the 14‐kb genomic clones which were isolated contain a long open reading frame (>2000 bp in length) which codes for the entire C‐terminal non‐collagenous (NC1) domain, the entire collagenous (COL) domain and part of the N‐terminal non‐collagenous (NC1) domain, the entire collagenous (COL) domain and part of the N‐terminal non‐collagenous (NC2) domain of the α1(X) collagen chain. The human genomic clone contains the major part of the COL10A1 gene, in addition to the region we have previously cloned, and is highly similar to the corresponding portions of the mouse genomic clone (84.5% similarity at the nucleotide level, and 86.1% at the level of the conceptual translation product). The identification of the mouse genomic clone as the α1(X) collagen gene (Col10a1) was confirmed by in situ hybridization of a fragment of the mouse genomic clone to sections from newborn mic. Hybridization was restricted to the hypertrophic chondrocytes of developing chondroepiphyses, being absent in small chondrocytes and in other tissues. Using interspecific backcross analysis, the locus for the mouse α1(X) collagen gene was assigned to chromosome 10. The cloning and chromosomal mapping of the human and mouse α1(X) collagen genes now permit the investigation of the possible role of type X collagen gene defects in the genesis of chondrodysplasias in both species and provide data essential for the generation of transgenic mice deficient in type X collagen.
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.
Small RNA helices as substrates for aminoacylation and their relationship to charging of transfer RNAs
Authors: FRANCKLYN Christopher , MUSIER‐FORSYTH Karin , SCHIMMEL Paul
Published: March 2005

RNA microhelices that reconstruct the acceptor stems of transfer RNAs can be aminoacylated. The anticodon‐independent aminoacylation is sequence‐specific and suggests a relationship between amino acids and nucleotide sequences which is different from that of the classical genetic code. The specific aminoacylation of RNA microhelices also suggests a highly differentiated adaptation of the structures of aminoacyl‐tRNA synthetases to sequences in the acceptor stems of transfer RNAs.
Effect of cavity‐modulating mutations on the stability of Escherichia coli ribonuclease HI
Authors: KIMURA Shigenobu , ODA Yasushi , NAKAI Takahisa , KATAYANAGI Katsuo , KITAKUNI Eiichi , NAKAI Chieko , NAKAMURA Haruki , IKEHARA Morio , KANAYA Shigenori
Published: March 2005

The size of the cavity around Ser68 of Escherichia coli ribonuclease HI was modulated by amino acid substitutions to examine the effects on the stability of the enzyme. Five mutant proteins, Ser68→Gly, Ser68→Ala, Ser68→Thr, Ser68→Val and Ser68→Leu, were constructed. Each of the mutant proteins exhibited at least 40% of the enzyme activity of the wild‐type protein. The stabilities of the mutant proteins were determined from urea‐denaturation and thermal‐denaturation curves. Among the five mutations, only the Ser→Val mutation resulted in an increase in the stability of the enzyme. The melting temperature, tm, at pH 3.0 of the mutant protein Ser68→Val was increased by 1.9°C. Its free‐energy change of unfolding in the absence of urea, G(H2O), and the midpoint of the denaturation curve, [D]1/2, were also increased by 5.4 kJ/mol and 0.18 M, respectively. The increase in the stability of the enzyme is probably due to the filling of the cavity space around Ser68 by valine. However, the mutation of Ser68 to glycine or leucine residues resulted in a considerable decrease in stability. In these cases, some conformational changes occur, as suggested by the CD and 1H‐NMR spectra of these mutant proteins.
Purification and characterization of a thermostable carboxypeptidase from the extreme thermophilic archaebacterium Sulfolobus solfataricus
Authors: COLOMBO Sonia , D'AURIA Sabato , FUSI Paola , ZECCA Laura , RAIA Carlo Antonio , TORTORA Paolo
Published: March 2005

A carboxypeptidase was purified to electrophoretic homogeneity from the thermoacidophilic archaebacterium Sulfolobus solfataricus. Molecular masses assessed by SDS/PAGE and gel filtration were 42 kDa and 170 kDa, respectively, which points to a tetrameric structure for the molecule. An isoelectric point of 5.9 was also determined. The enzyme was proven to be a metalloprotease, as shown by the inhibitory effects exerted by EDTA and o‐phenanthroline; furthermore, dialysis against EDTA led to a complete loss of activity, which could be restored by addition of Zn2+ in the micromolar range, and, to a lesser extent, by Co2+. The enzyme was endowed with a broad substrate specificity, as shown by its ability to release basic, acidic and aromatic amino acids from the respective benzoylglycylated and benzyloxycarbonylated amino acids. An esterase activity of the carboxypeptidase was also demonstrated on different esterified amino acids and dipeptides blocked at the N‐terminus. The enzyme displayed broad pH optima ranging over 5.5–7.0, or 5.5–9.0, when using an acidic or a basic benzyloxycarbonylated amino acid, respectively. With regard to thermostability, it was proven to be completely stable on incubation for 15 min at 85°C. Furthermore, thanks to its relatively low activation energy, i.e. 31.0 kJ/mol, it was still significantly active at room temperature. At 40°C, the enzyme could withstand 0.1% SDS and different organic solvents: particularly ethanol up to 99%. Amino acid and N‐terminal sequence analysis did not evidence any similarity to carboxypeptidases A nor thermolysin. A weak similarity was only found with bovine carboxypeptidase B.
Biochemical and photochemical properties of the photophobic receptors from Halobacterium halobium and Natronobacterium pharaonis
Authors: SCHARF Birgit , PEVEC Bernd , HESS Benno , ENGELHARD Martin
Published: March 2005

The phototaxis of Halobacterium halobium is initiated by two photoreceptors, the sensory rhodopsins sR‐I and sR‐II. An sR‐II‐like pigment has also been described in Natronobacterium pharaonis. In this work it was shown that N. pharaonis cells are repelled by light with a wavelength of 500 nm. A further comparison of membrane preparations from H. halobium (mutant D1) containing only sR‐II and from N. pharaonis [strain SP1(28)] with a chromophoric protein (psR‐II) resembling sR‐II revealed substantial similarities. The biochemical and photochemical properties of the pigments are quite similar, with psR‐II being more stable to external conditions such as pH and ionic strength of the buffer. Both pigments are bleached by low concentrations of hydroxylamine and can be reconstituted by the addition of all‐trans‐retinal. The absorption spectrum of psR‐II is quite similar to sR‐II including the shoulder on the short‐wavelength side. After light excitation sR‐II and psR‐II undergo photocycles with at least three intermediates. The earliest intermediate has an absorption maximum above 520 nm and decays to a species which has a characteristic absorption (∼ 380 nm) of a deprotonated Schiff base. The final step is the regeneration of the original ground state via a red‐shifted intermediate absorbing around 540 nm. From this cumulative evidence it can be concluded that, not only sR‐II, but also the pigment from N. pharaonis is a photophobic photoreceptor.
Effects of angiotensins on cellular hypertrophy and c‐fos expression in cultured arterial smooth muscle cells
Authors: MILLET Dominique , DESGRANGES Claude , CAMPAN Michel , GADEAU Alain‐Pierre , COSTEROUSSE Olivier
Published: March 2005

An increase in cell size and protein content was observed when quiescent arterial smooth muscle cells in culture were incubated with angiotensin II or III. These effects were inhibited by the specific angiotensin type‐1 receptor antagonist losartan (DuP 753) but not by CGP 42112A. In parallel, a transient and dose‐dependent induction of c‐fos was demonstrated not only with angiotensins II and III but also with angiotensin I. Both angiotensins II and III exerted their maximal effect at 1 μM, while angiotensin I needed a tenfold‐higher concentration to exert an identical effect. As for hypertrophy, losartan also inhibits angiotensin‐induced c‐fos expression, suggesting that this gene may be involved into the hypertrophic process. Angiotensin‐I‐mediated c‐fos induction is partially inhibited by the angiotensin‐converting enzyme inhibitors captopril and trandolaprilate; given that an angiotensin‐converting enzyme activity was detected in these smooth muscle cell cultures, these results suggest that angiotensin‐I‐induced c‐fos expression is mediated in part via angiotensin‐I conversion to angiotensin II, but also by other unidentified pathway(s). Angiotensin I could essentially induce smooth muscle cell hypertrophy by indirect mechanisms, while angiotensins II and III act directly on smooth muscle cells.
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.
Murine endothelial leukocyte‐adhesion molecule 1 is a close structral and functional homologue of the human protein
Authors: BECKER‐ANDRÉ Michael , VAN HUIJSDUIJENEN Rob Hoft , LOSBERGER Christophe , WHELAN James , DELAMARTER John F.
Published: March 2005

Human endothelial leukocyte‐adhesion molecule 1 (ELAM‐1), a cell‐surface glycoprotein expressed solely on cytokine‐activated endothelial, cells, mediates the adhesion of bolld neutrophils, memory T‐cells and some monocytes. ELAM‐1, also known as E‐selectin or leukocyte endohtlial‐cell‐adhesion molecule 2, is a member of the lectin/epidermal‐growth‐factor/complement‐regulatory‐protein‐like cell‐adhesion molecule family, which include structurally related molecules referred to as selectins. They are all involved in cell/cell adhesion, playing roles in leukocyte trafficking which are currently only partially defined. We report here the isolation and characterization of the murine equivalent of human ELAM‐1. Murine ELAM‐1 is encoded by a sigle‐copy gene, spanning about 13kb, which is structurally organized into 14 exons and 13 introns; very similar to that of its human counterpart. The exon/intron architecture exactly parallels the domain structure of the encoded protien. a murine ELAM‐1‐specific cDNA was cloned from heart tissue of an interleukin‐1‐(IL‐1)‐treated mouse. its nucleotide sequence shows an overall similarity of 70% to human ELAM‐1‐cDNA. Transiently expressed in Cos cells, the encoded protein promotes the adhesion between recombinant cells and both human polymorphic nuclear cells, as well as HL60 cells expressing the S‐Lewis−xsugar moiety. Northern blot studies revealed by far the highest expression of the murine ELAM‐1 gene in heart tissue and only low expression in lung tissue of IL‐1‐treated mice. Within the promoter, most of the recently identified regulatory elements are conserved. An exception is the nuclear factor (NF)kB box sequence, which, in the murine ELAM‐1 promoter, does not correspond to the consensus NFkB sequence (Lenardo and Baltimore 1989). Band‐shift analyses show no binding to NFkB‐like proteins. However, fusion of the murine ELAM‐1 promoter to a chloramphenicol acetyltransferase reporter confers cytokine‐inducible transcription, although at a lower level, when compared to the human ELAM‐1 promoter. Our results demonstrate the existence of a murine homologue of the human gene and demonstrate for adhesion functional equvalence between the homologous proteins from the two species. In addition, we provide the first evidence fo the utility of the murine model in addressing biological questions about the role which ELAM‐1 plays in inflammation.
A novel photoactivatable cross‐linker for the functionally‐directed region‐specific fluorescent labeling of proteins
Authors: THEVENIN Benard J.‐M. , SHAHROKH Zahra , WILLIARD Renee L. , FUJIMOTO Edward K. , KANG Jaw‐Jou , IKEMOTO Noriaki , SHOHET Stephen B.
Published: March 2005

A cleavable cross‐linking reagent, sulfosuccinimidyl‐2(7‐azido‐4‐methycoumarin‐3‐acetamido)‐ethyl‐1,3′‐dithiopropionate (SAED), was synthesized for the selective transfer of a coumarin fluorophore from a ‘donor’ protein to a position near the binding site of an interacting ‘target’ protein. SAED contains a terminal N‐sulfoscuccinimidyl ester for conjugation to the donor, a terminal photoactivatable azido‐coumarin species for cross‐linking with the interacting target, and a central disculfide spacer for the release of the labeled target after cleavage. To evaluate the effectiveness of this labeling reagent, soynean trypsin inhibitor (STI) was derivatized (∼ 0.5 mol/mol) with SAED and then photolyzed in the presence of trypsin. A single fluorescent cross–linked species (6–7 mol% of total STI) was observed by SDS/PAGE and, after refuctive cleavage, was shown to be a 1: 1 STI‐trypsin complex. This complex was not detected without photolysis or with an inactivated cross‐linker. Importantly, complex formation was inhibited by an excess of unmodified STI and prevented by substitution of a non‐interacting protein for trypsin. Cleavage of the cross‐linked complex revealed that the trypsin, but not the STI, was fluorescent; the uncomplexed trypsin fraction remained unlabeled. These results demonstrated the specificty of the labeling of trypsin by fluorescent‐transfer cross‐linking with SAED. An efficiency of about 15% for this cross‐linking mediated labeling of trypsin was calculated. The short cross‐linking span of SAED (≤1.8nm) strictly limited the labeling to the vicinity of the contact region of trypsin with STI. thus, this novel cross‐linker permits the region‐specific targeting of a fluorophore near a functionally important binding site.
Cloning and nucleotide sequence of the pst A gene of Wolinella succinogenes polysulphide reductase
Authors: KRAFFT Torsten , BOKRANZ Martin , KLIMMEK Oliver , SCHRÖDER Imke , FAHRENHOLZ Falk , KOJRO Elzbieta , KRÖGER Achim
Published: March 2005

The polysulphide reductase (formerly sulphur reductase) of Wolinella succinogenes is a component of the phosphorylative electron transport system with polysulphid as the terminal acceptor. Using an antiserum raised against the major subunit (PsrA, 85 kDa) of the enzyme, the corresponding gene (psr A) was cloned from a λ‐gene bank. The N‐terminal amino acid sequence of PsrA mapped within the psr A gene product, which also contained an apparent signal peptide. Downstream of the Psr A gene two more open reading frames (psrB and psrC) were found. the three genes may form a transcriptional unit with the transcription start site in front of psr A. The three genes were present only once on the genome. PsrA is a hydrophilic protein homologous to the largest subunits of six prokaryotic molybdoenzymes. PsrB is predicted to be hydrophilic, to contain ferredoxin‐like cysteine clusters and to be homologous to the smaller hydrophilic subunits of four molybdoenzymes. PsrC is predicted to be a hydrophobic protein that could possibly serve as the membrane anchor of the enzyme.
Dye‐linked dehydrogenase activities for formate and formate esters in Amycolatopsis methanolica
Authors: VAN OPHEM Peter W. , BYSTRYKH Leonid V. , DUINE Johannis A.
Published: March 2005

Cell‐free extracts of methanol‐grown Amycolatopsis methanolica contain dye‐linked dehydrogenase activities for formate and methyl formate. Fractionation of the extracts revealed that the (unstable) activity for formate resides in membrane particles, while that for methyl formate belongs to a soluble enzyme that was purified and characterized. The enzyme, indicated as formate‐ester dehydrogenase, appeared to be a molybdoprotein (4 Fe, 3 or 4 S, 1 Mo and 1 FAD were fond for each enzyme molecule), with a molecular mass of 186 kDa and consisting of two subunits of equal size. Product identification suggests that the formate moiety in the ester becomes hydroxylated to a carbonate group after which the unstable alkyl carbonate decomposes into CO2 and the alcohol moiety. Based on structural and catalytic characteristics, the enzyme appears to be very similar to an enzyme isolated from Comamonas testosteroni [Poels, P. A., Groen, B. W. & Duine, J. A. 1987 Eur. J. Biochem. 166, 575–579] which was at that time considered to be an aldehyde dehydrogenase. Formate‐ester dehydrogenase activity appeared to be present in several other bacteria. Possible roles for the A. methanolica enzyme in C1 dissimilation (oxidation of methyl formate to methanol and Co2 or a factor‐formate adduct to factor plus CO2) or in general aldehyde oxidation, are discussed.