bluuebunny/crossref_metadata_2025 huggingface dataset:
Modulation of extracellular ATP-induced Ca2+ responses: role of protein kinases
Authors: Tenneti L , Talamo B R
Published: August 2015

Evidence for the modulation of the P2z-purinoceptor for extracellular ATP in dissociated rat parotid cells is presented in studies using compounds that inhibit protein kinases. Preincubation of acinar cells with the protein kinase catalytic-site inhibitors K-252a and staurosporine, as well as with the regulatory-domain inhibitor sphingosine, specifically potentiates the elevation in cytosolic Ca2+ concentration ([Ca2+]i) mediated by extracellular ATP, but has no effect on the [Ca2+]i elevation mediated by muscarinic receptors through phospholipase C activation. Phorbol dibutyrate (PDBu), which activates protein kinase C (PKC), has no modulatory effect on ATP-mediated [Ca2+]i elevation. Further, pretreatment with PDBu does not reverse or block the effects of K-252a or sphinogosine, arguing against the involvement of PKC. Other pharmacological manipulations indicate that neither calmodulin-dependent nor cyclic-AMP-dependent kinases are involved. Neither the peak intracellular Ca2+ mobilization nor the sustained Ca2+ entry in response to carbachol or to a Ca2+ ionophore (4-bromo-A23187) is altered by the kinase inhibitors that potentiate the [Ca2+]i response to ATP, indicating that effects on the ATP response are not due to non-specific permeability changes, nor to decreased Ca2+ removal from the cytosol. ATP-mediated influx of Mn2+ as well as ATP-induced membrane depolarization are potentiated in cells preincubated with K-252a, directly demonstrating that cation influx is enhanced through a P2z-specific route. These results show that P2z responses (or purinoceptors) can be modulated and suggest that phosphorylation events are involved.
Molecular cloning and deduced amino acid sequences of the γ-subunits of rat and monkey NAD+-isocitrate dehydrogenases
Authors: Nichols B J , Hall L , Perry A C F , Denton R M
Published: August 2015

A 600 bp cDNA fragment encoding part of the gamma-subunit of pig heart NAD(+)-isocitrate dehydrogenase (ICDH gamma) was amplified by PCR using redundant oligonucleotide primers based on partial peptide sequence data [Huang and Colman (1990) Biochemistry 29, 8266-8273]. This PCR fragment was then used as a probe to isolate clones encoding the complete mature forms of the gamma-subunit from rat epididymis and monkey testis cDNA libraries. Comparison of the deduced amino acid sequences of the rat and monkey subunits and the partial sequence of the pig heart enzyme revealed a remarkably high level of sequence identity. The relationship between the deduced amino acid sequences of the NAD(+)-ICDH gamma-subunits and those of nonmammalian NAD(+)- and NADP(+)-ICDH subunits is discussed.
Non-proteoglycan forms of biglycan increase with age in human articular cartilage
Authors: Roughley P J , White R J , Magny M C , Liu J , Pearce R H , Mort J S
Published: August 2015

Polyclonal anti-peptide antibodies were raised to the C-terminal regions of human biglycan and decorin. These antibodies were used in immunoblotting to study structural variations with age in the proteoglycan core proteins present in extracts of human articular cartilage and intervertebral disc. Three forms of the biglycan core protein were identified. The largest form was detected only after chondroitinase treatment and represents the proteoglycan form of the molecule from which the glycosaminoglycan chains have been removed. However, chondroitinase treatment did not alter the electrophoretic mobility of the two smaller proteins, which appear to represent non-proteoglycan forms of the molecule, resulting either from a failure to substitute the intact proteoglycan core protein with glycosaminoglycan chains during its synthesis or from proteolytic processing of the intact proteoglycan causing removal of the N-terminal region bearing the glycosaminoglycan chains. The non-proteoglycan forms constituted a minor proportion of biglycan in the newborn, but were the major components in the adult. A similar trend was seen in both articular cartilage and intervertebral disc. In comparison, decorin appears to exist predominantly as a proteoglycan at all ages, with two core protein sizes being present after chondroitinase treatment. Non-proteoglycan forms were detected in the adult, but they were always a minor constituent.
Optimization of molecular design in the evolution of metabolism: the glycogen molecule
Authors: Meléndez-Hevia E , Waddell T G , Shelton E D
Published: August 2015

The animal glycogen molecule has to be designed in accordance with its metabolic function as a very effective fuel store allowing quick release of large amounts of glucose. In addition, the design should account for a high capacity of glucose storage in the least possible space. We have studied the optimization of these variables by means of a mathematical model of the glycogen molecule. Our results demonstrate that the structure is optimized to maximize (a) the total glucose stored in the smallest possible volume, (b) the proportion of it that can be directly released by phosphorylase before any debranching occurs, and (c) the number of non-reducing ends (points of attack for phosphorylase), which maximizes the speed of fuel release. The optimization of these four variables is achieved with appropriate values for two key parameters in glycogen design: the degree of branching and the length of the chains. The optimal values of these two parameters are precisely those found in cellular glycogen.
Evidence that the glutamine-stimulated loss of nitrate reductase protein from the yeast Candida nitratophila is not the result of inducer exclusion
Authors: Hipkin C R , Kau D A , Cannons A C
Published: August 2015

Synthesis of nitrate reductase protein and increases in nitrate reductase activity occurred in cultures of the yeast Candida nitratophila when they were incubated in medium containing ammonium nitrate. Similar treatment with glutamine plus nitrate resulted in little increase in nitrate reductase activity, in cultures grown previously with reduced nitrogen compounds, and decreases in enzyme activity, in cultures adapted to nitrate. Labelling studies conducted in vivo revealed a rapid cessation of de novo nitrate reductase synthesis when glutamine was supplied to nitrate-adapted cultures in the presence of nitrate. Intracellular glutamine concentrations increased rapidly under these conditions and these cultures exhibited high glutamine: glutamate ratios. As nitrate was taken up in the presence of glutamine in these experiments, it is concluded that the glutamine-stimulated inhibition of nitrate reductase synthesis is a consequence of repression and rapid turnover of nitrate reductase mRNA and not inducer (nitrate) exclusion.
Interactions between active-site-serine β-lactamases and mechanism-based inactivators: a kinetic study and an overview
Authors: Matagne A , Ghuysen M F , Frère J M
Published: August 2015

The interactions between three class A beta-lactamases and three beta-lactamase inactivators (clavulanic acid, sulbactam and olivanic acid MM13902) were studied. Interestingly, the interaction between the Streptomyces cacaoi beta-lactamase and clavulanate indicated little irreversible inactivation. With sulbactam, irreversible inactivation was found to occur with the three studied enzymes, but no evidence for transiently inactivated adducts was found. Irreversible inactivation of the S. albus G and S. cacaoi enzymes was particularly slow. With olivanate, irreversible inactivation was also observed with the three enzymes, but with the S. cacaoi enzyme, no hydrolysis could be detected. A tentative summary of the results found in the literature is also presented (including 6 beta-halogenopenicillanates), and the general conclusions underline the diversity of the mechanisms and the wide variations of the rate constants observed when class A beta-lactamases interact with beta-lactamase inactivators, in agreement with the behaviours of the same enzymes towards their good and poor substrates.
Structural features responsible for kinetic thermal stability of a carboxypeptidase from the archaebacterium Sulfolobus solfataricus
Authors: Villa A , Zecca L , Fusi P , Colombo S , Tedeschi G , Tortora P
Published: August 2015

Investigations were performed on the structural features responsible for kinetic thermal stability of a thermostable carboxypeptidase from the thermoacidophilic archaebacterium Sulfolobus solfataricus which had been purified previously and identified as a zinc metalloprotease [Colombo, D'Auria, Fusi, Zecca, Raia and Tortora (1992) Eur. J. Biochem. 206, 349-357]. Removal of Zn2+ by dialysis led to reversible activity loss, which was promptly restored by addition of 80 microM ZnCl2 to the assay mixture. For the first-order irreversible thermal inactivation the metal-depleted enzyme showed an activation energy value of 205.6 kJ.mol-1, which is considerably lower than that of the holoenzyme (494.4 kJ.mol-1). The values of activation free energies, enthalpies and entropies also dropped with metal removal. Thermal inactivation of the apoenzyme was very quick at 80 degrees C, whereas the holoenzyme was stable at the same temperature. These findings suggest a major stabilizing role for the bivalent cation. Chaotropic salts strongly destabilized the holoenzyme, showing that hydrophobic interactions are involved in maintaining the native conformation of the enzyme. However, the inactivation rate was also increased by sodium sulphate, acetate and chloride, which are not chaotropes, indicating that one or more salt bridges concur in stabilizing the active enzyme. Furthermore, at the extremes of the pH-stability curve, NaCl did not affect the inactivation rate, confirming the stabilizing role of intramolecular ionic bonds, as a pH-dependent decrease in stability is likely to occur from breaking of salt bridges involved in maintaining the native conformation of the protein.
Inactivation of glycogen synthase kinase-3β by phosphorylation: new kinase connections in insulin and growth-factor signalling
Authors: Sutherland C , Leighton I A , Cohen P
Published: August 2015

The beta-isoform of glycogen synthase kinase-3 (GSK3 beta) isolated from rabbit skeletal muscle was inactivated 90-95% following incubation with MgATP and either MAP kinase-activated protein kinase-1 (MAPKAP kinase-1, also termed RSK-2) or p70 S6 kinase (p70S6K), and re-activated with protein phosphatase 2A. MAPKAP kinase-1 and p70S6K phosphorylated the same tryptic peptide on GSK3 beta, and the site of phosphorylation was identified as the serine located nine residues from the N-terminus of the protein. The inhibitory effect of Ser-9 phosphorylation on GSK3 beta activity was observed with three substrates, (inhibitor-2, c-jun and a synthetic peptide), and also with glycogen synthase provided that 0.15 M KCl was added to the assays. The results suggest that Ser-9 phosphorylation underlies the reported inhibition of GSK3 beta by insulin and that GSK3 may represent a point of convergence of two major growth-factor-stimulated protein kinase cascades.
Modulation by fatty acids of Ca2+ fluxes in sarcoplasmic-reticulum vesicles
Authors: Cardoso C M , De Meis L
Published: August 2015

The fatty acids palmitic (C16:0), stearic (C18:0), arachidic (C20:0) and arachidonic (C20:4) acids inhibit Ca2+ uptake and enhance Ca2+ efflux measured in vesicles derived from the sarcoplasmic reticulum of skeletal muscle. These effects of the fatty acids are impaired by the Ca(2+)-ATPase ligands Mg2+, Ca2+ and K+, and by drugs that block the leakage of Ca2+ through the Ca(2+)-ATPase such as Ruthenium Red, spermine [de Meis (1991) J. Biol. Chem. 266, 5736-5742] and thapsigargin [de Meis and Inesi (1992) FEBS Lett. 299, 33-35].
Salmonella typhimurium neuraminidase acts with inversion of configuration
Authors: Guo X , Sinnott M L
Published: August 2015

When the time course of the hydrolysis of identical solutions of p-nitrophenyl N-acetyl-alpha-D-neuraminide by Salmonella typhimurium neuraminidase is monitored by u.v. and by its optical rotation, the rotation change is synchronous with, or even marginally in advance of, the absorbance change. In experiments under the same conditions with influenza-virus neuraminidase, known to react with retention of configuration [Chong, Pegg, Taylor and von Itzstein (1992) Eur. J. Biochem. 207, 335-343], the rotation change is much slower than the absorbance change. The inverting, presumably single-displacement, mode of action of the S. typhimurium enzyme follows from these observations, and the position (92.5% beta) of the slowly established mutarotational equilibrium of N-acetylneuraminic acid [Friebolin, Kunzelmann, Supp, Brossmer, Keilich and Ziegler (1981) Tetrahedron Lett. 22, 1383-1386].
Preparation and characterization of fragments from the N-terminal end of bovine serum albumin under native conditions
Authors: Saeed T , Salahuddin A
Published: August 2015

The domain I of BSA, containing residues 1-183 of the protein sequence, was isolated by CNBr treatment. It was further reductively cleaved into two subfragments, N1 and N2, in 8 M urea; the subfragments were regenerated in GSH and GSSG. The fragment N and subfragments N1 and N2 were found to be homogeneous with respect to size and charge. Results for amino acid composition, N-terminal amino acid sequence, thiol groups and M(r) suggested that the fragments N1 and N2 contain residues 88-183 and 1-87 of the intact BSA respectively. Optical studies, intrinsic-viscosity measurements, gel-filtration data and derived hydrodynamic parameters, taken together with the results on proteolytic digestion, showed that fragment N, as well as its subfragments N1 and N2, exist in compact and globular conformation and that the conformation of N2 fragment is more compact than that of the N1 fragment.
Activation of NADPH oxidase of human neutrophils involves the phosphorylation and the translocation of cytosolic p67phox
Authors: Dusi S , Rossi F
Published: August 2015

Activation of human neutrophil NADPH oxidase requires the interaction of cytosolic and membrane-associated components. Evidence has been accumulated that in phorbol 12-myristate 13-acetate (PMA)-stimulated neutrophils, the translocation to the plasma membrane of the cytosolic components p47phox and p67phox and the phosphorylation of p47phox are essential steps in activation of NADPH oxidase. No direct evidence has been presented to date as to whether p67phox is also phosphorylated. To address this problem we have immunoprecipitated p67phox from neutrophil cytosol and membrane fractions. The results indicate that, very soon after activation with PMA (20 s), p67phox was present in a phosphorylated form in the cytosol and in the membranes. At later times (1-3 min) the extent of p67phox phosphorylation continuously increased both in the cytosol and in the membrane fraction, while oxygen consumption reached the maximal rate within 40 s, and then remained linear. p67phox was also phosphorylated in formyl-methionyl-leucyl-phenylalanine-activated neutrophils. That the phosphorylated p67 protein we identified in immunoprecipitation experiments was p67phox was confirmed by the observation that no phosphorylated band of 67 kDa was immunoprecipitated from the cytosol and membranes of PMA-stimulated neutrophils from a p67phox-deficient chronic granulomatous disease patient. In this case, p47phox was normally phosphorylated. These data demonstrate that: (1) the phosphorylation of p67phox is correlated with activation of NADPH oxidase, and (2) continuous phosphorylation of p67phox is required in order to maintain the linearity of the respiratory burst.
Phosphoglucomutase 1: a gene with two promoters and a duplicated first exon
Authors: Putt W , Ives J H , Hollyoake M , Hopkinson D A , Whitehouse D B , Edwards Y H
Published: August 2015

In view of its central role in glycolysis and gluconeogenesis and its polymorphic genetic variability, the phosphoglucomutase 1 (PGM1) gene in man has been the target of protein structural studies and genetic analysis for more than 25 years. We have now isolated genomic clones containing the complete PGM1 gene and have shown that it spans over 65 kb and contains 11 exons. We have also shown that the sites of the two mutations which form the molecular basis for the common PGM1 protein polymorphism lie in exons 4 and 8 and are 18 kb apart. Within this region there is a site of intragenic recombination. We have discovered two alternatively spliced first exons, one of which, exon 1A, is transcribed in a wide variety of cell types; the other, exon 1B, is transcribed in fast muscle. Exon 1A is transcribed from a promoter which has the structural hallmarks of a housekeeping promoter but lies more than 35 kb upstream of exon 2. Exon 1B lies 6 kb upstream of exon 2 within the large first intron of the ubiquitously expressed PGM1 transcript. The fast-muscle form of PGM1 is characterized by 18 extra amino acid residues at its N-terminal end. Sequence comparisons show that exons 1A and 1B are structurally related and have arisen by duplication.
Characterization of the 3-dehydroquinase domain of the pentafunctional AROM protein, and the quinate dehydrogenase from Aspergillus nidulans, and the overproduction of the type II 3-dehydroquinase from neurospora crassa
Authors: Hawkins A R , Moore J D , Adeokun A M
Published: August 2015

The AROM protein of Aspergillus nidulans is a multidomain pentafunctional polypeptide that is active as a dimer and catalyses steps 2-6 in the prechorismate section of the shikimate pathway. The three C-terminal domains (including the type I 3-dehydroquinase) of the AROM protein are homologous with the qutR-encoded QUTR protein that represses transcription of the eight genes comprising the quinic acid utilization (qut) gene cluster, and the two N-terminal domains are homologous with the qutA-encoded QUTA protein that transcribes the qut genes. As part of a larger research programme designed to compare the structures of the three proteins and to probe the domain structure and interaction within each protein, we have overproduced and purified the 3-dehydroquinase domain of the AROM protein. Additionally we have overproduced and purified the qutB-encoded quinate dehydrogenase and overproduced the qa-2 encoded type II 3-dehydroquinase of Neurospora crassa. We report that the AROM 3-dehydroquinase domain has a monomeric native state, with an apparent kcat./Km ratio that is approx. 160-fold lower than the value for the native N. crassa AROM protein. The AROM protein 3-dehydroquinase domain is sensitive to inactivation by borohydride in the presence of the substrate 3-dehydroquinate, confirming that it is a typical type I 3-dehydroquinase. The purified quinate dehydrogenase is bifunctional, being able to metabolize shikimate as a substrate. The apparent Km values for quinate (450 microM), shikimate (1.7 mM) and NAD+ (150 microM) are all similar to values reported for the qa-3-encoded enzyme from N. crassa.
Structure of the glycosyl-phosphatidylinositol membrane anchor of acetylcholinesterase from the electric organ of the electric-fish, Torpedo californica
Authors: Mehlert A , Varon L , Silman I , Homans S W , Ferguson M A J
Published: August 2015

The structure of the glycan moiety of the glycosyl-phosphatidylinositol (GPI) membrane anchor from Torpedo californica (electric fish) electric-organ acetylcholinesterase was solved using n.m.r., methylation analysis and chemical and enzymic micro-sequencing. Two structures were found to be present: Glc alpha 1-2Man alpha 1-2Man alpha 1-6Man alpha 1-4GlcN alpha 1-6myo-inositol and Glc alpha 1-2Man alpha 1-2Man alpha 1-6(GalNAc beta 1-4)Man alpha 1-4GlcN alpha 1-6myo-inositol. The presence of glucose in this GPI anchor structure is a novel feature. The anchor was also shown to contain 2.3 residues of ethanolamine per molecule.
Reconstitution of the folding pathway of collagen in a cell-free system: formation of correctly aligned and hydroxylated triple helices
Authors: Middleton R B , Bulleid N J
Published: August 2015

We describe here a cell-free system which will carry out the initial stages in the synthesis, post-translational modification and assembly of type-X collagen. The mRNA coding for bovine type-X collagen was synthesized in vitro and translated in a rabbit reticulocyte lysate to yield a protein that was collagenase sensitive and could be immunoprecipitated with antibodies raised to purified avian type-X collagen. When type-X collagen was synthesized in the absence of added microsomes or in the presence of canine pancreas microsomes, the translation products showed partial resistance to digestion with pepsin but were completely degraded with a mixture of chymotrypsin and trypsin, suggesting that only incorrectly aligned non-native collagen molecules were synthesized under these conditions. When the protein was synthesized in the presence of microsomes derived from avian fibroblasts or a human fibrosarcoma cell line, the translocated product migrated as a diffuse band characteristic of hydroxylated collagen. The synthesized polypeptides were also resistant to both pepsin and trypsin/chymotrypsin digestion, demonstrating the formation of correctly aligned native collagen. Furthermore, the collagen polypeptides assembled into higher-order structures, possibly trimers, which were stabilized by interchain disulphide bonds. The collagen helix synthesized in vitro had a melting temperature of 41 degrees C which is comparable with the protein synthesized in vivo, further demonstrating that the polypeptides were hydroxylated and that the triple helix formed was correctly aligned.
Effect of selective thiol-group derivatization on enzyme kinetics of (R)-3-hydroxybutyrate dehydrogenase
Authors: Dalton L A , McIntyre J O , Fleischer S
Published: August 2015

(R)-3-Hydroxybutyrate dehydrogenase (BDH) is a phosphatidylcholine-requiring tetrameric enzyme with two thiol groups (SH-1 and SH-2) per protomer. By first protecting the more rapidly reacting thiol group (SH-1) with diamide [1,1′-azobis-(NN′-dimethylformamide), DM] to form DM(SH-1)BDH, SH-2 can be selectively derivatized by reaction with maleimide reagents such as 4-maleimido-2,2,6,6-tetramethyl-piperidine-N-oxyl (MSL), which gives DM(SH-1)MSL(SH-2)BDH. Reduction with dithiothreitol (DTT) regenerates SH-1, yielding MAL(SH-2)BDH (where MAL is the diamagnetic reduction product of MSL-BDH and DTT). The enzymic activity of DM(SH-1)BDH is decreased to approx. 4% relative to the native purified enzyme, and the apparent Km for substrate, KmBOH, is increased approx. 100-fold. Reduction of DM(SH-1)BDH with DTT regenerates SH-1 and restores normal enzymic function. Modification of SH-2 with piperidinylmaleimide [MAL(SH-2)BDH] diminishes enzymic activity to approx. 35% of its original value, but has no significant effect on apparent KmBOH. The doubly derivatized enzyme, DM(SH-1)MSL(SH-2)BDH, has lower enzymic activity [about half that for DM(SH-2)BDH] and a yet higher apparent KmBOH than DM(SH-1)BDH. Derivatization of SH-2 with different maleimide reagents results in diminished activity approximately proportional to the size of the maleimide substituent, suggesting that this inhibition is steric. Whereas modification of SH-1 results in marked changes in kinetic parameters (increased apparent Km and reduced apparent Vmax), derivatization of SH-2 has a lesser effect on enzymic function. Thus SH-1 is postulated to be closer to the active centre than is SH-2, although neither is involved in catalysis, since: (1) the activity of the derivatized enzyme is not abolished; and (2) activity can be enhanced by increasing substrate (and cofactor) concentrations.
Topology of nucleotide-sugar:dolichyl phosphate glycosyltransferases involved in the dolichol pathway for protein glycosylation in native rat liver microsomes
Authors: Bossuyt X , Blanckaert N
Published: August 2015

Activities of nucleotide-sugar:dolichyl phosphate glycosyltransferases (UDP-N-acetylglucosamine:dolichyl phosphate N-acetylglucosaminyl 1-phosphotransferase, UDP-glucose:dolichyl phosphate glucosyltransferase and GDP-mannose:dolichyl phosphate mannosyltransferase) are not fully expressed in native microsomes and can be enhanced by pretreatment of the microsomes with detergent. To examine whether the latency of dolichyl phosphate glycosyltransferases in native microsomes reflects a lumenal orientation of the catalytic centre, we examined the effect of proteinase treatment of native microsomes on enzymic activity and investigated the relationship between enzymic activity and alteration of the permeability of the microsomal membrane barrier. The enzymic activities catalysing transfer of N-acetylglucosamine and glucose to lipid acceptors were proteinase-sensitive in native sealed microsomes. When various detergents were used to disrupt the membrane barrier, we found no relationship between activity of dolichyl phosphate glycosyltransferases and the latency of mannose-6-phosphatase, which is a marker of the permeability properties of the microsomal membrane. Permeabilization of the endoplasmic reticulum membrane by the pore-forming Staphylococcus aureus alpha-toxin did not affect glycosyltransferase activities. These results do not support the hypothesis that latency of the transferase activities is dependent on the permeability properties of the endoplasmic-reticulum membrane. Collectively our findings can best be explained by postulating that the active centres of the transferases are cytoplasmically oriented, while activation by detergent may be conformation-dependent.
Analysis, by electrospray ionization mass spectrometry, of several forms of Clostridium pasteurianum rubredoxin
Authors: Petillot Y , Forest E , Mathieu I , Meyer J , Moulis J M
Published: August 2015

Clostridium pasteurianum rubredoxin and its recombinant counterpart purified from Escherichia coli have been analysed by electrospray ionization m.s. (e.s.i.m.s.). Whereas the N-terminal methionine of the native protein is formylated, the recombinant one has a free N-terminal methionine. E. coli cells also produce a colourless protein from the cloned gene. This protein is absent from C. pasteurianum and was shown to be zinc-substituted rubredoxin. The molecular forms of rubredoxin detected by e.s.i.m.s. depended on the experimental conditions used. Significant conversion into apo-rubredoxin occurred when the proteins were ionized at acidic pH and detected in the positive-ion mode. This conversion was quantitative in the case of Zn-rubredoxin. In contrast, when the proteins were analysed at neutral pH in the negative-ion mode, only the holoproteins, i.e. the species initially present in the solutions, were detected in the spectra. The e.s.i.m.s. experimental conditions set up here may prove useful for the analysis of other acidic metalloproteins with weakly bound metals.
Characterization of two novel casein transcripts in rabbit mammary gland
Authors: Dawson S P , Wilde C J , Tighe P J , Mayer R J
Published: August 2015

Two clones were isolated from a cDNA library corresponding to mRNAs which accumulate in mid-lactating (14 day) rabbit mammary gland and characterized by DNA sequencing. The two clones sequenced corresponded to two novel casein transcripts (pBRM5 and pBRM42). Relative mRNA abundances for the two clones were assessed by dot-blot analysis. Phylogenetic analysis and comparison of both pBRM5 and pBRM42 with other members of the casein family revealed that the rabbit may be unique among mammals in expressing two alpha s2-casein genes. The presence of two alpha s2-casein genes in the rabbit may be the result of a relatively recent intergenic duplication event.
Nicotinamide inhibits nitric oxide synthase mRNA induction in activated macrophages
Authors: Pellat-Deceunynck C , Wietzerbin J , Drapier J C
Published: August 2015

Nitric oxide (NO) is a potent mediator involved in many biological functions including inflammation and non-specific immunity. Murine macrophages possess the prototype of high-output NO synthase which is not constitutively expressed but induced within a few hours by immunological stimuli. In this study, we explored the possibility of controlling the activity of the inducible NO synthase by interfering with the transduction signal which triggers its induction, in the RAW 264.7 macrophage cell line. We found that nicotinamide, an inhibitor of ADP-ribosylation, prevented NO synthase induction in RAW 264.7 cells after stimulation with interferon gamma (IFN-gamma) and lipopolysaccharide (LPS). Furthermore, the level of NO synthase mRNA was measured by Northern-blot analysis and we found that nicotinamide prevents expression of NO synthase mRNA in IFN-gamma- and LPS-stimulated cells. Nicotinamide was also found to inhibit other macrophage functions expressed in response to IFN-gamma, i.e. tumour necrosis factor secretion and the expression of the Ia antigen of the major histocompatibility complex. Analysis of the pattern of ADP-ribosylated proteins revealed that nicotinamide as well as cholera toxin prevented the ADP-ribosylation of a 107-117 kDa protein found constitutively ADP-ribosylated in stimulated and non-stimulated macrophage extracts. Together, our results indicate ADP-ribosylation as a crucial point of the signalling pathway which leads to NO synthase mRNA induction.
N-[2-bromocinnamyl(amino)ethyl]-5-isoquinolinesulphonamide (H-89) inhibits incorporation of choline into phosphatidylcholine via inhibition of choline kinase and has no effect on the phosphorylation of CTP:phosphocholine cytidylyltransferase
Authors: Wieprecht M , Wieder T , Geilen C C
Published: August 2015

We have shown previously that N-[2-bromocinnamyl(amino)-ethyl]-5-isoquinolinesulphonamide (H-89), a selective inhibitor of cyclic-AMP-dependent protein kinase (PKA), inhibits phosphatidylcholine biosynthesis in HeLa cells. In the present study, we elucidated the mechanism underlying the described inhibition. Treatment of cells with 10 microM H-89 had no effect on the phosphorylation of CTP:phosphocholine cytidylyltransferase. However, H-89 slightly affected the distribution of cytidylyltransferase between cytosol and membranes, but the cellular 1,2-diacylglycerol content was not influenced. Furthermore, pulse-chase experiments revealed that H-89 did not affect cytidylyltransferase activity. Instead, H-89 inhibited choline kinase, the enzyme catalysing the first step in the CDP-choline pathway. In the presence of 10 microM H-89, choline kinase activity was inhibited by 36 +/- 7.6% in vitro. Additionally, the phosphorylation of choline to phosphocholine was inhibited by 30 +/- 3% in cell-culture experiments. This inhibitory effect could be partly prevented by simultaneous addition of 10 microM forskolin, indicating that choline kinase is regulated in part by PKA activity.
Constitutive expression of a saturable transport system for non-esterified fatty acids in Xenopus laevis oocytes
Authors: Zhou S L , Stump D , Isola L , Berk P D
Published: August 2015

In the presence of 150 microM BSA, uptake of [3H]oleate by Xenopus laevis oocytes was a saturable function of the unbound oleate concentration (Vmax. 110 +/- 4 pmol/h per oocyte; Km 193 +/- 11 nM unbound oleate). Oleate uptake was three orders of magnitude faster than that of another test substance, [35S]bromosulphophthalein, and was competitively inhibited by 55 nM unbound palmitate (Vmax. 111 +/- 14 pmol/h per oocyte; Km 424 +/- 63 nM unbound oleate) (P < 0.01). Oleate uptake was also inhibited by antibodies to a 43 kDa rat liver plasma-membrane fatty acid-binding protein, a putative transporter of long-chain fatty acids in mammalian cells; uptake of the medium-chain fatty acid [14C]octanoate was unaffected. Immunofluorescence and immunoblotting demonstrated that the antiserum reacted with a single 43 kDa protein on the oocyte surface. Hence a protein related to the mammalian plasma-membrane fatty acid-binding protein may play a role in saturable uptake of long-chain fatty acids by Xenopus oocytes.
Steady-state kinetics of malonyl-CoA synthetase from Bradyrhizobium japonicum and evidence for malonyl-AMP formation in the reaction
Authors: Kim Y S , Kang S W
Published: August 2015

Malonyl-CoA synthetase catalyses the formation of malonyl-CoA directly from malonate and CoA with hydrolysis of ATP into AMP and PP1. The catalytic mechanism of malonyl-CoA synthetase from Bradyrhizobium japonicum was investigated by steady-state kinetics. Initial-velocity studies and the product-inhibition studies with AMP and PPi strongly suggested ordered Bi Uni Uni Bi Ping Pong Ter Ter system as the most probable steady-state kinetic mechanism of malonyl-CoA synthetase. Michaelis constants were 61 microM, 260 microM and 42 microM for ATP, malonate and CoA respectively, and the value for Vmax, was 11.2 microM/min. The t.l.c. analysis of the 32P-labelled products in a reaction mixture containing [gamma-32P]ATP in the absence of CoA showed that PPi was produced after the sequential addition of ATP and malonate. Formation of malonyl-AMP, suggested as an intermediate in the kinetically deduced mechanism, was confirmed by the analysis of 31P-n.m.r. spectra of an AMP product isolated from the 18O-transfer experiment using [18O]malonate. The 31P-n.m.r. signal of the AMP product appeared at 0.024 p.p.m. apart from that of [16O4]AMP, indicating that one atom of 18O transferred from [18O]malonate to AMP through the formation of malonyl-AMP. Formation of malonyl-AMP was also confirmed through the t.l.c. analysis of reaction mixture containing [alpha-32P]ATP. These results strongly support the ordered Bi Uni Uni Bi Pin Pong Ter Ter mechanism deduced from initial-velocity and product-inhibition studies.
Association of the insulin receptor and phosphatidylinositol 3-kinase requires a third component
Authors: Liu R , Livingston J N
Published: August 2015

We have studied the interactions between the insulin receptor and PtdIns 3-kinase by a reconstitution system in vitro composed of highly purified PtdIns 3-kinase from rat liver and highly purified insulin receptors bound to insulin-agarose or to antibodies against insulin receptors. As a positive control, receptors for platelet-derived growth factor, which bind and phosphorylate PtdIns 3-kinase, were studied in parallel with insulin receptors. Our results indicate that the insulin receptor, regardless of its phosphorylation state, does not directly associate with purified PtdIns 3-kinase, whereas the autophosphorylated receptor does associate with PtdIns 3-kinase present in the crude CHO-cell lysate. Also, we could not detect phosphorylation of PtdIns 3-kinase by the insulin receptor, even through the receptor readily underwent autophosphorylation and phosphorylated an insulin-receptor substrate, poly(Glu-Tyr) (4:1). These findings argue that one or more cytosolic components link the receptor and the enzyme. Insulin-receptor substrate-1 (IRS-1) was evaluated as a potential linking protein. In the absence of ATP, IRS-1 did not facilitate the coupling of the phosphorylated insulin receptor to PtdIns 3-kinase. Thus IRS-1 is unlikely to be the component in crude CHO-cell lysate that couples PtdIns 3-kinase to the phosphorylated insulin receptor. However, the addition of ATP, which allows phosphorylation of IRS-1 by the insulin receptor, also enhances the coupling of PtdIns 3-kinase to the insulin receptor. In support of this idea, immunoprecipitates of IRS-1 from insulin-treated CHO cells were found to contain both the insulin receptor and PtdIns 3-kinase. In conclusion, the insulin receptor does not appear to phosphorylate or bind directly to PtdIns 3-kinase, regardless of the receptor's state of phosphorylation. Association of PtdIns 3-kinase with the insulin receptor is mediated by one or more components, one of which may involve an unidentified factor in cell lysate and another that apparently involves phosphorylated IRS-1.
Some taste substances are direct activators of G-proteins
Authors: Naim M , Seifert R , Nürnberg B , Grünbaum L , Schultz G
Published: August 2015

Amphiphilic substances may stimulate cellular events through direct activation of G-proteins. The present experiments indicate that several amphiphilic sweeteners and the bitter tastant, quinine, activate transducin and Gi/Go-proteins. Concentrations of taste substances required to activate G-proteins in vitro correlated with those used to elicit taste. These data support the hypothesis that amphiphilic taste substances may elicit taste through direct activation of G-proteins.
Functional compartments of sulphatide metabolism in cultured living cells: evidence for the involvement of a novel sulphatide-degrading pathway
Authors: Tempesta M C , Salvayre R , Levade T
Published: August 2015

The modes of uptake and degradation of radiolabelled cerebroside sulphate (CS or sulphatide) were investigated in cultured living skin fibroblasts and Epstein-Barr virus-transformed lymphoblastoid cell lines established from control individuals and patients affected with metachromatic leucodystrophy (cerebroside sulphatase deficiency), multiple sulphatase deficiency and low-density-lipoprotein-receptor-negative familial hypercholesterolaemia. In both cell types, CS was taken up through a non-receptor-mediated process. In fibroblasts, CS degradation occurred intralysosomally as was evident from the findings that fibroblasts from metachromatic leucodystrophic patients accumulated the sulphatide and that chloroquine inhibited its degradation by normal cells. In contrast, under similar conditions of CS availability, lymphoblastoid cell lines from patients with metachromatic leucodystrophy could degrade the incorporated sulphatide exactly as their normal counterparts. This metabolic pathway was also fully active in lymphoblastoid cells from patients with multiple sulphatase deficiency and was not inhibited by chloroquine treatment. These data are consistent with a non-lysosomal type of hydrolysis. In addition to the lysosomal and non-lysosomal compartments, a third compartment was identified in the two cell types which is probably formed by the pool of the sulphatide molecules incorporated into the plasma membrane. This is the first report on the existence of a CS-degrading pathway in intact cells with deficient lysosomal cerebroside sulphatase activity.
Phosphorylation of adenosine in renal brush-border membrane vesicles by an exchange reaction catalysed by adenosine kinase
Authors: Sayós J , Solsona C , Mallol J , Lluis C , Franco R
Published: August 2015

Uptake of [3H]adenosine in brush-border membrane (BBM) vesicles from either rat or pig kidney leads to an accumulation of intravesicular [3H]AMP. The lack of significant levels of ATP and the presence of AMP in BBM indicated that a phosphotransfer between [3H]adenosine and AMP occurs. The phosphotransfer activity is inhibited by iodotubercidin, which suggests that it is performed by adenosine kinase acting in an ATP-independent manner. The existence of a similar phosphotransferase activity was demonstrated in membrane-free extracts from pig kidney. From the compounds tested it was shown that a variety of mononucleotides could act as phosphate donors. The results suggest that phosphotransfer reactions may be physiologically relevant in kidney.
Phorbol esters inhibit the glucocorticoid-mediated stimulation of cytosolic aspartate aminotransferase gene transcription
Authors: Feilleux-Duché S , Garlatti M , Aggerbeck M , Bouguet J , Hanoune J , Barouki R
Published: August 2015

The regulation of cytosolic aspartate aminotransferase (cAspAT) gene expression by phorbol esters was investigated in the highly differentiated hepatoma cell line Fao. Phorbol 12,13-dibutyrate (PdBu) had no effect on basal activity but partially inhibited the induction of cAspAT by dexamethasone. The extent of inhibition (40%) was similar to that obtained with insulin or vanadate. The inhibitory effects of PdBu and vanadate were additive. In the case of PdBu, the inhibitory effects could be eliminated by first incubating the cells with PdBu, which down-regulates protein kinase C. In contrast, inhibition by insulin was not modified by this treatment. The molecular mechanism of PdBu action was investigated. Northern blot analysis showed that the steady-state mRNA levels of cAspAT were decreased by PdBu in the presence of dexamethasone. In addition, the transcription rate, as measured by run-on experiments, was also decreased under the same conditions. Finally, a 2.4 kb promoter fragment driving the chloramphenicol acetyltransferase gene was stably transfected into the Fao cells. The regulation of the activity of this promoter fragment by dexamethasone and PdBu was similar to the regulation of the endogenous cAspAT activity. We conclude that PdBu acts by regulating the promoter activity of the cAsPAT gene.
Turnover of acyl-CoA-binding protein in four different cell lines measured by using two-dimensional polyacrylamide-gel electrophoresis
Authors: Buus C L , Kristiansen K , Knudsen J
Published: August 2015

Acyl-CoA-binding protein (ACBP), also named diazepam-binding inhibitor or endozepine, is a 10 kDa protein for which a surprisingly large number of biological activities has been suggested. Some of these would seem to require a rapid intracellular turnover of the protein. In this paper we report on the turnover of ACBP in cell lines derived from mouse, rat and man. ACBP was identified in two-dimensional gels by using specific antibodies. Cells were labelled with [35S]methionine and chased for various periods of time. Total protein was extracted, subjected to two-dimensional PAGE, and radioactivity in the spot containing ACBP was determined by liquid-scintillation counting. ACBP half-life was determined, and varied from 25 to 53 h depending on the cell line and the growth conditions. In all cases, radioactivity in ACBP was lost slightly faster than radioactivity in total protein. These results are discussed in relation to the possible function suggested for ACBP.
Partial purification of a diacylglycerol lipase from bovine aorta
Authors: Lee M W , Severson D L
Published: August 2015

A diacylglycerol (DG) lipase has been purified from a soluble subcellular fraction of bovine aorta by (NH4)2SO4 precipitation in the presence of 5.0% (w/v) Triton X-100, followed by chromatography on DEAE-Sephacel, heparin-Sepharose and octyl-Sepharose in the presence of either CHAPS or Triton X-100 detergents. Under basal conditions, the hydrolysis of a short-chain [3H]dioctanoylglycerol ([3H]diC8) substrate was much greater than that of a long-chain 1-[1-14C]palmitoyl-2-oleoyl-sn-glycerol (1-[14C]POG) substrate. Lipase activity measured with 1-[14C]POG was markedly enhanced by Triton X-100. In the presence of 0.1% Triton X-100, specific enzyme activities in the octyl-Sepharose fraction determined with 1-[14C]POG or 1-stearoyl-2-[1-14C]-arachidonoyl-sn-glycerol as substrates were the same as that measured with [3H]diC8. MgCl2 (5mM) or CaCl2 (2 mM) also selectively stimulated lipase activity (up to 10-13-fold) measured with the long-chain (1-[14C]POG) substrate only. The increase in relative specific activity in the octyl-Sepharose fraction was 60-fold and 155-fold, based on hydrolysis of [3H]diC8 and 1-[14C]POG (+ Triton X-100), respectively. Unlabelled diC8 was a competitive inhibitor of 1-[14C]POG hydrolysis, suggesting that a single lipase hydrolyses both the short-chain and long-chain DG substrates; selective stimulatory effects of non-ionic detergents and bivalent cations on the hydrolysis of 1-[14C]POG may be due to effects on the physical properties of the substrate preparation. Monoacylglycerol lipase, DG kinase and cholesterol esterase activities could not be detected in the partially purified lipase preparation.
Functional interaction between different isoforms of the Oct-2 transcription factor expressed in neuronal cells
Authors: Lillycrop K A , Estridge J K , Latchman D S
Published: August 2015

The predominant neuronal isoforms of the Oct-2 transcription factor, Oct 2.4 and Oct 2.5, repress the herpes simplex virus immediate-early promoter both in neuronal cells and in fibroblasts normally lacking Oct-2. In contrast, the predominant B lymphocyte form Oct 2.1, which is present at a lower level in neuronal cells, activates the immediate-early promoter in fibroblasts but represses it in neuronal cells. We show here that both Oct 2.4 and Oct 2.5 can functionally interact with Oct 2.1 and convert it from an activator into a repressor. Hence, the cell type-specific activity of Oct 2.1 results from the presence of Oct 2.4 and 2.5 in neuronal cells and their absence in other cell types. The significance of this effect is discussed in terms of the role of Oct-2 in the regulation of viral and cellular gene expression in neuronal cells.
Determination of control coefficients in intact metabolic systems
Authors: Cornish-Bowden A , Hofmeyr J H S
Published: August 2015

The control structure of a metabolic system can in principle be determined without the need for purification of the component enzymes and study of their kinetic properties, provided that their activities can be perturbed by amounts sufficient to produce measurable changes in the steady-state variables, i.e. the fluxes through the system and the concentrations of the intermediates. Each perturbation is characterized in terms of the co-response coefficients of all pairs of variables, i.e. the slopes of the lines produced when the logarithm of one variable is plotted against the logarithm of another, both varying in response to the same perturbation. If all the co-response coefficients are assembled into a matrix, the inverse of this matrix can be transformed into a matrix containing all the component elasticities, which can be inverted to provide the complete matrix of control coefficients. In a simple three-enzyme pathway studied, the analysis proves not to require unrealistically high accuracy in the original co-response measurements: even with errors with standard deviation +/- 5.77 degrees in the angles to the horizontal of the lines in the co-response plots (equivalent at best to errors of +/- 20% in the corresponding co-response coefficients), the final control coefficient matrix may be adequate for assessing the control structure of the system. Examination of literature data from studies of mitochondrial respiration and of gluconeogenesis indicates that considerably higher precision than this is achievable.
The post-translational processing of chromogranin A in the pancreatic islet: involvement of the eukaryote subtilisin PC2
Authors: Arden S D , Rutherford N G , Guest P C , Curry W J , Bailyes E M , Johnston C F , Hutton J C
Published: August 2015

The post-translational processing of chromogranin A (CGA) and the nature of the enzyme(s) involved were investigated in rat pancreatic islet and insulinoma tissue. Pulse-chase radiolabelling experiments using sequence-specific antisera showed that the 98 kDa (determined by SDS/PAGE) precursor was processed to an N-terminal 21 kDa peptide, a C-terminal 14 kDa peptide and a 45 kDa centrally located peptide with a rapid time course (t1/2 approx. 30 min) after an initial delay of 30-60 min. The 45 kDa peptide was, in turn, converted partially into a 5 kDa peptide with pancreastatin immunoreactivity and a 3 kDa peptide with WE-14 immunoreactivity over a longer time period. Incubation of bovine CGA with rat insulinoma secretory-granule lysate produced peptides of 18, 16 and 40 kDa via intermediates of 65 and 55 kDa. N-terminal sequence analysis indicated that cleavage occurred at the conserved paired basic sites Lys114-Arg115 and Lys330-Arg331, suggesting that cleavage of the equivalent sites (Lys129-Arg130 and Lys357-Arg358) in the rat molecule produced the initial post-translational products observed in intact pancreatic beta-cells. The enzyme activity responsible for the cleavage of bovine CGA co-chromatographed on DEAE-cellulose with the type-2 proinsulin endopeptidase and with PC2 immunoreactivity. The type-1 enzyme (PC1/3) appeared inactive towards CGA. The requirement for Ca2+ ions and an acidic pH for conversion was consistent with the involvement of a member of the eukaryote subtilisin family, and the composition of the released peptides in pulse-chase and secretion studies suggested that conversion occurred in the secretory-granule compartment. The overall catalytic rate as well as the relative susceptibilities of the Lys114-Arg115 and Lys330-Arg331 sites to cleavage were affected by pH, suggesting that the ionic environment of the processing compartment may play a role in the differential processing of CGA which is evident in various neuroendocrine cells.