bluuebunny/crossref_metadata_2025 huggingface dataset:
Identity of the core proteins of the large chondroitin sulphate proteoglycans synthesized by skeletal muscle and prechondrogenic mesenchyme
Authors: Carrino D A , Dennis J E , Drushel R F , Haynesworth S E , Caplan A I
Published: August 2015

Large, chondroitin sulphate-containing proteoglycans are synthesized by three prominent tissue in the embryonic chick limb. One of these proteoglycans is aggrecan, the phenotype-specific proteoglycan of cartilage. Another, PG-M, is produced by prechondrogenic mesenchymal cells. The third, M-CSPG, is made by developing skeletal muscle cells. While the carbohydrate components of PG-M and M-CSPG share some similarities, both of these proteoglycans clearly have different carbohydrate moieties from those of aggrecan. To compare these three proteoglycans at another level, their core protein structures were analysed in three ways: by the presence or absence of monoclonal antibody epitopes, by one-dimensional peptide display of the cyanogen bromide-cleaved core proteins and by electron microscopic imaging of the molecules. Monoclonal antibodies whose epitopes are present in aggrecan core protein were tested with core protein preparations from M-CSPG and PG-M. One of these, 7D1, recognizes both PG-M and M-CSPG, while another, 1C6, shows no reactivity for the non-cartilage proteoglycans. The absence of 1C6 reactivity is of interest, as its epitope is in a region of the aggrecan core protein known to have a functional homologue in the core proteins of PG-M and M-CSPG. The cyanogen bromide-fragmented peptide pattern of M-CSPG is the same as that of PG-M, and both are different from that of aggrecan. The aggrecan pattern has one prominent large band (molecular mass 130 kDa), some less prominent large bands (molecular mass 70-100 kDa) and several smaller bands. In contrast, the PG-M and M-CSPG patterns show no bands with molecular masses > 73 kDa, and the smaller bands (molecular mass < 40 kDa) have a different pattern to that of the smaller bands from aggrecan. The electron microscopic images of aggrecan show a core protein with one end having two globular regions separated by a short linear segment; adjacent to this is a long linear segment, which sometimes contains a third globular region at the end of the core protein opposite the end with the double-globe structure. M-CSPG and PG-M core proteins never show images with the double-globe structure. Instead, one end of the molecule has a single globular domain, and a second globular region is variably present at the opposite end of the core protein. Thus, by all three methods, the core proteins of PG-M and M-CSPG appear to be the same and both differ from the core protein of aggrecan.
Molecular variants of β2-microglobulin in renal insufficiency
Authors: Vincent C , Dennoroy L , Revillard J P
Published: August 2015

Many patients with renal insufficiency treated by dialysis for more than 10 years have tissue deposits of amyloid material containing polymerized beta 2-microglobulin (beta 2m). The mechanisms of beta 2m polymerization and degradation remain unknown. In biological fluids (serum and urine) from haemodialysis patients and in dialysis fluids from patients treated by chronic ambulatory peritoneal dialysis (CAPD), we have characterized different molecular forms of beta 2m, including proteolytic split products. beta 2m isoforms of pI 5.7, 5.3 and 4.5-5.0 were isolated from urine and CAPD fluid. The pI 5.3 beta 2m, but not the other forms, was recovered both as monomers and as dimers. Such dimers were also detected in serum from patients but not from healthy controls. pI 5.3 and 5.7 beta 2m isoforms were found to be nearly identical by mass spectrometry and by their amino acid sequences. The amino acid sequence of the 43 N-terminal amino acids of beta 2m of pI 5.0 showed identity with the corresponding region of pI 5.7 beta 2m. Fragments recovered from CAPD fluid were similar to proteolytic fragments generated from pure pI 5.7 beta 2m by incubation in mouse ascitic fluid at acidic pH. Furthermore, pure pI 5.7 beta 2m was converted into more acidic forms of 12 kDa upon incubation in mouse ascitic fluid at acid pH. beta 2m dimers found in serum may represent a precursor of amyloid fibrils.
Rapid turnover of phosphatidylinositol 3-phosphate in the green alga Chlamydomonas eugametos: signs of a phosphatidylinositide 3-kinase signalling pathway in lower plants?
Authors: Munnik T , Irvine R F , Musgrave A
Published: August 2015

When Chlamydomonas eugametos gametes were incubated in carrier-free [32P]P1, the label was rapidly incorporated into PtdInsP and PtdInsP2 and, after reaching a maximum within minutes, was chased out by recirculating unlabelled P1 in the cell. This pulse-chase labelling pattern reflects their rapid turnover. In contrast, 32P incorporation into the structural lipids was slow and continued for hours. Of the radioactivity in the PtdInsP spot, 15% was in PtdIns3P and the rest in PtdIns4P, and of that in the PtdInsP2 spot, 1% was in PtdIns(3,4)P2 and the rest in PtdIns(4,5)P2, confirming the findings by Irvine, Letcher, Stephens and Musgrave [(1992) Biochem. J. 281, 269-266]. When cells were labelled with carrier-free [32P]P1, both PtdInsP isomers incorporated label in a pulse-chase-type pattern, demonstrating for the first time in a plant or animal system that D-3 poly-phosphoinositides turn over rapidly in non-stimulated cells, with kinetics similar to those shown by the D-4 isomers. In animal systems such lipids are already established as signalling molecules, and the data suggest that a similar role must be sought for them in lower plants such as Chlamydomonas.
Autoinduction of activin genes in early Xenopus embryos
Authors: Suzuki A , Nagai T , Nishimatsu S I , Sugino H , Eto Y , Shibai H , Murakami K , Ueno N
Published: August 2015

Activin exhibits a potent mesoderm inducing activity towards the ectodermal tissue (animal cap) of Xenopus laevis blastulae. Thus in order to investigate the role of activin in morphogenesis of early Xenopus embryos, activation of genes for activin beta A and beta B was examined by the reverse transcription polymerase chain reaction. In vivo, activin beta B mRNA appears to be present in embryonic stage 1 whereas beta A mRNA is undetectable prior to gastrulation. beta B and beta A mRNAs were noted to accumulate after stages 9 and 15 respectively. Activin gene expression in Xenopus animal caps was examined after treatment with various concentrations of activin A. Under these treatment conditions, both activin beta A and beta B mRNAs accumulated in a dose-dependent fashion after 24 h. The same effect was noted for treatment with similar concentrations of activin B. Accumulation of mRNAs was inhibited by the addition of cycloheximide to the culture medium, consistent with the proposition that activin gene expression requires certain protein factors. In total, therefore, these data suggest that an autoinduction mechanism is involved in the regulation of activin mRNA levels in normal Xenopus embryos and that this mechanism may play a pivotal role during early embryonic development.
Calcium promotes membrane association of reticulocyte 15-lipoxygenase
Authors: Watson A , Doherty F J
Published: August 2015

The reticulocyte 15-lipoxygenase (linoleate:oxygen oxidoreductase, EC 1.13.11.12) is implicated in oxidative damage to reticulocyte mitochondria before their elimination by degradation during maturation to the erythrocyte. A proportion of the 15-lipoxygenase sediments with the mitochondrial-rich stromal fraction of density-gradient-fractionated rabbit reticulocytes suggesting a physical association with mitochondria before their elimination. Ca2+ promotes binding of reticulocyte 15-lipoxygenase to isolated rat liver and reticulocyte mitochondria and 15-lipoxygenase-mediated lipid peroxidation of mitochondrial lipids and free linoleic acid. Association of reticulocyte 15-lipoxygenase with isolated mitochondria is not simply a consequence of Ca(2+)-induced swelling, but implies that Ca2+ mediates translocation of soluble lipoxygenase to mitochondrial membranes. Therefore, Ca2+ may have an important physiological role in the regulation of 15-lipoxygenase-mediated targeting of reticulocyte mitochondria for degradation.
Characterization of calmodulin-dependent cyclic nucleotide phosphodiesterase isoenzymes
Authors: Sharma R K , Kalra J
Published: August 2015

Calmodulin-dependent phosphodiesterase (CaMPDE) is one of the key enzymes involved in the complex interactions which occur between the cyclic-nucleotide and Ca2+ second-messenger systems. Calmodulin-dependent phosphodiesterase exists in different isoenzymic forms, which exhibit distinct molecular and/or catalytic properties. The kinetic properties suggest that the 63 kDa brain isoenzyme is distinct from the brain 60 kDa and heart and lung CaMPDE isoenzymes. The CaMPDE isoenzymes of 60 kDa from brain, heart and lung are regulated by calmodulin, but the affinities for calmodulin are different. At identical concentrations of calmodulin, the bovine heart CaMPDE isoenzyme is stimulated at a much lower Ca2+ concentration than the bovine brain or lung isoenzymes. The bovine lung CaMPDE isoenzyme contains calmodulin as a tightly bound subunit, so that a change in calmodulin concentration had no effect on the [Ca2+]-dependence of activation of this isoenzyme. These observations are consistent with the notion that differential regulation by calmodulin and Ca2+ is an important function of these isoenzymes, which provide fine-tuning mechanisms for calmodulin action.
Phosphorylation by protein kinase C and cyclic AMP-dependent protein kinase of synthetic peptides derived from the linker region of human P-glycoprotein
Authors: Chambers T C , Pohl J , Glass D B , Kuo J F
Published: August 2015

Specific sites in the linker region of human P-glycoprotein phosphorylated by protein kinase C (PKC) were identified by means of a synthetic peptide substrate, PG-2, corresponding to residues 656-689 from this region of the molecule. As PG-2 has several sequences of the type recognized by the cyclic AMP-dependent protein kinase (PKA), PG-2 was also tested as a substrate for PKA. PG-2 was phosphorylated by purified PKC in a Ca2+/phospholipid-dependent manner, with a Km of 1.3 microM, and to a maximum stoichiometry of 2.9 +/- 0.1 mol of phosphate/mol of peptide. Sequence analysis of tryptic fragments of PG-2 phosphorylated by PKC identified Ser-661, Ser-667 and Ser-671 as the three sites of phosphorylation. PG-2 was also found to be phosphorylated by purified PKA in a cyclic AMP-dependent manner, with a Km of 21 microM, and to a maximum stoichiometry of 2.6 +/- 0.2 mol of phosphate/mol of peptide. Ser-667, Ser-671 and Ser-683 were phosphorylated by PKA. Truncated peptides of PG-2 were utilized to confirm that Ser-661 was PKC-specific and Ser-683 was PKA-specific. Further studies showed that PG-2 acted as a competitive substrate for the P-glycoprotein kinase present in membranes from multidrug-resistant human KB cells. The membrane kinase phosphorylated PG-2 mainly on Ser-661, Ser-667 and Ser-671. These results show that human P-glycoprotein can be phosphorylated by at least two protein kinases, stimulated by different second-messenger systems, which exhibit both overlapping and unique specificities for phosphorylation of multiple sites in the linker region of the molecule.
Electrostatic control of oxidative deamination catalysed by bovine serum amine oxidase
Authors: Stevanato R , Mondovi B , Befani O , Scarpa M , Rigo A
Published: August 2015

The ionic-strength-dependence of steady-state kinetic parameters (kc and Km') for non-biogenic (benzylamine, butylamine) and biogenic (spermine, spermidine) amines has been measured in the bovine serum amine oxidase reaction. The catalytic rate constant (kc) values are similar (0.9-2.5 s-1) for all the substrates studied and are almost constant over the experimental ionic strength range (24-155 mM). In contrast, Km' values are in the range 6-2300 microM and undergo a 4-12-fold increase with increasing ionic strength, parallelled by a decrease in catalytic efficiency. From an analysis of the kc and Km' values and their dependence on ionic strength, we conclude that more than one negative site is involved in the binding of these amines and that the relative dielectric constant of the binding site is lower than that of aqueous solutions.
The significance of denaturant titrations of protein stability: a comparison of rat and baker's yeast cytochrome c and their site-directed asparagine-52-to-isoleucine mutants
Authors: Koshy T I , Luntz T L , Plotkin B , Schejter A , Margoliash E
Published: August 2015

The residue asparagine-52 of rat cytochrome c and baker's yeast iso-1-cytochrome c was mutated to isoleucine by site-directed mutagenesis, and the unfolding of the wild-type and mutant proteins in urea or guanidinium chloride solutions was studied. Whereas the yeast mutant cytochrome unfolded in 4-7 M urea with a rate constant (k) of 1.7 x 10(-2) s-1, the rat mutant protein unfolded with k = 5.0 x 10(-2) s-1, followed by a slow partial refolding with k = 5.0 x 10(-4) s-1. Denaturant titrations indicated that the mutation increased the stability of the yeast cytochrome by 6.3 kJ (1.5 kcal)/mol, while it decreased that of the rat protein by 11.7 kJ (2.8 kcal)/mol. These results probably reflect structural differences between yeast iso-1 and vertebrate cytochromes c in the vicinity of the Asn-52 side chain.
Zinc is required for the expression of ornithine decarboxylase in a difluoromethylornithine-resistant cell line
Authors: Flamigni F , Campana G , Carboni L , Guarnieri C , Spampinato S
Published: August 2015

Dilution of quiescent L1210-DFMOr (difluoromethylornithine-resistant) cells in fresh medium containing serum led to the induction of ornithine decarboxylase (ODC) and to the expression of its mRNA, as determined by a sensitive solution-hybridization-RNase-protection assay. Addition of the chelating agent diethylenetriaminepentaacetic acid (DTPA) at seeding time caused an inhibition of the induction of ODC activity by up to 90%, and only Zn2+ of the bivalent metal ions tested was effective in reversing this effect. The inhibition of the induction of ODC activity was accompanied by a marked decrease, prevented by Zn2+ supplementation, of the accumulation of immunoreactive ODC protein and ODC mRNA. DTPA treatment also caused a slight acceleration of ODC turnover. These results indicate that a restricted Zn2+ availability in L1210-DFMOr cells impairs ODC induction remarkably, mainly by affecting the expression of the messenger.
Collagen type IV stimulates an increase in intracellular Ca2+ in pancreatic acinar cells via activation of phospholipase C
Authors: Somogyi L , Lasić Z , Vukičević S , Banfić H
Published: August 2015

Intracellular Ca2+ responses to extracellular matrix molecules were studied in suspensions of pancreatic acinar cells loaded with Fura-2. Collagen type I, laminin, fibrinogen and fibronectin were unable to raise cytosolic free Ca2+ concentration ([Ca2+]i), whereas collagen type IV, at concentrations from 5 to 50 micrograms/ml, significantly increased it. The effect of collagen type IV was not due to possible contamination with type-I transforming growth factor beta or plasminogen, as neither of these agents was able to increase [Ca2+]i. Using highly specific mass assays, concentrations of inositol lipids, 1,2-diacylglycerol (DAG) and Ins(1,4,5) P3 were measured in pancreatic acinar cells stimulated with collagen type IV. A decrease in the concentrations of PtdIns(4,5) P2 and PtdIns4 P with a concomitant increase in the concentrations of DAG and InsP3 mass were observed, showing that collagen type IV increases [Ca2+]i by activation of phospholipase C. The observed [Ca2+]i signals had two components, the first resulting from Ca2+ release from the intracellular stores, and the second resulting from Ca2+ flux from the extracellular medium through the verapamil-insensitive channels. A tyrosine kinase inhibitor (tyrphostine) was able to block inositol lipid signalling caused by collagen type IV, which together with the insensitivity of this pathway to cholera toxin and pertussis toxin or to preactivation of protein kinase C, the longer duration of the increase in [Ca2+]i and a longer lag period needed for observation of increases in DAG and InsP3 concentration with collagen type IV than with carbachol (50 mM) suggest that activation of phospholipase C by collagen type IV is caused by tyrosine kinase activation. Inositol lipid signalling and increases in [Ca2+]i were also observed with Arg-Gly-Asp (RGD)-containing peptide but not with Arg-Asp-Gly (RDG)-containing peptide. Collagen type IV and RGD-containing peptide, but not carbachol, competed in increasing [Ca2+]i and DAG concentration, suggesting that the binding site of collagen type IV responsible for phospholipase C activation contains the RGD sequence. Together the present results suggest that, in pancreatic acinar cells, RGD sequence(s) within collagen type IV molecules cause activation of tyrosine kinase, probably through one of the integrin receptors, which then stimulates phospholipase C and increases [Ca2+]i.
Micromolar calcium decreases affinity of inositol trisphosphate receptor in vascular smooth muscle
Authors: Benevolensky D , Moraru I I , Watras J
Published: August 2015

The mechanism by which Ca2+ inhibits InsP3-induced Ca2+ release from sarcoplasmic reticulum of vascular smooth muscle was investigated. InsP3 binding to sarcoplasmic-reticulum vesicles from dog aortic smooth muscle was inhibited by 51 +/- 6% by 2 microM Ca2+ in the presence of 10 nM [3H]InsP3. Scatchard analysis indicated the presence of two InsP3-binding sites in the absence of Ca2+ (Kd = 2.5 +/- 0.9 and 49 +/- 8 nM InsP3), though the low-affinity site was more prevalent (representing 92 +/- 3% of the total number of binding sites). Ca2+ (2 microM) did not alter InsP3 binding to the high-affinity site (P > 0.05), but increased the Kd of the low-affinity site 3-fold (Kd = 155 +/- 4 nM InsP3; P < 0.001). The possibility that the apparent decrease in InsP3 affinity was caused by Ca(2+)-dependent activation of an endogenous phospholipase C could be excluded, because the Ca(2+)-dependent inhibition of InsP3 binding was completely reversible and insensitive to an inhibitor of phospholipase C. Moreover, Ca2+ did not inhibit InsP3 binding to InsP3 receptor partially purified by heparin-Sepharose chromatography, though another fraction (devoid of InsP3 receptor) restored Ca(2+)-sensitivity of the partially purified InsP3 receptor. Thus Ca2+ binding to a Ca(2+)-sensitizing factor associated with the InsP3 receptor decreases the affinity of the receptor complex for InsP3. This Ca(2+)-sensitizing factor may provide a negative-feedback mechanism for regulating the rise in cytosolic Ca2+ concentration in vascular smooth muscle after hormone activation of the phosphoinositide cascade.
Immunological analysis of developmental changes in ecdysone 20-mono-oxygenase expression in the cotton leafworm, Spodoptera littoralis
Authors: Chen J H , Hara T , Fisher M J , Rees H H
Published: August 2015

The developmental changes in ecdysone 20-mono-oxygenase during the sixth larval instar of the cotton leafworm, Spodoptera littoralis, were investigated. The specific activity of mitochondrial ecdysone 20-mono-oxygenase in the fat-body exhibited a distinct peak at 72 h, at which time the larvae stop feeding. Immunoblot analyses, using antibodies raised against components of vertebrate mitochondrial steroidogenic enzyme systems [anti-(cytochrome P-450scc), anti-(cytochrome P-450(11) beta), anti-adrenodoxin and anti-(adrenodoxin reductase) antibodies], revealed the presence of specific immunoreactive polypeptides in fat-body mitochondrial extracts. In addition, these antibodies effectively inhibited fat-body mitochondrial ecdysone 20-mono-oxygenase activity. This suggests that the S. littoralis steroid-hydroxylating system(s) may contain polypeptide components analogous to those present in vertebrates. A close correlation between developmental changes in mitochondrial ecdysone 20-mono-oxygenase activity and the abundance of polypeptides (approx. 66 kDa and 50 kDa) recognized by the anti-(cytochrome P-450(11) beta) antibody and a polypeptide (approx. 52 kDa) recognized by the anti-(adrenodoxin reductase) antibody were observed in both fat-body and midgut. These results suggest that developmental changes in the abundance of components of the ecdysone 20-mono-oxygenase system may play an important role in the developmental regulation of the enzyme expression and, hence, of 20-hydroxyecdysone titre.
Dietary omega-3 and polyunsaturated fatty acids modify fatty acyl composition and insulin binding in skeletal-muscle sarcolemma.
Authors: Liu S , Baracos V E , Quinney H A , Clandinin M T
Published: August 2015

Feeding animals with diets high in saturated fat induces insulin resistance, and replacing saturated fat isocalorically with poly-unsaturated fat, especially long-chain omega-3 fatty acids, will prevent the development of insulin resistance in skeletal-muscle tissue. To investigate the mechanism, rats were fed on high-fat (20%, w/w) semipurified diets for 6 weeks. Diets containing ratios of polyunsaturated/saturated (P/S) fatty acid of 0.25 (low-P/S diet) and 1.0 (high-P/S diet) were used to study the effect of the level of saturated fat. To study the effects of omega-3 fatty acids, diets with a low-P/S ratio containing either 0 (low-omega-3 diet) or 3.3% (high-omega-3 diet) long-chain omega-3 fatty acids from fish oil were fed. Plasma membrane from skeletal muscle was purified. The content of fatty acids in sarcolemmal phospholipid was significantly related to the dietary composition. Insulin binding to intact sarcolemmal vesicles prepared from rats fed on diets high in omega-3 fatty acids increased 14-fold compared with animals fed on the low-omega-3 diet (P < 0.0001). Feeding rats on a diet with a high P/S ratio increased sarcolemmal insulin binding by 2.3-fold (P < 0.05). Increased insulin binding was due to increased receptor number at the low-affinity high-capacity binding site. Dietary effects on insulin binding were eliminated when studies were carried out on detergent-solubilized membranes, indicating the importance of the phospholipid fatty acyl composition for insulin binding. The results suggest that dietary omega-3 and polyunsaturated fatty acids increase insulin binding to sarcolemma by changing the fatty acyl composition of phospholipid surrounding the insulin receptor, and this might be the mechanism by which dietary fatty acids modify insulin action.
Uptake of protoporphyrin and continuous spectrophotometric assay for magnesium chelatase in Rhodobacter spheroides.
Authors: Gorchein A
Published: August 2015

Uptake of protoporphyrin was shown by Rhodobacter spheroides under anaerobic conditions in the dark. The process was not energy-dependent but required EGTA and was markedly stimulated by Methyl Viologen. Kinetic studies were consistent with a saturable process with Kd = 5-10 microM and Bmax. = 2.2 nmol of protoporphyrin bound/mg dry weight of cells. Bound protoporphyrin could be converted into magnesium protoporphyrin monomethyl ester under anaerobic conditions in the light or at low pO2 (6.3%) in the dark. This formed the basis of a sensitive continuous spectrophotometric assay for magnesium chelatase, which avoids the need to extract the product into organic solvent, and may facilitate the development of a cell-free system for magnesium chelatase in photosynthetic bacteria. It is proposed also that the uptake mechanism shown for exogenous protoporphyrin may indicate the existence of a ligand or carrier system for endogenously produced protoporphyrin essential for magnesium chelatase activity.
Expression of rat endopeptidase-24.18 in COS-1 cells: membrane topology and activity
Authors: Milhiet P E , Corbeil D , Simon V , Kenny A J , Crine P , Boileau G
Published: August 2015

Endopeptidase-24.18 (E-24.18; EC 3.4.24.18) is a metallopeptidase of the astacin family and is highly expressed in kidney brush-border membranes of rodents. Rat E-24.18 consists of two disulphide-linked alpha/beta dimers [(alpha/beta)2]. In order to investigate the mechanisms of assembly and the importance of each subunit in the enzymic process, the cloned cDNAs for the rat alpha and beta subunits were transiently expressed either alone or together in COS-1 cells. Immunoblotting of cell extracts and spent culture media showed that, when expressed alone, the alpha subunit is secreted, whereas the beta subunit is membrane-bound. In alpha/beta-transfected cells, the alpha subunit remained membrane-bound, but could be released from the cell surface after papain treatment or after incubation with 10 mM dithiothreitol. Furthermore, mutants of the alpha subunit in which the putative C-terminal anchor domain was deleted could still form cell-associated alpha/beta dimers. These results are consistent with a topological model of E-24.18 in which the beta subunit is anchored in the plasma membrane and the alpha subunit is retained at the cell surface through disulphide bridge(s) with the beta subunit. Both the alpha and beta recombinant subunits expressed in COS-1 cells showed little azocasein-degrading activity. However, activity of either individual subunits of alpha/beta dimers was increased after mild trypsin digestion, suggesting that in COS-1 cells the enzymes are synthesized as zymogens. Finally, inactivation of the alpha subunit by site-directed mutagenesis of Glu-157, which is believed to play a role in catalysis, showed that both subunits participate in the enzymic activity of the heterodimer.
Site-directed mutagenesis of rat muscle 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase: role of Asp-130 in the 2-kinase domain
Authors: Rider M H , Crepin K M , De Cloedt M , Bertrand L , Hue L
Published: August 2015

Asp-130 of the recombinant skeletal-muscle 6-phosphofructo-2-kinase (PFK-2)/fructose-2,6-bisphosphatase was mutated into Ala in order to study its role in catalysis and/or substrate binding. The D130A mutant displayed a 30- to 140-fold decreased 2-kinase Vmax, depending on the pH, and a 30- and 60-fold increase in Km for MgATP and Fru-6-P respectively at pH 8.5 compared with the wild-type. Mutagenesis of Asp-130 to Ala had no effect on the 2-phosphatase activity, and fluorescence measurements indicated that the changes in kinetic properties of PFK-2 in the D130A mutant were not due to instability. The role of Asp-130 in the 2-kinase reaction is discussed and compared with that of Asp-103 of 6-phosphofructo-1-kinase from Escherichia coli, which binds Mg2+.
ADP-ribosylation of Rho proteins by Clostridium botulinum exoenzyme C3 is influenced by phosphorylation of Rho-associated factors
Authors: Fritz G , Aktories K
Published: August 2015

Specific [32P]ADP-ribosylation by Clostridium botulinum exoenzyme C3 was used to study the involvement of phosphorylation in the regulation of the low-molecular-mass GTP-binding protein Rho. Dephosphorylation of CHO cell extracts by alkaline phosphatase treatment resulted in a 80-90% reduction in the C3-catalysed [32P]ADP-ribosylation of Rho proteins in both cytosolic and membrane fractions. Similar results were obtained after dephosphorylation with protein phosphatase type-1 from bovine retina, whereas type-2B and type-2C phosphatases had no effect on the level of subsequent [32P]ADP-ribosylation of Rho by C3. Incubation of CHO cell lysate under phosphorylation conditions increased the subsequent C3-mediated [32P]ADP-ribosylation of Rho proteins. The protein kinase inhibitors H7 and H9 had no effect on [32P]ADP-ribosylation at concentrations which are specific for inhibition of protein kinase A or C. Recombinant glutathione S-transferase-RhoA fusion protein (GST-RhoA) was phosphorylated by protein kinase A; however, the phosphorylation had no stimulatory effect on the ADP-ribosylation of GST-RhoA by C3. An approx. 48 kDa phosphoprotein was identified which bound specifically to recombinant GST-RhoA fusion protein. By gel-permeation chromatography, Rho-containing complexes of approx. 50 kDa and 130-170 kDa were detected. The ADP-ribosylation of Rho in the 130-170 kDa complex was reduced by alkaline phosphatase pretreatment. The data suggest that Rho activity is influenced by phosphorylation of Rho-associated regulatory factors. Phosphorylation/dephosphorylation of these Rho-regulating factors appears to alter the ability of Rho to serve as a substrate for C3-induced [32P]ADP-ribosylation.
The green alga Scenedesmus obliquus contains both diadenosine 5′,5‴-P1,P4-tetraphosphate (asymmetrical) pyrophosphohydrolase and phosphorylase activities
Authors: McLennan A G , Mayers E , Hankin S , Thorne N M H , Prescott M , Powls R
Published: August 2015

Diadenosine 5′,5‴-P1,P4-tetraphosphate (Ap4A) phosphorylase and Ap4A pyrophosphohydrolase activities have been purified from extracts of the green alga Scenedesmus obliquus. Both activities were also detected in Scenedesmus brasiliensis, Scenedesmus quadricauda and in Chlorella vulgaris. This is the first time that both types of enzyme have been detected in the same species. The Ap4A phosphorylase has a molecular mass of 46-48 kDa, a broad pH optimum between 7.5 and 9.5, and requires a divalent ion for activity (Mg2+ > Co2+ > Ca2+ = Mn2+ = Cd2+ > Zn2+). It degrades substrates with at least four phosphate groups and always produces a nucleoside 5′-diphosphate product. The Km values for Ap4A and Pi are 5.3 microM and 160 microM, respectively, and kcat. = 1.8 s-1. Arsenate, vanadate, molybdate, chromate and tungstate can substitute for phosphate. The enzyme also catalyses Ap4A synthesis (Keq. = [Ap4A] [Pi]/[ATP][ADP] = 9 x 10(-4)) and ADP arsenolysis. The Ap4A hydrolase has a molecular mass of 26-28 kDa, an alkaline pH optimum of 8.8-9.8, and prefers Zn2+ as the stimulatory ion (Zn2+ > Mg2+ > Mn2+ > Co2+ > Cd2+). It degrades substrates with at least four phosphate groups, having a slight preference for Ap5A, and always produces a nucleoside 5′-triphosphate product. The Km value for Ap4A is 6.6 microM and kcat. = 1.3 s-1. It is inhibited competitively by adenosine 5′-tetraphosphate (Ki = 0.67 microM) and non-competitively by fluoride (Ki = 150 microM). A 50-54 kDa dinucleoside 5′,5‴-P1,P3-triphosphate (Ap3A) pyrophosphohydrolase was also detected in S. obliquus, S. quadricauda and C. vulgaris. The corresponding enzyme in S. brasiliensis (> 100 kDa) may be a dimer
ATP citrate-lyase and glycogen synthase kinase-3β in 3T3-L1 cells during differentiation into adipocytes
Authors: Benjamin W B , Pentyala S N , Woodgett J R , Hod Y , Marshak D
Published: August 2015

ATP citrate-lyase (CL), acetyl-CoA carboxylase (ACC) and glycogen synthase kinase-3 beta (GSK-3 beta) levels were measured in cytosol from 3T3-L1 cells during differentiation from fibroblasts into fat-cells. Protein levels were estimated from immunoblots using specific antisera. Cytosol from confluent cells contain significant amounts of GSK-3 beta, which fell during differentiation of these cells into adipocytes. CL from confluent cells was found to be mostly in the form of a single protein band of apparent mass 110 kDa. Levels of CL and ACC increased during cell differentiation into adipocytes. During the first 3 days of differentiation, CL migration changed, and it was expressed as a complex of protein bands of apparent mass 110 kDa, 113 kDa and 115 kDa. At later stages of differentiation, when these cells had assumed the phenotype of fat-cells, they expressed CL mainly as protein bands of 110 and 113 kDa. When samples containing these bands were treated with alkaline phosphatase, the 113 kDa protein band collapsed into the 110 kDa species. This suggests that the slower-migrating species of CL is a higher-order phosphorylation state of the same protein. Furthermore, when purified CL, mostly expressed as the 110 kDa species, was phosphorylated with cyclic AMP-dependent protein kinase alone or together with GSK-3 and resolved by SDS/PAGE, the phosphorylated CL now migrated more slowly as the 113 kDa and 115 kDa forms. CL phosphorylation was hormone-regulated, since, in samples from fat-cells that had the complex two-band pattern, when cultured in medium without serum or hormones, CL migration reverted to a single band of 110 kDa, similar to confluent cells. Treatment of these ‘down-regulated’ cells with insulin rapidly induced substantial amounts of the 113 kDa species, with a concomitant decrease in the 110 kDa species.
Human leucocyte glycosylasparaginase is an α/β-heterodimer of 19 kDa α-subunit and 17 and 18 kDa β-subunit
Authors: Tollersrud O K , Heiskanen T , Peltonen L
Published: August 2015

Human lysosomal glycosylasparaginase (AGA; EC 3.5.1.26) consists of two glycosylated subunits, alpha and beta. Treatment with 3% SDS at 45 degrees C as part of a new purification scheme did not affect enzyme activity, but the alpha-subunit migrated an apparent 19 kDa peptide on SDS/PAGE instead of as a 24 kDa peptide, as observed without this SDS treatment. The N-terminal sequence was similar to that of the 24 kDa form, and, after reversed-phase h.p.l.c., the 19 kDa form was transformed to an apparent 24 kDa peptide on SDS/PAGE, indicating that their primary structures were identical. As the molecular mass of the alpha-subunit deduced from its cDNA was 19.5 kDa, the variation might be due to incomplete SDS coating of the 24 kDa form. This was confirmed by the tendency of the 24 kDa variant to polymerize even in the presence of SDS. The molecular mass of the beta-subunit was 17 and 18 kDa in accordance with previous reports. Chemical cross-linking with 1-ethyl-3-(3-dimethylaminopropyl)carbodi-imide resulted in the appearance of a 38 kDa peptide on SDS/PAGE which reacted with both the subunit-specific antisera on Western-blot analysis. On SDS/PAGE at pH 10.2 the active enzyme migrated as an apparent 43 kDa peptide. These results indicate that native human glycosylasparaginase is a heterodimer.
Selective down-regulation of protein kinase c-ε by carcinogens does not prevent stimulation of phospholipase D by phorbol ester and platelet-derived growth factor
Authors: Kiss Z , Anderson W H
Published: August 2015

It is well established that activators of protein kinase C (PKC) also enhance the activity of phospholipase D (PLD), and that this regulatory mechanism is altered in transformed cells. Here we used the C3H/10T1/2 mouse embryo fibroblast line, a cellular model for the study of carcinogenesis, to examine possible effects of carcinogens on the PKC isoenzyme pattern and on the regulation of PLD by the PKC activators phorbol 12-myristate 13-acetate (PMA) and platelet-derived growth factor (PDGF). Treatment of these fibroblasts with 0.5 microgram/ml 7,12-dimethyl-benz[a]anthracene or benzo[a]pyrene for 24 h greatly decreased (> 80%) the amount of immunoreactive PKC-epsilon. Of the remaining three isoenzymes identified, carcinogens alone had no effect on the cellular status of PKC-alpha and PKC-delta, although they appeared to promote slightly PMA-induced membrane translocation of the cytosolic forms of these isoenzymes in exponentially growing cells. Carcinogens and/or PMA had no effects on the cellular content or distribution of PKC-zeta. Chronic (24 h) treatments with carcinogens resulted in increased or decreased release of [14C]ethanolamine or [14C]choline from the appropriate prelabelled phospholipids, respectively. However, carcinogens failed to block the stimulatory effects of PMA and PDGF on the hydrolysis of phosphatidylethanolamine and phosphatidylcholine or on the synthesis of phosphatidylethanol mediated by PLD. These data indicate that in fibroblasts PKC-epsilon is not a major regulator of PLD activity.
The effect of replacing the conserved active-site residues His-264, Asp-312 and Arg-314 on the binding and catalytic properties of Escherichia coli citrate synthase
Authors: Man W J , Li Y , O'Connor C D , Wilton D C
Published: August 2015

The first step in the overall catalytic mechanism of citrate synthase is the binding and polarization of oxaloacetate. Active-site residues Arg-314, Asp-312 and His-264 in Escherichia coli citrate synthase, which are involved in oxaloacetate binding, were converted by site-directed mutagenesis to Gln-314, Asn-312 and Asn-264 respectively. The R314Q and D312N mutants expressed negligible overall catalytic activity at pH 8.0, the normal assay pH, but substantial activities for the partial reactions that reflect the cleavage and hydrolysis of the substrate intermediate citryl-CoA. However, when the pH was lowered to 7.0, the overall reaction of the mutants became significant, in contrast to the wild-type enzyme, whereas the two mutants exhibited reduced activities for the partial reactions. This result is consistent with the existence of a rate-limiting step between the two partial reactions for these mutants that is pH-dependent. The Km for oxaloacetate for the two mutants was increased 10-fold and was paralleled by an increase in the Km for citryl-CoA, whereas the Km for acetyl-CoA was increased only 2-fold. Overall, there was a striking parallel between the results obtained for these two mutants, which suggests that they are functionally linked in the E. coli enzyme. The equivalent of these two residues form a salt bridge in the pig heart citrate synthase crystal structure. The H264N mutant, in which the amide nitrogen of asparagine should mimic the delta-nitrogen of histidine, showed negligible activity in terms of both overall and partial catalysis, which may result from a hindrance of conformational change upon oxaloacetate binding. The affinity of this mutant for oxaloacetate appeared to be greatly reduced when investigated using indirect fluorescence and chemical modification techniques.
The effect of iron overload on rat plasma and liver oxidant status in vivo
Authors: Dabbagh A J , Mannion T , Lynch S M , Frei B
Published: August 2015

There is ample evidence implicating reactive oxygen species in a number of human degenerative diseases such as atherosclerosis and haemochromatosis. Although lipid peroxidation underlies many of the toxic effects of oxidative stress, there is a lack of a sensitive and reliable method for its assessment in vivo. To understand the implications of oxidative stress in vivo, we have used dietary iron overload (IO) in the rat. Oxidant status in these animals was determined by assessing depletion of endogenous antioxidants and formation of various lipid peroxidation products, including acylated F2-isoprostanes, a novel class of free-radical-derived prostaglandin-F2-like compounds. IO led to a significant decrease in the concentration of the antioxidants alpha-tocopherol and ascorbic acid in plasma, and alpha-tocopherol, beta-carotene and ubiquinol-10 in liver. Whereas there was no significant lipid peroxidation in plasma, hepatic F2-isoprostane levels were moderately but significantly increased in IO. In addition, IO caused a significant increase in plasma total and high-density lipoprotein cholesterol levels, an effect that was correlated with depletion of plasma ascorbic acid but not alpha-tocopherol. The data demonstrate that IO causes lipid metabolism disturbances and oxidative stress which is associated with substantial depletion of endogenous antioxidants and moderate lipid peroxidative damage.
The kinetics of the oxidation of cytochrome c by Paracoccus cytochrome c peroxidase
Authors: Gilmour R , Goodhew C F , Pettigrew G W , Prazeres S , Moura J J , Moura I
Published: August 2015

In work that is complementary to our investigation of the spectroscopic features of the cytochrome c peroxidase from Paracoccus denitrificans [Gilmour, Goodhew, Pettigrew, Prazeres, Moura and Moura (1993) Biochem. J. 294, 745-752], we have studied the kinetics of oxidation of cytochrome c by this enzyme. The enzyme, as isolated, is in the fully oxidized form and is relatively inactive. Reduction of the high-potential haem at pH 6 with ascorbate results in partial activation of the enzyme. Full activation is achieved by addition of 1 mM CaCl2. Enzyme activation is associated with formation of a high-spin state at the oxidized low-potential haem. EGTA treatment of the oxidized enzyme prevents activation after reduction with ascorbate, while treatment with EGTA of the reduced, partially activated, form abolishes the activity. We conclude that the active enzyme is a mixed-valence form with the low-potential haem in a high-spin state that is stabilized by Ca2+. Dilution of the enzyme results in a progressive loss of activity, the extent of which depends on the degree of dilution. Most of the activity lost upon dilution can be recovered after reconcentration. The M(r) of the enzyme on molecular-exclusion chromatography is concentration-dependent, with a shift to lower values at lower concentrations. Values of M(r) obtained are intermediate between those of a monomer (39,565) and a dimer. We propose that the active form of the enzyme is a dimer which dissociates at high dilution to give inactive monomers. From the activity of the enzyme at different dilutions, a KD of 0.8 microM can be calculated for the monomerdimer equilibrium. The cytochrome c peroxidase oxidizes horse ferrocytochrome c with first-order kinetics, even at high ferrocytochrome c concentrations. The maximal catalytic-centre activity (‘turnover number’) under the assay conditions used is 62,000 min-1, with a half-saturating ferrocytochrome c concentration of 3.3 microM. The corresponding values for the Paracoccus cytochrome c-550 (presumed to be the physiological substrate) are 85,000 min-1 and 13 microM. However, in this case, the kinetics deviate from first-order progress curves at all ferrocytochrome c concentrations. Consideration of the periplasmic environment in Paracoccus denitrificans leads us to propose that the enzyme will be present as the fully active dimer supplied with saturating ferrocytochrome c-550.
Assisted refolding of recombinant prochymosin with the aid of protein disulphide isomerase
Authors: Tang B , Zhang S , Yang K
Published: August 2015

Protein disulphide isomerase (PDI) was shown to be able to accelerate the refolding of unfolded recombinant prochymosin and to enhance the overall yield of active protein. Unlike previous reports in this study PDI was found to be active at pH values as high as 11. The coincidence of the similar apparent optimum pH values of uncatalysed and PDI-catalysed reactions suggests that conditions favourable to spontaneous refolding of proteins may help PDI to catalyse thiol/disulphide interchange. Under the conditions described here no exogenously added dithiothreitol was required for PDI-catalysed renaturation, implying that the disulphide form of PDI was reduced to its active form by the free thiol groups in prochymosin molecules.
Regulation of the GLUT1 glucose transporter in cultured myocytes: total number and subcellular distribution as determined by photoaffinity labelling
Authors: el-Kebbi I M , Roser S , Pollet R J , Cushman S W , Wilson C M
Published: August 2015

We have used the impermeant photoaffinity label 2-N-4-(1-azi-2,2,2-trifluoroethyl)benzoyl-[2-3H] 1,3-bis-(D-mannos-4-yloxy)-2-propylamine (ATB-[2-3H]BMPA) to identify and quantify the glucose transporters on the surface of BC3H-1 cells, a continuously cultured skeletal-muscle cell line lacking the MyoD transcription factor required for cell fusion. ATB-[2-3H]BMPA was used in combination with immunoprecipitation of the GLUT1 glucose transporter, the only isoform expressed in these cells. The total cellular GLUT1 content was also determined by photolabelling and immunoprecipitation after cell permeabilization with digitonin (0.025%). In glucose-starved cells, 85% of the glucose transporters were present at the cell surface in the basal state, with little change in response to insulin (200 nM), correlating with lack of additional 2-deoxyglucose uptake in response to insulin. Feeding the cells with glucose (25 mM) for 24 h resulted in an 80% decrease in the total GLUT1 content relative to starved cells, of which only 25% were present on the cell surface. This was associated with an 85% decrease in 2-deoxyglucose uptake. In addition, acute stimulation of the fed cells with insulin or phorbol 12-myristate 13-acetate (PMA) led to an increase in GLUT1 at the cell surface, and, in correspondence, an increase in 2-deoxyglucose uptake by approx. 2- and 4-fold respectively. We conclude that exofacial photoaffinity labelling of glucose transporters with ATB-[2-3H]BMPA in the presence and absence of digitonin, followed by specific immunoprecipitation, provides an accurate measure of total and cell-surface glucose transporters in differentiated BC3H-1 muscle cells. This technique demonstrates that glucose pre-feeding (1) decreases the total number of GLUT1 and (2) redistributes the majority of the remaining transporters to an intracellular site, where they can now be translocated to the cell surface in response to insulin and PMA.
Conversion of epidermal growth factor (EGF) into a stimulatory ligand for A431-cell growth by herbimycin A by decreasing the level of expression of EGF receptor
Authors: Murakami Y , Fukazawa H , Mizuno S , Uehara Y
Published: August 2015

We examined effect of the tyrosine kinase inhibitor herbimycin A on A431 human epidermoid carcinoma cells which over-express epidermal growth factor (EGF) receptor. Herbimycin A inhibited the autophosphorylation of EGF-stimulated receptors in intact cells both time- and dose-dependently. The inhibition was found to be due to a decrease in the level of expression of the receptor amount, because herbimycin A equally decreased the receptor quantity and EGF-stimulated receptor kinase activity in intact cells, but did not exhibit a direct inhibitory effect on EGF receptor kinase activity in vitro. The decrease of the level of EGF receptor was also confirmed by 125I-EGF binding to herbimycin A-treated cells, and Scatchard analysis showed that the decrease in the receptor number occurred in the major population of the low-affinity binding ones, whereas the number with high-affinity binding was unaffected. Interestingly, although the proliferation of A431 cells was inhibited by EGF, herbimycin A converted EGF into a stimulatory ligand for cell growth, as determined by both cell number and DNA synthesis. These findings indicated that herbimycin A decreased the level of expression of EGF receptor by a mechanism other than inactivation of the receptor kinase and reversed the transformed phenotype of A431 cells to a normal one in the proliferative response to EGF.
Induction of an inactivation pathway for ecdysteroids in larvae of the cotton leafworm, Spodoptera littoralis
Authors: Chen J H , Kabbouh M , Fisher M J , Rees H H
Published: August 2015

Treatment of the last-instar larvae of the cotton leafworm (Spodoptera littoralis) with ecdysteroids (moulting hormones) results in the induction of an ecdysteroid-inactivation pathway. Administration of ecdysone, 20-hydroxyecdysone or an ecdysteroid agonist, RH 5849, leads to induction of an ecdysteroid 26-hydroxylase activity. This induction occurred in both early sixth-instar larvae and in older larvae which had been head-ligated to prevent the normal developmental increase in ecdysone 20-mono-oxygenase activity. The induction of 26-hydroxylase activity requires both RNA and protein synthesis, as demonstrated by experiments involving actinomycin D and cycloheximide. The 26-aldehyde derivative of ecdysone and ecdyson-26-oic acid were also formed from ecdysone in the RH 5849-induced systems. Formation of the aldehyde and the corresponding 26-oic acid (ecdysonoic acid) from 26-hydroxyecdysone was directly demonstrated in a cell-free system, thus establishing the following inactivation pathway: Ecdysteroid-->26-hydroxyecdysteroid-->ecdysteroid 26-aldehyde-->ecdysteroid 26-oic acid.