bluuebunny/crossref_metadata_2025 huggingface dataset:
Artemia purine phosphoribosyltransferases. Purification and characterization
Authors: Montero C , Llorente P
Published: August 2015

Adenine phosphoribosyltransferase (APRTase) and hypoxanthine-guanine phosphoribosyltransferase (HGPRTase) have been purified from Artemia cysts and nauplii to apparent homogeneity, as determined by SDS-PAGE. The purification includes affinity chromatography on AMP-Sepharose, which binds both enzymes, and they are eluted at different 5-phospho-alpha-D-ribosyl diphosphate (PP-Rib-P) concentrations. The purified enzymes from Artemia cysts were similar to nauplii enzymes with respect to Mr in denaturing gel electrophoresis and gel filtration, pH and cation dependence and kinetic constants for substrates and inhibitors. By Sephadex G-100 filtration, the native Mr of the adenine and hypoxanthine-guanine enzymes was estimated to be Mr 28,000 and 66,000, respectively. Analysis by SDS-PAGE revealed that the APRTase was a dimer of Mr 15,000 sub-units and the HGPRTase, a tetramer of four identical Mr 19,000 sub-units. The pH profile of the HGPRTase shows two apparent buffer-independent pH optima, at 7.0 and 9.5, while the APRTase has just one, at about pH 8-9. The purine phosphoribosyltransferase activity with adenine was highest, about tenfold the HGPRTase activity with hypoxanthine and fivefold that with guanine. Both enzymes exhibited similar requirements for divalent cations, either Mg2+, Mn2+ or Zn2+, while Ca2+ is highly inhibitory. The Km values of APRTase for adenine and PP-Rib-P are 2 and 30 microM, respectively, and the Km values of HGPRTase for hypoxanthine, guanine and PP-Rib-P are less than 1, less than 1 and 15 microM, respectively. Plots of the reciprocal enzyme activities versus reciprocal concentrations of one substrate at several fixed levels of the second one yield a pattern of inhibition by guanine and hypoxanthine. Product-inhibition studies indicated that AMP is a competitive inhibitor with respect to PP-Rib-P in the APRTase reaction, while the HGPRTase shows a mixed inhibition by GMP.
Comparison of the activities of protein disulphide-isomerase and thioredoxin in catalysing disulphide isomerization in a protein substrate
Authors: Hawkins H C , Blackburn E C , Freedman R B
Published: August 2015

1. The activities of protein disulphide-isomerase (PDI) and thioredoxin in catalysing disulphide bond isomerization in a protein substrate were compared by using the standard assay, namely the re-activation of ‘scrambled’ RNAase. 2. The specific activity of PDI was 25-fold greater than that of thioredoxin. 3. The greater efficiency of PDI compared with thioredoxin is considered to be due more to the presence of multiple catalytic domains in PDI than to differences in their active-site sequences. 4. Data and procedures were defined for expressing enzyme activity in standard units, i.e. mumol of active RNAase generated/min.
The inositol phosphate/diacylglycerol signalling pathway in Trypanosoma cruzi
Authors: Docampo R , Pignataro O P
Published: August 2015

Using [32P]Pi and [3H]inositol as precursors, we have detected the presence of phosphatidylinositol, phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate, and their derivatives inositol phosphate, inositol 1,4-bisphosphate and inositol 1,4,5-trisphosphate respectively, in Trypanosoma cruzi epimastigotes. Using digitonin-permeabilized cells it was possible to detect a stimulation in the formation of inositol 1,4,5-trisphosphate and inositol 1,4-bisphosphate as well as an increased generation of diacylglycerol in the presence of 1 mM-CaCl2. These results are consistent with the operation of a functional inositol phosphate/diacylglycerol pathway in T. cruzi, and constitute the first demonstration of the presence and activation of this pathway in a parasitic protozoan. These results also indicate that this pathway is conserved during evolution from lower to higher eukaryotic organisms.
Molecular structure of the human muscle-specific enolase gene (ENO3)
Authors: Peshavaria M , Day I N
Published: August 2015

The single human gene for muscle-specific enolase was isolated and its structure was characterized, from which the mature mRNA transcript and encoded protein were also deduced. The gene contains 12 exons, spans approx. 6 kb and encodes a protein of 433 residues. The gene structure is similar to that found for the rat neuron-specific enolase gene, and the deduced protein aligns precisely with other enolase sequences, including the sequence of the only published crystallized enolase, yeast eno-1. The 5′ boundary of the gene includes a 5′ non-coding exon and is characterized by an upstream TATA-like box and CpG-rich region. This region contains potential recognition motifs for general transcriptional regulation involving Sp1, activator protein 1 and 2, CCAAT box transcription factor/nuclear factor I and cyclic AMP, and for muscle-specific transcriptional regulation involving a CC(A + T-rich)6GG box, M-CAT-box CAATCCT and two myocyte-specific enhancer-binding factor 1 boxes.
Inhibition of prolyl hydroxylation and procollagen processing in chick-embryo calvaria by a derivative of pyridine-2,4-dicarboxylate. Characterization of the diethyl ester as a proinhibitor
Authors: Tschank G , Brocks D G , Engelbart K , Mohr J , Baader E , Günzler V , Hanauske-Abel H M
Published: August 2015

The biochemical and morphological consequences of procollagen prolyl 4-hydroxylase inhibition by pyridine-2,4-dicarboxylic acid (2,4-PDCA) and its diethyl ester (diethyl-2,4-PDC) were studied in chick-embryo calvaria, which predominantly synthesize type I collagen. Half-maximal inhibition of tissue hydroxyproline formation required 650 microM-2,4-PDCA, whereas the Ki with respect to chicken prolyl 4-hydroxylase in vitro was 2 microM. In contrast, half-maximal inhibition was caused by 10 microM-diethyl-2,4-PDC in the intact calvaria, although chicken prolyl 4-hydroxylase in vitro was not inhibited even at 1 mM. The collagenous material produced in the presence of diethyl-2,4-PDC showed an altered ‘melting’ profile and a lowering of the transition temperature by 10 degrees C, indicating misalignment and thermal instability of its triple-helical structure. Amount and electrophoretic mobility of procollagen type I chains were increased in a dose-dependent manner. The amounts of partially processed species and alpha-chains were decreased, without change in mobility. This marked effect on procollagen-collagen conversion in the intact calvaria suggests that the underhydroxylated collagenous material generated in the presence of diethyl-2,4-PDC is resistant to or acts as endogenous secondary inhibitor of type I procollagen N-proteinase. Electron microscopy of treated calvaria cells showed dilated rough endoplasmic reticulum and numerous phagolysosomes, indicating intracellular retention and lysosomal degradation of the newly synthesized underhydroxylated collagenous material. In summary, these results identify 2,4-PDCA and diethyl-2,4-PDC as the first prolyl 4-hydroxylase-directed inhibitor/proinhibitor pair that affects intra- and extra-cellular events during collagen formation.
Cyclosporin A blocks bile acid synthesis in cultured hepatocytes by specific inhibition of chenodeoxycholic acid synthesis
Authors: Princen H M , Meijer P , Wolthers B G , Vonk R J , Kuipers F
Published: August 2015

Bile acid synthesis, determined by conversion of [4-14C]cholesterol into bile acids in rat and human hepatocytes and by measurement of mass production of bile acids in rat hepatocytes, was dose-dependently decreased by cyclosporin A, with 52% (rat) and 45% (human) inhibition of 10 microM. The decreased bile acid production in rat hepatocytes was due only to a fall in the synthesis of beta-muricholic and chenodeoxycholic acids (-64% at 10 microM-cyclosporin A), with no change in the formation of cholic acid. In isolated rat liver mitochondria, 26-hydroxylation of cholesterol was potently inhibited by the drug (concn. giving half-maximal inhibition = 4 microM). These results suggest that cyclosporin A blocks the alternative pathway in bile acid synthesis, which leads preferentially to the formation of chenodeoxycholic acid.
Independent segregation of glutathione S-transferase and fatty acid ethyl ester synthase from pancreas and other human tissues
Authors: Sharma R , Gupta S , Singhal S S , Ahmad H , Haque A , Awasthi Y C
Published: August 2015

Glutathione S-transferase (GST) isoenzymes of human pancreas were purified, characterized and evaluated for their possible role in the metabolism of ethanol. Human pancreas has at least two GST isoenzymes belonging to the Alpha class (pI 8.8 and 8.1), one belonging to the Mu class (pI 6.4) and one belonging to the Pi class (pI 4.9). During the purification of GSTs from pancreas as well as from heart, liver, lung, brain and muscle, the fatty acid ethyl ester synthase (FAEES) activity was monitored in order to evaluate the role of GSTs in metabolism of ethanol, as suggested in earlier studies. Both t.l.c. and h.p.l.c. were used to identify ethyl oleate in reaction mixtures to monitor FAEES activity. During the purification of GSTs with the use of affinity chromatography on GSH linked to epoxy-activated Sepharose 6B, FAEES and GST activities from each of these tissues segregated independently. Purified GST isoenzymes from these tissues did not exhibit any FAEES activity. Antibodies raised against Pi-class GST, as expected, immunoprecipitated most of the GST activity of brain and heart without precipitating FAEES activity. These results suggest that human GST isoenzymes belonging to the Alpha, Mu and Pi classes do not express FAEES activity. The independent segregation of GST and FAEES activities was further demonstrated by monitoring GST activity during the purification of FAEES from pancreas. It was found that purified FAEES had no GST activity towards 1-chloro-2,4-dinitrobenzene and a number of other electrophilic substrates. Results of these studies demonstrate that FAEES and GSTs are distinct proteins.
Amino acid distributions around O-linked glycosylation sites
Authors: Wilson I B , Gavel Y , von Heijne G
Published: August 2015

To study the sequence requirements for addition of O-linked N-acetylgalactosamine to proteins, amino acid distributions around 174 O-glycosylation sites were compared with distributions around non-glycosylated sites. In comparison with non-glycosylated serine and threonine residues, the most prominent feature in the vicinity of O-glycosylated sites is a significantly increased frequency of proline residues, especially at positions -1 and +3 relative to the glycosylated residues. Alanine, serine and threonine are also significantly increased. The high serine and threonine content of O-glycosylated regions is due to the presence of clusters of several closely spaced glycosylated hydroxy amino acids in many O-glycosylated proteins. Such clusters can be predicted from the primary sequence in some cases, but there is no apparent possibility of predicting isolated O-glycosylation sites from primary sequence data.
Formation of neutrophil-activating peptide 2 from platelet-derived connective-tissue-activating peptide III by different tissue proteinases
Authors: Car B D , Baggiolini M , Walz A
Published: August 2015

Neutrophil-activating peptide 2 (NAP-2) is generated by cleavage of two inactive precursors, connective-tissue-activating peptide III (CTAP-III) and platelet basic protein (PBP), which are stored in the alpha-granules of blood platelets. Using highly purified CTAP-III as the substrate we studied the generation of NAP-2 by several neutral tissue proteinases. CTAP-III was rapidly cleaved by chymotrypsin, cathepsin G and trypsin, yielding products with neutrophil-stimulating activity. This activity remained unchanged for 24 h in the presence of chymotrypsin, decreased only slowly in the presence of cathepsin G, but was rapidly destroyed by trypsin. CTAP-III was also degraded by human neutrophil elastase and porcine pancreatic elastase, but no active fragments were obtained. By contrast, no degradation of CTAP-III was observed with thrombin, plasmin or ‘granzymes’ from cytolytic T-lymphocyte granules. Two active fragments of CTAP-III, generated by chymotrypsin or cathepsin G, were purified and partially sequenced, and were found to have the same N-terminal sequence as NAP-2. These results indicate that both proteinases cleave preferentially the bond between amino acids 15 (Tyr) and 16 (Ala) of CTAP-III. We conclude that chymotrypsin-like proteolytic activity in the vicinity of activated platelets may generate NAP-2 intravascularly. Due to its presence in the primary granules of neutrophils and monocytes cathepsin G is likely to be involved in this process.
Characterization and immunological properties of selenium-containing glutathione peroxidase induced by selenite in Chlamydomonas reinhardtii
Authors: Shigeoka S , Takeda T , Hanaoka T
Published: August 2015

The selenite-induced glutathione peroxidase in Chlamydomonas reinhardtii has been purified about 323-fold with a 10% yield, as judged by PAGE. The native enzyme had an Mr of 67,000 and was composed of four identical subunits of Mr 17,000. Glutathione was the only electron donor, giving a specific activity of 193.6 mumol/min per mg of protein. L-Ascorbate, NADH, NADPH, pyrogallol, guaiacol and o-dianisidine did not donate electrons to the enzyme. In addition to H2O2, organic hydroperoxides were reduced by the enzyme. The Km values for glutathione and H2O2 were 3.7 mM and 0.24 mM respectively. The enzyme reaction proceeded by a Ping Pong Bi Bi mechanism. Cyanide and azide had no effect on the activity. The enzyme contained approx. 3.5 atoms of selenium per mol of protein. On immunoprecipitation, Chlamydomonas glutathione peroxidase was precipitated and its activity was inhibited about 90% by the antibody raised against bovine erythrocyte glutathione peroxidase. The antibody also cross-reacted with the subunits of Chlamydomonas glutathione peroxidase in Western blotting SDS/PAGE. In terms of enzymic, physico-chemical and immunological properties, the experimental results demonstrate clearly that Chlamydomonas glutathione peroxidase resembles other well-characterized glutathione peroxidases from animal sources that contain selenium.
Purification, properties and influence of dietary copper on accumulation and functional activity of lysyl oxidase in rat skin
Authors: Romero-Chapman N , Lee J , Tinker D , Uriu-Hare J Y , Keen C L , Rucker R R
Published: August 2015

Lysyl oxidase (protein-lysine 6-oxidase; EC 1.4.3.13) is a copper-containing enzyme that functions extracellularly and catalyses the oxidative deamination of peptidyl lysine. Lysyl oxidase was purified 150-175-fold from urea extracts of rat skin and uteri. Features of the enzyme were similar to those reported previously for lysyl oxidase obtained from rat aorta and bovine ligamenture. However, both approximately 40 and approximately 32 kDa polypeptide chains could be isolated from rat skin with apparent lysyl oxidase activity. Antibodies raised in chickens against the approximately 40 kDa form of lysyl oxidase detected the approximately 32 kDa form in immunoblots. Consequently it is inferred that the approximately 32 kDa form of lysyl oxidase is processed from the approximately 40 kDa form of the enzyme. The antibodies were also used to prepare anti(rat lysyl oxidase) affinity columns to facilitate the separation of lysyl oxidase from other proteins in studies to assess the extent to which lysyl oxidase serves as a reservoir for skin copper. At 16 h after an oral dose of copper, as 67Cu, about 6-8% of the total 67Cu incorporated into rat skin was found in association with lysyl oxidase. The lysyl oxidase concentration in rat skin was 2.5-7.5 nmol/g (determined by e.l.i.s.a.). Changing the copper status of rats by feeding a diet deficient in copper did not appear to influence lysyl oxidase accumulation in skin nor the percentage of incorporation of 67Cu in skin as lysyl oxidase. However, when rats were deprived of copper, the functional activity of lysyl oxidase in skin was one-third to one-half the normal values.
Activation of smooth muscle myosin light chain kinase activity by a monoclonal antibody which recognizes the calmodulin-binding region
Authors: Araki Y , Ikebe M
Published: August 2015

The regulatory domain of smooth muscle myosin light chain kinase (MLCK) was studied using monoclonal antibodies. Of the 22 monoclonal antibodies tested, a monoclonal antibody designated LKH-18 was found to activate MLCK in the absence of Ca2+/calmodulin. This activation was even greater when an Fab fragment of LKH-18 was used. Consequently, the actin-dependent smooth muscle myosin ATPase activity and the superprecipitation of actomyosin were significantly activated by MLCK plus LKH-18, even in the absence of Ca2+/calmodulin. The antibody-binding site was studied using proteolytic fragments and synthetic peptide analogues of MLCK. Immunoblot analysis revealed that LKH-18 reacted with the 66 kDa calmodulin-dependent active fragment but not with the 64 kDa inactive fragment or with the 61 kDa calmodulin-independent active fragment. Furthermore, LKH-18 reacted with MLCK-(796-815)-peptide but not with MLCK-(786-801)-peptide or with MLCK-(796-807)-peptide. Therefore the LKH-18-binding site was assigned to amino acid residues 808-815 of MLCK, which are thought to be a part of the calmodulin-binding site. The present results suggest that the binding of ligand to this region induces a conformation change in MLCK and that this abolishes the action of the inhibitory region which exists next to the N-terminal side of the calmodulin-binding site.
The octameric structure of β-glucosidase from Botryodiplodia theobromae Pat
Authors: Umezurike G M
Published: August 2015

1. Whereas only beta-glucosidase A (beta-D-glucoside glucohydrolase, EC 3.2.1.21) was produced by the tropical fungus Botryodiplodia theobromae Pat. (I.M.I. 115626; A.T.C.C. 26123) in young cultures containing D-cellobiose as carbon source, lower-Mr forms (B, C and D) were found in older cultures when the pH had drifted from the initial value of pH 6.2 to pH 7.9. 2. The Michaelis constants (Km) of the various molecular forms of the enzyme were 0.30 +/- 0.03 mM-, 0.26 +/- 0.01 mM-, 0.20 +/- 0.02 mM- and 0.16 +/- 0.01 mM-o-nitrophenyl beta-D-glucopyranoside for beta-glucosidase forms A (Mr 320,000), B (Mr 160,000), C (Mr 80,000) and D (Mr 40,000) respectively. 3. Only beta-glucosidase D showed substrate inhibition. 4. Only L-arginine was found as the N-terminal residue, and beta-glucosidase A contained 31.7 +/- 0.6 mol of N-terminal L-arginine/mol of the enzyme. 5. Storage of purified beta-glucosidase A under mildly alkaline conditions caused its dissociation into the lower-Mr forms, whereas adjustment of the pH of a solution of beta-glucosidase A to pH 12.0 with 1 M-NaOH led to complete inactivation on incubation at 40 degrees C for 1 h and to the release of 25.2 +/- 1.5 mol of inorganic phosphate/mol of the enzyme. 6. O-Phospho-L-serine was isolated from the acid-hydrolysis product of beta-glucosidase A but not from that of beta-glucosidase D. 7. Reduction and carboxamidomethylation of the various forms of beta-glucosidase gave only one enzymically inactive protein with an Mr of 10,000-11,000. 8. After partial succinylation (3-carboxypropionylation) of beta-glucosidase D at pH 5.0 and removal of the precipitated protein formed, the supernatant solution contained beta-glucosidase components similar to the other molecular forms (A, B and C) and an aggregate (beta-glucosidase Xs) that gave a positive result in the alkaline hydroxylamine test, whereas N-succinylated beta-glucosidase D, an aggregate (form Xp) that behaved like beta-glucosidase Xs and traces of forms A, B and C were found by gel filtration of the solution of the precipitate solubilized at neutral pH (7.0-7.7). 9. These observations are discussed in terms of the proposed octameric structure of beta-glucosidase A based on the result of electron microscopy [Umezurike (1975) Biochem. J. 145, 361-368].
Alkylation of glyceraldehyde-3-phosphate dehydrogenase with haloacetylphosphonates. An unusual pH-dependence
Authors: Li Y K , Boggaram J , Byers L D
Published: August 2015

Two new alkylating reagents, chloro- and bromo-acetylphosphonate, were found to be very effective thiol-blocking reagents. The pH-dependence of the reaction of BAP with 2,4-dinitrothiophenol (25 degrees C, I 0.5) shows a tailing bell-shaped curve (with a plateau at high pH) characteristic of two ionizing groups: the thiol group (pKa 3.2) and the phosphonate group (pKa2 4.6). The rate constant for the reaction of the monoanionic inhibitor with dinitrothiophenolate (k2 = 7 M-1.s-1) is 120 times larger than that of the dianionic species. The haloacetylphosphonates were found to be irreversible inhibitors of glyceraldehyde-3-phosphate dehydrogenase from a variety of sources. They react with the active-site thiol group (Cys-149) and are half-site reagents with yeast glyceraldehyde-3-phosphate dehydrogenase. Thus, when two of the identical four subunits are modified the enzyme is catalytically inactive. The effects of pH (7-10), 2H2O and NAD+ on the reaction with the yeast enzyme were examined in detail. NAD+ enhances the alkylation rates. The second-order rate constant does not show a simple sigmoidal dependence on pH but rather a tailing bell-shaped curve (pKa 7.0 and 8.4) qualitatively similar to that obtained with dinitrothiophenol. There is no significant solvent isotope effect on the limiting rate constants and a normal isotope effect on the two pKa values. The results are consistent with the more reactive enzyme species containing a thiolate and an acidic group that may either donate a proton to the dianionic haloacetylphosphonate or orient the inhibitor.
Phosphonate monoester inhibitors of class A β-lactamases
Authors: Rahil J , Pratt R F
Published: August 2015

Phosphonate monoesters with the general structure: [formula: see text] are inhibitors of representative class A and class C beta-lactamases. This result extends the range of this type of inhibitor to the class A enzymes. Compounds where X is an electron-withdrawing substituent are better inhibitors than the unsubstituted analogue (X = H), and enzyme inhibition is concerted with stoichiometric release of the substituted phenol. Slow turnover of the phosphonates also occurs. These observations support the proposition that the mechanism of action of these inhibitors involves phosphorylation of the beta-lactamase active site. The inhibitory ability of these phosphonates suggests that the beta-lactamase active site is very effective at stabilizing negatively charged transition states. One of the compounds described also inactivated the Streptomyces R61 D-alanyl-D-alanine carboxypeptidase/transpeptidase.
Computer program for the kinetic equations of enzyme reactions. The case in which more than one enzyme species is present at the onset of the reaction
Authors: Varón R , Havsteen B H , García M , García-Canóvas F , Tudela J
Published: August 2015

This paper presents an extension of the program developed by Varón, Havsteen, García, García-Cánovas & Tudela [(1990) Biochem. J. 270, 825-828] for the expression of the transient-phase and steady-state kinetic equations of a general enzyme system in which the only enzyme species present at the onset of the reaction is the free enzyme. The program has been extended to situations in which more than one enzyme species may be present at the onset of the reaction. The program is given in Supplementary Publication SUP50165 (5 pages), which has been deposited at the British Library Document Supply Centre, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1991) 273, 5.
The expression of glycogen phosphorylase in normal and dystrophic muscle
Authors: Leyland D M , Beynon R J
Published: August 2015

Specific cofactor labelling was employed to determine the degradation rate of glycogen phosphorylase in normal adult C57BL/6J mice and their dystrophic counterparts (C57BL/6Jdy/dy). The rate constant for the decay of phosphorylase-bound label was 0.125 day-1 in normal muscle and 0.49 day-1 in dystrophic muscle, i.e. a lower rate of catabolism of phosphorylase in dystrophic muscle. Quantitative Northern-blot analyses of total RNA isolated from normal and dystrophic muscle indicated that the abundance of phosphorylase mRNA as a percentage of total RNA was approx. 40% lower in dystrophic muscle. The specific activity of phosphorylase in dystrophic muscle is approx. 60% lower than in normal muscle, and is elicited by a lower rate of turnover of the enzyme, i.e. both synthesis and degradation are decreased.
Effects of macrophage colony-stimulating factor and phorbol myristate acetate on 2-d-deoxyglucose transport and superoxide production in rat peritoneal macrophages
Authors: Rist R J , Jones G E , Naftalin R J
Published: August 2015

2-D-Deoxyglucose (2-dGlc) uptake and accumulation into rat peritoneal macrophages was increased by colony-stimulating factor (mCSF) by stimulating the coupling between endofacial hexokinase activity and the sugar transporter. The evidence for this is as follows: (1) mCSF significantly decreased the Km for zero-trans uptake (P less than 0.05), without altering Vmax.; (2) the accumulation of free 2-dGlc was increased by mCSF (P less than 0.05); (3) mCSF retarded the rate of exit of accumulated free 2-dGlc. The mCSF-dependent increase in 2-dGlc uptake by macrophages was enhanced by preincubation of the cells in mCSF-free solution. The activity of the hexose monophosphate shunt (HMPS) measured by the differential uptake of 2-d[1-3H]Glc and 2-d[2,6-3H]Glc was not stimulated by mCSF. Also, in quiescent cells, superoxide production, as determined by cytochrome c reduction, was unaffected by mCSF. Phorbol myristate acetate (PMA; 40 nM) stimulated both the HMPS activity and superoxide production. Both these effects were dependent on the uptake of external sugar (2-dGlc). Incubation of the macrophages with mCSF enhanced the sugar transport and PMA-dependent stimulation of HMPS activity and superoxide production, indicating a role for mCSF in the ‘priming’ of macrophage functions. Both HMPS activity and superoxide production are entirely dependent on uptake of exogenous sugar, since the potent sugar-transport inhibitor cytochalasin B competitively inhibited 2-dGlc uptake, HMPS activity and superoxide generation in PMA-activated cells (Ki approximately 0.3 microM for all three processes). Over a wide range of 2-dGlc concentrations, 4 mol of superoxide were generated/mol of 2-dGlc metabolized in the HMPS pathway, indicating coupling between these processes. The Km of 2-d[2,6-3H]Glc uptake in PMA-treated cells was 0.45 +/- 0.07 mM, and Vmax. was 1.32 +/- 0.05 mumol.min-1.ml of cell water-1. It is evident that there is a large degree of slippage between HMPS activity and membrane-associated hexokinase activity, since the Km for HMPS activity was 0.06 +/- 0.02 mM and the Vmax. was 0.10 +/- 0.03 mumol.min-1.ml of cell water-1.
Altered affinity of insulin-like growth factor II (IGF-II) for receptors and IGF-binding proteins, resulting from limited modifications of the IGF-II molecule
Authors: Oh Y , Beukers M W , Pham H M , Smanik P A , Smith M C , Rosenfeld R G
Published: August 2015

The binding affinities of seven analogues of recombinant human insulin-like growth factor II (hIGF-II) were characterized for the IGF type-I and type-II receptors and insulin receptors, as well as for IGF-binding protein (IGFBP)-1, IGFBP-2, IGFPB-3 and human serum IGFBPs. A switch of two of the three cysteine bridges in hIGF-II, 9-47 and 46-51 to 9-46 and 47-51, severely impaired the binding of this analogue to all receptors and to the IGFBPs. The affinities for the IGF type-I receptor and the IGFBPs were decreased over 100-fold, while the binding to the insulin receptor and the IGF type-II receptor was less affected, with a 6-10-fold decrease in affinity. Slight modifications of the N-terminus had only minor effects upon the binding of hIGF-II to the IGFBPs or to the receptors. Deletion of both the N-terminal amino acid and the two C-terminal amino acids resulted in moderate decreases in affinity, with a 60% decrease in affinity for IGFBP-1 and the IGF type-I receptor. Acetylation of the N-terminus of Ala1 and the epsilon-nitrogen of Lys65 decreased the affinity, by 60-90%, of hIGF-II for all of the IGFBPs and receptors. The experiments involving acetylation of IGF-II or switching of its cysteine bridges indicated that these modifications (no substitution, deletion or addition of any of the 67 amino acids of hIGF-II) may lead to a severe impairment of the binding affinity of IGF-II for both the IGFBPs and the receptors. Acetylation of the epsilon-nitrogen of Lys65, which causes a charge change, or alteration of the three-dimensional structure, as shown by the cysteine bridge switch, lead to a severe impairment of the binding affinity for the binding proteins and for the receptors. In general, care should be taken with the synthesis of analogues and the interpretation of resulting binding data, since affinity alterations ascribed to amino acid changes may instead be caused by alterations of the charge or the three-dimensional structure of the protein.
The relationship between cytosolic Ca2+, sn-1,2-diacylglycerol and inositol 1,4,5-trisphosphate elevation in platelet-activating-factor-stimulated rabbit platelets. Influence of protein kinase C on production of signal molecules
Authors: Murphy C T , Elmore M , Kellie S , Westwick J
Published: August 2015

The temporal and dose-response relationships of platelet-activating-factor (PAF)-induced changes in the concentrations of cytosolic Ca2+ ([Ca2+]i), Ins(1,4,5)P3 and 1,2-diacylglycerol (DAG) were examined. In addition, phosphorylation of protein kinase C (PKC) substrate (40-47 kDa protein) was determined. In high-dose PAF-activated platelets, all three signal molecules increased rapidly and transiently, with the peak Ins(1,4,5)P3 concentration preceding maximal elevation of [Ca2+]i by 5 s. In low-dose PAF-activated platelets there were large increases in [Ca2+]i and dense-granule release, without any increase in Ins(1,4,5)P3 and DAG or 40-47 kDa protein phosphorylation. Staurosporine, a non-specific PKC inhibitor, produced enhanced elevations in the concentrations of Ins(1,4,5)P3, DAG and thromboxane B2, and the duration of the Ca2+ signal in platelets stimulated with a high dose, but not a low dose, of PAF. These results suggest there are both phospholipase C-dependent and -independent changes in Ca2+ homoeostasis. Endogenously activated PKC regulates the formation of signal molecules.
Expression in Escherichia coli and characterization of the fatty-acid-binding protein from human muscle
Authors: Peeters R A , Ena J M , Veerkamp J H
Published: August 2015

The coding part of the cDNA encoding human muscle fatty-acid-binding protein (FABP) was ligated in the pET8c vector and expressed under the control of the lacUV5 promoter. After induction with isopropyl beta-D-thiogalactopyranoside, almost 12% of the cytoplasmic proteins consisted of FABP. The protein could be isolated after sonication of the bacterial pellet followed by (NH4)2SO4 precipitations, anion-exchange chromatography and gel filtration. The muscle FABP produced in Escherichia coli has an isoelectric point of 5.3 and is recognized by anti-(human muscle FABP) antiserum after Western blotting. The purified FABP has a preference for binding to palmitic acid and C18-C22 (poly)unsaturated fatty acids, and no affinity to palmitoyl-CoA or other hydrophobic ligands tested. The dissociation constant for oleic acid is 0.58 microM, with a binding stoichiometry of 0.72 mol of fatty acid/mol of protein. The physicochemical and binding characteristics of the protein were in complete agreement with those of FABP isolated from human skeletal muscle.
Dietary fat modifies thromboxane A2-induced stimulation of rat platelets
Authors: Heemskerk J W M , Feijge M A H , Kester A , Hornstra G
Published: August 2015

Diets containing high levels of monounsaturated, n-6 polyunsaturated and n-3 polyunsaturated fatty acids were fed to Wistar rats. This resulted in decreases in the arachidonate content in platelet phospholipids to 91%, 79% and 51% respectively of the level found after feeding a diet rich in saturated fatty acids. In the presence of CaCl2, collagen- and thrombin-induced aggregation of washed platelets from the saturated-fat dietary group (with highest level of arachidonate) was low compared with that of platelets from the other dietary groups, despite a relatively high production of thromboxane B2. On the other hand, n-3 polyunsaturated fatty acids in the diet resulted in platelets aggregating actively, but producing low levels of levels of thromboxane B2. When indomethacin-treated rat platelets were activated with the thromboxane A2 analogue U46619, the presence of a second agonist such as collagen. ADP or thrombin was necessary for aggregate formation. U46619-induced aggregation in combination with either co-activator was relatively low in arachidonate-rich platelets, and was higher in platelets with a low arachidonate content. Similarly, phospholipase C-catalysed formation of L-myo-inositol phosphates was higher in platelets with a low arachidonate content. We conclude that the ability of platelets to react with thromboxane A2 is modified by diet in such a way that a decreased substrate-limited generation of thromboxane A2 is compensated for by an increased response to thromboxane, and vice versa. No significant differences were detected in the binding of U46619 or SQ29548 to platelets from the various dietary groups. Therefore the changed response seems not to be caused by modified properties of the thromboxane A2/prostaglandin H2 receptors, but by altered transduction of the thromboxane signal.
Solubilization of a functionally active platelet-activating factor receptor from rabbit platelets
Authors: Rogers J E , Duronio V , Wong S I , McNeil M , Salari H
Published: August 2015

Binding of platelet-activating factor (PAF) to a specific high-affinity membrane receptor has been demonstrated in numerous cell types, but very little is known about the molecular nature of this receptor. The receptor from rabbit platelets was solubilized using CHAPS, digitonin, octyl glucoside, Nonidet P-40 or sodium cholate, either with pre-bound [3H]PAF or in the absence of ligand. We have been able to demonstrate for the first time that the receptor solubilized with CHAPS, in the absence of ligand, could retain its binding activity. It migrated as a high molecular mass complex (greater than 350 kDa) on a Bio-Gel A-0.5 m gel filtration column. Binding to solubilized receptor rapidly reached an equilibrium at room temperature, but was much slower at 0 degrees C. Scatchard plots were used to calculate the number (approx. 100 per cell) and the affinity (Kd 2.5 +/- 1.4 nM) of the solubilized receptors. These values were comparable with those obtained from whole-cell binding experiments. Competition by PAF antagonists also verified that the assay was measuring PAF receptor binding activity. The presence of a protein in the receptor complex was demonstrated by heat and trypsin inactivation of binding activity. Trypsin had no effect on binding of PAF to whole cells, but was able to decrease binding activity in solubilized receptor preparations. Attempts to demonstrate the involvement of a glycoprotein by use of various lectin columns proved unsuccessful. The latter results are consistent with findings suggesting that the binding site of the PAF receptor may not be exposed at the cell surface.
A highly sensitive e.l.i.s.a. for endopeptidase-24.11, the common acute-lymphoblastic-leukaemia antigen (CALLA, CD-10), applicable to material of porcine and human origin
Authors: Howell S , Murray H , Scott C S , Turner A J , Kenny A J
Published: August 2015

Endopeptidase-24.11 is a widely distributed cell-surface enzyme with a key role in the metabolism of neuropeptides. It is now known to be identical with CD-10, the common acute-lymphoblastic-leukaemia antigen (CALLA). An e.l.i.s.a. is described which utilizes two antibodies, one monoclonal, the other polyclonal, generated to pig endopeptidase-24.11. These antibodies cross-reacted with human endopeptidase-24.11, thus making the assay applicable to both species. By using optimum conditions for the e.l.i.s.a., as little as 25 pg of pure pig endopeptidase-24.11 could be quantified at 95% confidence limits. E.l.i.s.a. of tissue homogenates from a variety of pig tissues and of human kidney correlated well with enzymic assays. However, the use of detergents to solubilize the antigen greatly decreased the sensitivity of the e.l.i.s.a. The e.l.i.s.a. is 1000-fold more sensitive than the immunoradiometric assay and has advantages in specificity over enzymic assays. Daudi cells, some leukaemic cells shown to be CALLA-positive, and Caco-2 cells, could also be assayed, but N2 cavitation was necessary to fragment the cells, and only part of the total endopeptidase-24.11 activity in Daudi cells was recognized by the e.l.i.s.a.
Identification, characterization and purification to near-homogeneity of a novel 67 kDa phosphotyrosyl protein phosphatase associated with pig lung annexin extract
Authors: Vicendo P , Fauvel J , Ragab-Thomas J M F , Chap H
Published: August 2015

During the purification of annexin VI from pig lung, we previously reported the isolation of another 67 kDa protein (protein 67E) differing from the former by immunological reactivity, amino acid composition, inability to interact with anionic phospholipids in the presence of Ca2+ and inability to inhibit phospholipase A2 [Fauvel, Vicendo, Roques, Ragab-Thomas, Granier, Vilgrain, Chambaz, Rochat, Chap & Douste-Blazy (1987) FEBS Lett. 221, 397-402]. Attempts to phosphorylate protein 67E by the protein tyrosine kinase of epidermal-growth-factor receptor revealed a dramatic inhibition of receptor autophosphorylation, which was also observed with insulin receptor. This inhibitory effect was found to be supported by a phosphatase active towards p-nitrophenyl phosphate, phosphotyrosine, [32P]phosphotyrosyl histones and [32P]phosphotyrosyl poly(Glu,Tyr), but inactive towards phosphoserine, phosphothreonine and [32P]phosphoseryl histones. Although not purified to complete homogeneity, the enzyme was purified 273-fold over EGTA extracts from pig lung and corresponded to a monomeric protein displaying an apparent molecular mass of 67 kDa. With [32P]phosphotyrosyl poly(Glu,Tyr) as substrate, the purified enzyme displayed Km and Vmax. values of 10 microM and 1.93 mumol/min per mg respectively, which compare reasonably well with other recently described phosphotyrosyl protein phosphatases. From these data and from its sensitivity to various inhibitors, it is concluded that protein fraction 67E contains a novel phosphotyrosyl protein phosphatase, the association of which with annexin extract might offer a clue to the understanding of its possible targeting to membrane substrates.
Mammalian cartilage synthesizes both proteoglycan and non-proteoglycan forms of type IX collagen
Authors: Ayad S , Marriott A , Brierley V H , Grant M E
Published: August 2015

Bovine epiphysial cartilage synthesizes both proteoglycan (PG) and non-PG forms of type IX collagen in a ratio of approx. 2:1. The PG form with its attached glycosaminoglycan on the alpha 2(IX) chain is the major form in the medium, whereas both forms are found in the tissue. The results are discussed with regard to cartilage matrix organization.
Protein kinase C regulates the stimulated accumulation of 3-phosphorylated phosphoinositides in platelets
Authors: King W G , Kucera G L , Sorisky A , Zhang J , Rittenhouse S E
Published: August 2015

We have shown that platelets stimulated with thrombin or guanosine 5′-[gamma-thio]triphosphate (GTP[S]), both of which activate phospholipase C and protein kinase C (PKC), show enhancement of 3-phosphorylated phosphoinositide accumulation (3-PPI). We now report the following. (1) Inhibition of thrombin- or GTP[S]-stimulated PKC by pseudo-substrate peptide (RFARK) added to permeabilized platelets markedly inhibits 3-PPI, whereas the serine/threonine phosphatase inhibitor, okadaic acid, promotes 3-PPI. PKC activity, insufficient in itself for fully activating 3-PPI, appears crucial to receptor and post-receptor stimulation of 3-PPI, even when tyrosine phosphorylation is unimpaired. (2) Alteration of Gi by ADP-ribosylation only slightly affects the stimulation of 3-PPI by thrombin, and activation of the G-protein Gi by adrenaline has no effect on 3-PPI. (3) Inhibition of PKC blocks activated secretion of platelet-derived growth factor (PDGF). However, PDGF cannot promote platelet 3-PPI, and thus cannot account for the inhibitory effects of RFARK on 3-PPI.
Evidence that the androgen receptor mediates sexual differentiation of mouse renal cytochrome P450 expression
Authors: Henderson C J , Wolf C R
Published: August 2015

We have previously shown that sexual dimorphism in the expression of mouse renal cytochrome P450s is mediated by androgens, probably at a transcriptional level [Henderson, Scott, Yang & Wolf (1990), Biochem. J. 266, 675-681]. In the present study we show that this effect is already observed for most isoenzymes at only 2-3 weeks of age, as is the ability to induce or suppress expression with exogenous testosterone. The testosterone responsiveness did, however, exhibit age- as well as dose-dependency. Intriguingly, the effects of androgen took up to 8 days to become maximized, and the dose of testosterone needed to convert the female into the male phenotype was much higher than the circulating levels normally found in males. Studies using testicular feminized (Tfm) male mice, which carry an androgen receptor defect, showed them to have the female kidney cytochrome P450 phenotype, and these animals were not responsive to testosterone treatment. These data demonstrate the involvement of the androgen receptor in the regulation process. Taken together, our results indicate that the androgen receptor does not interact directly with the P450 genes, but initiates a cascade of events leading to the changes in cytochrome P450 gene expression. Significant differences were observed in the degree of sexual dimorphism in kidney P450 expression in other mammalian species. The significance of these findings in relation to the observed sexual dimorphism in other species is discussed.
Expression of the rabbit intestinal N2 Na+/nucleoside transporter in Xenopus laevis oocytes
Authors: Jarvis S M , Griffith D A
Published: August 2015

Polyadenylated [poly(A)+] mRNA isolated from rabbit small-intestinal mucosa was injected into Xenopus laevis oocytes, and expression of the N2 Na+/nucleoside co-transporter was assayed by measuring Na(+)-dependent thymidine uptake. Expression of Na(+)-dependent thymidine uptake steadily increased after mRNA injection and was on average increased 11-fold by day 6 over background. Na(+)-dependent thymidine uptake was saturable (apparent Km approximately 30 microM at 22 degrees C) and inhibited by uridine and cytidine, but not by guanosine and inosine. These properties of the expressed thymidine transport strongly suggest that the epithelial N2 Na+/nucleoside co-transporter can be expressed in X. laevis oocytes.
Alterations in the metabolism of very-low- and low-density lipoproteins after partial ileal-bypass surgery in the Watanabe heritable hyperlipidaemic rabbit
Authors: Mol M J T M , Stalenhoef A F H , Demacker P N M , van 't Laar A
Published: August 2015

The influence of interruption of bile-acid enterohepatic circulation by partial ileal bypass (PIB) surgery on serum lipids, lipoproteins and the turnover of very-low- (VLDL) and low-density (LDL) lipoproteins was investigated in Watanabe heritable hyperlipidaemic (WHHL) rabbits. Compared with controls, total serum cholesterol was 48% lower after PIB (16.88 +/- 1.57 versus 8.79 +/- 1.66 mmol/l; P less than 0.01), owing to lower levels of cholesterol in VLDL (-23%), intermediate-density lipoprotein (IDL; -39%) and LDL (-72%); serum triacylglycerols were 32% higher (3.86 +/- 1.35 versus 5.11 +/- 0.82 mmol/l). The ratio of the percentages of mass of cholesteryl esters to triacylglycerols was 71% lower in VLDL and LDL and 67% lower in IDL (P less than 0.01). Compared with controls, the secretion rate of LDL was 33% lower (31.1 +/- 7.2 versus 20.7 +/- 6.9 mg/day per kg; P less than 0.05) and the fractional catabolic rate (FCR) of LDL was 33% higher (0.46 +/- 0.06 versus 0.61 +/- 0.12 pool/day; P less than 0.02). The VLDL turnover showed that after PIB there was a higher secretion rate of VLDL apolipoprotein B (63.9 versus 167.4 mg/day per kg), a higher FCR (3.84 versus 8.61 pools/day), a higher direct uptake (38.8 versus 146.4 mg/day per kg) and a higher conversion of VLDL into LDL (4.8 versus 9.0 mg/day per kg). Some 82% of LDL originated from direct synthesis in controls, and after PIB this was 59%. In both controls and treated rabbits there was a direct LDL synthesis, which was 52% lower after PIB (26.3 versus 12.6 mg/day per kg). It is concluded that LDL-cholesterol lowering by PIB is due to an increased uptake of LDL, a decreased synthesis of LDL, and cholesterol depletion of the LDL particles; the decreased LDL synthesis is due to a decreased direct production of LDL, which exceeds the increased conversion of VLDL into LDL.
Antibody mapping of tissue factor implicates two different exon-encoded regions in function
Authors: Ruf W , Rehemtulla A , Edgington T S
Published: August 2015

Tissue Factor (TF), a small transmembrane glycoprotein, is the cellular receptor for the zymogen Factor VII and the serine protease Factor VIIa (VIIa). TF provides cofactor function for VIIa in the catalytically active (TF: VIIa) binary complex. To explore the structural loci of TF that are responsible for binding of VII and VIIa, monoclonal antibodies (MAbs) and sequence-specific polyclonal antibodies to the native TF protein were analysed for inhibition of VII binding. Two independent epitopes of MAbs were localized by reciprocal competition and by binding of the MAbs to different proteolytic fragments of TF. The epitopes were also characterized in part by progressive C-terminal deletional mutation of the TF protein. Reactivity of the anti-(locus II) MAb TF9-6G4 is consistent with epitope localization in residues Thr40-Val83, encoded by exon 3. In contrast, the anti-(locus I) MAb TF9-5G9 was reactive with fragments encompassing exon 4 (Thr106-Lys165). Antibodies to linear sequences encoded by the same two exons also inhibited VII binding. These data suggest a minimum requirement for two of the four exon-encoded regions of TF for the functional integrity of this receptor cofactor with respect to ligand recognition and high-affinity binding.
Immobilization of polynucleotides on magnetic particles. Factors influencing hybridization efficiency
Authors: Day P J , Flora P S , Fox J E , Walker M R
Published: August 2015

Immobilization of oligonucleotides containing 5′-terminal thiol groups on thiol-terminated paramagnetic Biomag beads via disulphide bond formation was investigated. Oligonucleotides are demonstrated to couple at high yields, the linkage is stable at 90 degrees C and is reversible, and the immobilized oligonucleotide is available for complementary, but not non-complementary, hybridization. Specific hybridization capacity per micrograms of immobilized oligonucleotide exceeds that achieved with other forms of immobilization chemistries employing random attachment and/or specific end attachment of the oligonucleotide to the solid support. Adsorption of DNA on the surface of the beads was decreased by incubation in 0.2% SDS; other non-specific blocking agents had no effect. Brief heating of the beads possessing immobilized oligonucleotides at 90 degrees C before hybridization increased the amount of specific hybridization dependent upon the inclusion of poly(dT) spacer sequences 5′ to the immobilized oligonucleotide and 3′ to the thiol group. Increasing lengths of spacers [up to a poly(dT16) spacer] linearly increased hybridization of complementary sequences.