bluuebunny/crossref_metadata_2025 huggingface dataset:
Purification and biosynthesis of cottonseed (Gossypium hirsutum L.) catalase
Authors: Kunce C M , Trelease R N , Turley R B
Published: August 2015

As part of our research on peroxisome biogenesis, catalase was purified from cotyledons of dark-grown cotton (Gossypium hirsutum L.) seedlings and monospecific antibodies were raised in rabbits. Purified catalase appeared as three distinct electrophoretic forms in non-denaturing gels and as a single protein band (with a subunit Mr of 57,000) on silver-stained SDS/polyacrylamide gels. Western blots of crude extracts and isolated peroxisomes from cotton revealed one immunoreactive polypeptide with the same Mr (57,000) as the purified enzyme, indicating that catalase did not undergo any detectable change in Mr during purification. Synthesis in vitro, directed by polyadenylated RNA isolated from either maturing seeds or cotyledons of dark-grown cotton seedlings, revealed a predominant immunoreactive translation product with a subunit Mr of 57,000 and an additional minor immunoreactive product with a subunit Mr of 64000. Labelling studies in vivo revealed newly synthesized monomers of both the 64000- and 57,000-Mr proteins present in the cytosol and incorporation of both proteins into the peroxisome without proteolytic processing. Within the peroxisome, the 57,000-Mr catalase was found as an 11S tetramer; whereas the 64,000-Mr protein was found as a relatively long-lived 20S aggregate (native Mr approx. 600,000-800,000). The results strongly indicate that the 64,000-Mr protein (catalase?) is not a precursor to the 57,000-Mr catalase and that cotton catalase is translated on cytosolic ribosomes without a cleavable transit or signal sequence.
Purification and characterization of two wheat-embryo protein phosphatases
Authors: Polya G M , Haritou M
Published: August 2015

Two protein phosphatases (enzymes I and II) were extensively purified from wheat embryo by a procedure involving chromatography on DEAE-cellulose, phenyl-Sepharose CL-4B, DEAE-Sephacel and Ultrogel AcA 44. Preparations of enzyme I (Mr 197,000) are heterogeneous. Preparations of enzyme II (Mr 35,000) contain only one major polypeptide (Mr 17,500), which exactly co-purifies with protein phosphatase II on gel filtration and is not present in preparations of enzyme I. However, this major polypeptide has been identified as calmodulin. Calmodulin and protein phosphatase II can be separated by further chromatography on phenyl-Sepharose CL-4B. Protein phosphatases I and II do not require Mg2+ or Ca2+ for activity. Both enzymes catalyse the dephosphorylation of phosphohistone H1 (phosphorylated by wheat-germ Ca2+-dependent protein kinase) and of phosphocasein (phosphorylated by wheat-germ Ca2+-independent casein kinase), but neither enzyme dephosphorylates a range of non-protein phosphomonoesters tested. Both enzymes are inhibited by Zn2+, Hg2+, vanadate, molybdate, F-, pyrophosphate and ATP.
Azide-binding studies reveal type 3 copper heterogeneity in ascorbate oxidase from the green zucchini squash (Cucurbita pepo)
Authors: Casella L , Gullotti M , Pallanza G , Pintar A , Marchesini A
Published: August 2015

Titration of native ascorbate oxidase from green zucchini squash (Cucurbita pepo) with azide in 0.1 M-phosphate buffer, pH 6.8, exhibits a biphasic spectral behaviour. Binding of the anion with ‘high affinity’ (K greater than 5000 M-1) produces a broad increase of absorption in the 400-500 nm region (delta epsilon approximately 1000 M-1.cm-1) and c.d. activity in the 300-450 nm region, whereas azide binding with ‘low affinity’ (K approximately 100 M-1) is characterized by an intense absorption band at 420 nm (delta epsilon = 6000 M-1.cm-1), corresponding to negative c.d. activity and a decrease of absorption at 330 nm (delta epsilon = -2000 M-1.cm-1). The high-affinity binding involves a minor fraction of the protein containing Type 3 copper in the reduced state, and the spectral features of this azide adduct can be eliminated by treatment of the native enzyme with small amounts of H2O2, followed by dialysis before azide addition. As shown by e.s.r. spectroscopy, Type 2 copper is involved in both types of binding, its signal being converted into that of a species with small hyperfine splitting constant [12 mT (approximately 120 G)] in the case of the low-affinity azide adduct. The spectral similarities of the two types of azide adducts with the corresponding adducts formed by native laccase, which also exhibits Type 3 copper heterogeneity, are discussed.
Oligosaccharide mapping of heparan sulphate by polyacrylamide-gradient-gel electrophoresis and electrotransfer to nylon membrane
Authors: Turnbull J E , Gallagher J T
Published: August 2015

A new method that we have called ‘oligosaccharide mapping’ is described for the analysis of radiolabelled heparan sulphate and other glycosaminoglycans. The method involves specific enzymic or chemical scission of polysaccharide chains followed by high-resolution separation of the degradation products by polyacrylamide-gradient-gel electrophoresis. The separated oligosaccharides are immobilized on charged nylon membranes by electrotransfer and detected by fluorography. A complex pattern of discrete bands is observed covering an oligosaccharide size range from degree of polymerization (d.p.) 2 (disaccharide) to approximately d.p. 40. Separation is due principally to differences in Mr, though the method also seems to detect variations in conformation of oligosaccharide isomers. Resolution of oligosaccharides is superior to that obtained with isocratic polyacrylamide-gel-electrophoresis systems or gel chromatography, and reveals structural details that are not accessible by other methods. For example, in this paper we demonstrate a distinctive repeating doublet pattern of iduronate-rich oligosaccharides in heparitinase digests of mouse fibroblast heparan sulphate. This pattern may be a general feature of mammalian heparan sulphates. Oligosaccharide mapping should be a valuable method for the analysis of fine structure and sequence of heparan sulphate and other complex polysaccharides, and for making rapid assessments of the molecular distinctions between heparan sulphates from different sources.
A 36 kDa monomeric protein and its complex with a 10 kDa protein both isolated from bovine aorta are calpactin-like proteins that differ in their Ca2+-dependent calmodulin-binding and actin-severing properties
Authors: Martin F , Derancourt J , Capony J P , Watrin A , Cavadore J C
Published: August 2015

Interaction of plasma membrane with the cytoskeleton involves a large number of proteins, among them a 36 kDa protein that was found to be involved in the interaction with actin filaments. We have isolated a 36 kDa protein from bovine aorta as a monomer and in a complex with a 10 kDa protein. Partial amino acid sequence determinations show that the 36 kDa and 10 kDa proteins isolated from bovine aorta are analogous to or identical with corresponding proteins purified from bovine intestine already described by Kristensen, Saris, Hunter, Hicks, Noonan, Glenney & Tack [(1986) Biochemistry 25, 4497-4503]. We report here that the association of the 10 kDa protein with the 36 kDa protein confers specific calmodulin-binding and actin-severing properties on the complex that are not possessed by the 36 kDa monomer alone. These findings suggest that the protein complex could be involved in thin-filament-related structures or could modulate some Ca2+-regulated events mediated by calmodulin.
Biosynthesis, import and processing of precursor polypeptides of mammalian mitochondrial pyruvate dehydrogenase complex
Authors: De Marcucci O G L , Gibb G M , Dick J , Lindsay J G
Published: August 2015

An immunological analysis has been conducted of early events in the biosynthesis, import and assembly of the mammalian pyruvate dehydrogenase complex (PDC). For this purpose, monospecific polyclonal antisera were produced against the intact assembly from ox heart, Mr 8.5 x 10(6), and each of its component polypeptides, E1 alpha, E1 beta, E2, E3 and protein X. Optimal detergent-based incubation mixtures were developed for obtaining clean immunoprecipitation of PDC polypeptides and their precursors from [35S]methionine-labelled extracts of PK-15 (pig kidney), NBL-1 (bovine kidney) and BRL (Buffalo Rat liver) cells. In PK-15 cells, independent higher Mr species, corresponding to precursors of the E2, E1 alpha and E1 beta subunits of PDC, could be detected by immune precipitation and fluorography after incubation of intact cells for 4 h with [35S]methionine and 1-2 mM-2,4-dinitrophenol or 10-15 microM-carbonyl cyanide p-trifluoromethoxyphenylhydrazone. Similar precursor states could be observed in uncoupler-treated BRL or NBL-1 cells. Pre-E1 alpha, pre-E1 beta and also pre-E3, have signal sequences in the Mr range 1500-3000 while pre-E2 contains a long additional segment of Mr 7000-9000. All of these forms exhibit similar kinetics of processing to the mature subunits with a transit time of 10-12 min. In NBL-1 cells, E3 is present in the immune complexes formed with anti-PDC serum whereas this is not the case in PK-15 cells. Thus, there are significant variations in the affinity of lipoamide dehydrogenase (E3) for the E2 core structure in different species. Pre-E1 alpha accumulates only poorly in PK-15 cells and is aberrantly processed on removal of uncoupler. This precursor is markedly more stable in NBL-1 and BRL cells. The lack of detection of a precursor form of component X is also discussed.
Isolation and characterization of fatty acid binding proteins from mammary tissue of lactating rats
Authors: Jones P D , Carne A , Bass N M , Grigor M R
Published: August 2015

A protein fraction with fatty acid binding activity has been isolated from mammary tissue from lactating rats by a process involving DEAE-cellulose ion-exchange chromatography, heat treatment, CM-cellulose ion-exchange chromatography and finally ammonium sulphate precipitation. The purified fraction migrated as a single band on SDS/polyacrylamide-gel electrophoresis with an apparent molecular mass of 14400. However, when this protein fraction was electrophoresed under non-dissociating conditions, two species were observed in a 4:1 ratio. The two components were separated using h.p.l.c. Both bind fatty acids and appear to have similar amino acid compositions although exhibiting different pI values of 4.8 and 4.9. The mammary fatty acid binding proteins appear to be very similar to the fatty acid binding protein isolated from rat heart based on the electrophoretic mobilities and amino acid composition. The major mammary form (pI 4.9) has been partially sequenced and the amino acid sequences obtained can be aligned with 67 residues of the revised rat heart amino acid sequence [Heuckeroth, Birkenmeier, Levin & Gordon (1987) J. Biol. Chem. 262, 9709-9717]. Both mammary species also showed immunochemical identity to rat heart fatty acid binding protein when tested with an anti-serum raised against the heart protein. Anti-sera raised against the minor mammary form (pI 4.8) specifically precipitated this form under non-denaturing conditions but both forms after they had been denatured. Quantitative immunoassays using the anti-(heart fatty acid binding protein) serum showed that concentrations of the fatty acid binding proteins present in mammary cytosols increase during lactation and increase further after feeding a high-fat diet.
Chemically synthesized non-radioactive biotinylated long-chain nucleic acid hybridization probes
Authors: al-Hakim A H , Hull R
Published: August 2015

A new method for the chemical labelling of nucleic acid with biotin to produce non-radioactive probes has been developed. NN'-Bis-(3-aminopropyl)butane-1,4-diamine (spermine) and long-chain diamino compounds (diaminohexane, diaminodecane and diaminododecane) were linked covalently to biotin and the resultant conjugates were attached to nucleic acid by using a cross-linking reagent (glutaraldehyde or diepoxyoctane). Iodoacetylation and biotinylation of the long-chain diamino compounds produced modified biotinylated conjugates that can be linked to DNA without the use of a cross-linking reagent. These types of probes attach one biotin molecule to each linker arm of spermine, diamino and iodoacetylated amino derivatives. Such probes have long linker arms separating the biotin moiety from the hybridization sites of the nucleic acid. These probes can detect 10 pg of target DNA by dot-blot hybridization.
Determination of molecular mass of the aroid alternative oxidase by radiation-inactivation analysis
Authors: Berthold D A , Fluke D J , Siedow J N
Published: August 2015

The functional molecular mass of the cyanide-resistant salicylhydroxamate-sensitive duroquinol oxidase activity from Sympocarpus foetidus (skunk cabbage) and Sauromatum guttatum spadix mitochondria was determined by radiation-inactivation analysis. The functional molecular mass for the oxidase activity was found to be 26,700 Da for skunk cabbage and 29,700 Da for Sauromatum guttatum mitochondria frozen at ∗70 degrees C. Irradiation of dried mitochondrial samples resulted in a larger target size of 38,000 Da, and in some cases, a stimulation of activity at low dose of radiation. The functional molecular mass of cytochrome c oxidase activity from skunk-cabbage and bovine heart mitochondria was also investigated. Dried and frozen mitochondrial samples from both species yielded similar target sizes, in the range 70,900-73,400 Da. Purified bovine heart cytochrome c oxidase was also irradiated, and yielded a functional molecular mass of 66,400 Da. The target size of cytochrome c oxidase agrees with literature values insofar as the target size is considerably smaller than the molecular mass of the entire complex.
The amino acid sequence of a gonococcal growth inhibitor from Staphylococcus haemolyticus
Authors: Watson D C , Yaguchi M , Bisaillon J G , Beaudet R , Morosoli R
Published: August 2015

A gonococcal inhibitor produced by Staphylococcus haemolyticus was separated into three components by reverse-phase h.p.l.c. The amino acid composition analysis of each of the three components indicated extensive similarities. N-Terminal sequence analysis of all three components allowed the identification of the first 27-30 residues of each. The complete primary structure of each component was determined from the sequence analysis of trypic peptides and peptides generated by mild acid hydrolysis. Each component is composed of 44 amino acid residues, with evidence suggesting the presence of an N-terminal formylmethionine residue in each. The components I, II and III have respectively 33, 29 and 33 identical amino acid residues in their sequences, which represents 75%, 65.9% and 75% homology. These components contain a high proportion of hydrophobic amino acids, and their hydrophobicity profiles are closely related. Also, each of the three components contains a positively charged residue (lysine) as the third residue, followed by a core of hydrophobic residues. These results suggest that the three components are possible signal sequences of one or more secreted or membrane-associated proteins.
Isolation, purification and characterization of an iron-binding protein from the horseshoe crab (Limulus polyphemus)
Authors: Topham R , Cooper B , Tesh S , Godette G , Bonaventura C , Bonaventura J
Published: August 2015

The presence of an iron-binding protein in the haemolymph of the horseshoe crab, Limulus polyphemus, was detected by gel filtration of 59Fe-labelled haemolymph. Lysis of amoebocytes did not change the amount of iron-binding protein in haemolymph samples. The protein was purified to homogeneity by ion-exchange chromatography. The molecular mass of the purified protein was estimated to be 282,000 +/- 10,000 Da by gel filtration and analytical ultracentrifugation. SDS/polyacrylamide-gel electrophoresis demonstrated that the protein is composed of ten subunits having a molecular mass of 28,000 +/- 2,000 Da. The purified, unlabelled protein efficiently sequestered 59Fe in the absence of haemolymph indicating that no other haemolymph factors are required for the incorporation of iron into the protein. No 59Fe was removed from the purified protein with EDTA or 2,2′-bipyridyl. Partial removal of 59Fe was achieved by dialysis with nitrilotriacetic acid or desferal. Analysis of the iron-loaded protein indicated that each subunit has the capacity to bind two iron atoms with high affinity. The isolation of an iron-binding protein from L. polyphemus supports the proposal that such proteins are an ancient evolutionary development not necessarily linked to the appearance of iron proteins which serve as oxygen carriers.
Interleukin-1 α modulates collagen gene expression in cultured synovial cells
Authors: Mauviel A , Teyton L , Bhatnagar R , Penfornis H , Laurent M , Hartmann D , Bonaventure J , Loyau G , Saklatvala J , Pujol J P
Published: August 2015

The effects of porcine interleukin-1 (IL-1) alpha on collagen production were studied in cultured human rheumatoid synovial cells. Addition of 0.05-5 ng of IL-1/ml into the cultures resulted in a dose-dependent decreased rate of collagen released into the medium over 24 h. To determine whether this inhibition was due to secondary action of prostaglandin E2 (PGE2) secreted in response to IL-1, cultures were incubated in presence of various inhibitors of arachidonate metabolism. Depending on the cell strains, these inhibitors were able to suppress or diminish the effect of IL-1, suggesting that PGE2 is involved in the mechanism. Depression of collagen production caused by IL-1 mainly affected type I collagen and therefore led to a change in the type I/type III collagen ratio in the extracellular medium. Steady-state levels of mRNA for types I and III procollagens were estimated by dot-blot hybridization and compared with the amounts of respective collagens produced in the same cultures. IL-1 generally increased procollagen type I mRNA, but to a variable extent, as did indomethacin (Indo). Depending on the cell strain, the combination of indo and IL-1 could elevate the mRNA level of type I procollagen compared with Indo alone. These results did not correlate with the production rate of collagen in the medium, which was diminished by exposure to IL-1. The level of mRNA for collagen type III was not greatly changed by incubation with IL-1, and a better correlation was generally observed with the amount of type III collagen found in the medium. These data suggest that an additional control mechanism at translational or post-translational level must exist, counterbalancing the stimulatory effect of IL-1 on collagen mRNA transcription. It is likely that IL-1 could modulate the production of collagen in synovial cells by an interplay of different mechanisms, some of them limiting the effect of primary elevation of the steady-state mRNA level.
Activation of sea urchin eggs by inositol phosphates is independent of external calcium
Authors: Crossley I , Swann K , Chambers E , Whitaker M
Published: August 2015

We investigated the contribution of external calcium ions to inositol phosphate-induced exocytosis in sea urchin eggs. We show that: (a) inositol phosphates activate eggs of the sea urchin species Lytechinus pictus and Lytechinus variegatus independently of external calcium ions; (b) the magnitude and duration of the inositol phosphate induced calcium changes are independent of external calcium; (c) in calcium-free seawater, increasing the volume of inositol trisphosphate solution injected decreased the extent of egg activation; (d) eggs in calcium-free sea water are more easily damaged by microinjection; microinjection of larger volumes increased leakage from eggs pre-loaded with fluorescent dye. We conclude that inositol phosphates do not require external calcium ions to activate sea urchin eggs. This is entirely consistent with their role as internal messengers at fertilization. The increased damage caused to eggs in calcium-free seawater injected with large volumes may allow the EGTA present in the seawater to enter the egg and chelate any calcium released by the inositol phosphates. This may explain the discrepancy between this and earlier reports.
The oxidation-state-dependent ATP-binding site of cytochrome c. Implication of an essential arginine residue and the effect of occupancy on the oxidation-reduction potential
Authors: Corthésy B E , Wallace C J
Published: August 2015

Arg-91 is not part of the active site of cytochrome c that mediates binding and electron transfer, yet it is absolutely conserved in eukaryotic cytochromes c, indicating a special function. The physicochemical properties of analogues are unaffected by the modification of this residue, so they can be used with confidence to study the role of Arg-91. We have established limiting conditions under which this residue alone is specifically modified by cyclohexane-1,2-dione, and have subsequently shown that ATP, and to a lesser extent ADP or Pi, protects it from the action of the reagent in an oxidation-state-dependent manner. These observations strongly support the idea that this site exerts a controlling influence on cytochrome c activity in the electron transport or other cellular redox systems, and we have commenced a study of how that influence might operate. We find that the redox potentials of both cytochrome c and analogue are little affected by changing ATP or Pi concentrations.
Iron K-edge X-ray absorption spectroscopy of the iron-molybdenum cofactor of nitrogenase from Klebsiella pneumoniae
Authors: Arber J M , Flood A C , Garner C D , Gormal C A , Hasnain S S , Smith B E
Published: August 2015

Iron K-edge X-ray absorption data for the iron-molybdenum cofactor (‘FeMoco’) from Klebsiella pneumoniae reported here provide the first evidence for long-range structural order in the cofactor [Fe…Fe(Mo) = 0.368 nm in addition to Fe…S = 0.22 nm and Fe…Fe(Mo) = 0.27 nm] and, in contrast with previously published data [Antonio, Teo, Orme-Johnson, Nelson, Groh, Lindahl, Kauzlarich & Averill (1982) J. Am. Chem. Soc. 104, 4703-4705], indicate that most of the iron centres are not co-ordinated to light (oxygen, nitrogen) atoms. This demonstrates that presently available chemical models for FeMoco are inadequate.
Hydroxyl-radical-induced iron-catalysed degradation of 2-deoxyribose. Quantitative determination of malondialdehyde
Authors: Cheeseman K H , Beavis A , Esterbauer H
Published: August 2015

The degradation of 2-deoxyribose to thiobarbituric acid-reactive material was investigated with two hydroxyl-radical-generating systems: (i) a defined gamma-radiolysis method and (ii) incubation with FeSO4 in phosphate buffer. In each case the thiobarbituric acid-reactive material can be accounted for by malondialdehyde, as measured by an h.p.l.c. method for free malondialdehyde. In the radiolysis system there is a large post-irradiation increase in free malondialdehyde if iron ions are added to the samples. It is proposed that this is due to iron ions catalysing the formation of hydroxyl radicals from radiolytically generated H2O2 as well as stimulating the breakdown of an intermediate deoxyribose degradation product. A mechanism for the formation of malondialdehyde during deoxyribose degradation is proposed.
A kinetic and equilibrium study of ligand binding to the monomeric and dimeric haem-containing globins of two chitons
Authors: Smith S E , Brittain T , Wells R M G
Published: August 2015

The radular muscles of the amphineuran molluscs Amaurochiton glaucus and Sipharochiton pelliserpentis contain both a dimeric and a monomeric form of myoglobin. The dimeric form of the protein is composed of two polypeptide chains covalently linked to each other via one or more disulphide bonds. The dimeric protein shows co-operative O2-binding curves. Kinetic investigations indicate that CO binding is co-operative in the dimeric protein, subsequent to full photolysis, but mono-exponential following 10% photolysis. O2 recombination following part photolysis is mono-exponential in the dimeric form, whereas O2 dissociation kinetics indicates the presence of chain heterogeneity. The monomeric form of the protein exhibits mono-exponential time courses in all the experimental situations explored. Although the rate constants associated with the reactions of individual dimer and monomer molecular species are very different, the two species of chiton investigated show remarkably similar properties when compared with each other. All the reactions studied are pH-independent in the range pH 6-8. Amino acid analysis indicates that the monomeric units that combine to form the dimeric species are not identical with the naturally occurring monomeric form. A comparison is made between the chiton myoglobins and other similar O2-binding proteins.
Effect of specific proteolytic cleavages on tubulin polymer formation
Authors: Serrano L , Wandosell F , de la Torre J , Avila J
Published: August 2015

The capacity for self-polymerization and shape of the tubulin polymers assembled after digestion with trypsin, Pronase, chymotrypsin, subtilisin, Staphylococcus aureus proteinase V8 and proteinase K were investigated. Digestion with trypsin, Pronase or chymotrypsin resulted in a decrease in the ability of tubulin for self-assembly, whereas limited proteolysis with subtilisin, S. aureus proteinase V8 or proteinase K resulted in an increase in such ability. The shape of the assembled polymers varied from typical microtubules (after the treatment with trypsin or Pronase) to sheets (after the treatment with chymotrypsin) and from hooked microtubules with a constant polarity (after the treatment with subtilisin) to the disappearance of a defined polarity of such polymers (after the treatment with S. aureus V8 proteinase or proteinase K). These results indicate that the tubulin C-terminal regions are involved in the regulation of microtubule polymerization, shape, directional growth and lateral interactions between tubulin protofilaments.
Occupancy of adenosine receptors raises cyclic AMP alone and in synergy with occupancy of chemoattractant receptors and inhibits membrane depolarization
Authors: Cronstein B N , Kramer S B , Rosenstein E D , Korchak H M , Weissmann G , Hirschhorn R
Published: August 2015

We have recently demonstrated that adenosine, acting via adenosine A2 receptors, inhibits generation of superoxide anions (O2-) by stimulated neutrophils. To determine the mechanism(s) by which adenosine inhibits O2- generation stimulated by the chemoattractant N-formylmethionylleucylphenylalanine (FMLP), we examined cyclic AMP (cAMP) concentrations, stimulated membrane depolarization and Ca2+ movements. Neither adenosine nor 5′-N-ethylcarboxamidoadenosine (NECA), the most potent agonist at adenosine A2 receptors, increases neutrophil cAMP content. However in the presence of the non-methylxanthine phosphodiesterase inhibitor, Ro-20-1724, both adenosine and NECA elicit a reversible increase in intracellular cAMP concentration. The chemoattractant FMLP also elicits an increment in the neutrophil cAMP content. NECA, in the presence of Ro-20-1724, synergistically enhances the increment in cAMP following stimulation by FMLP. However Ro-20-1724 does not potentiate the inhibition of O2- generation by NECA. Unlike other agents which increase neutrophil cAMP concentrations, NECA, even in the presence of a phosphodiesterase inhibitor, only trivially inhibits degranulation. We also found that adenosine markedly inhibits stimulated membrane depolarization but does not affect the stimulated increment in free ionized intracellular calcium. Moreover, inhibition by adenosine of O2- generation does not vary with the concentration of extracellular calcium. These results fulfil the last criterion for the demonstration of an A2 receptor on human neutrophils, and indicate that adenosine occupies an A2 receptor on neutrophils to raise intracellular cAMP in synergy with occupancy of the FMLP receptor. The results reported here also indicate that cAMP is not the second messenger for inhibition of O2- generation by adenosine and its analogues.
Import of rat liver mitochondrial transhydrogenase
Authors: Wu L N Y , Lubin I M , Fisher R R
Published: August 2015

The biosynthesis of pyridine dinucleotide transhydrogenase has been studied in isolated rat hepatocytes and in a rabbit reticulocyte-lysate translation system supplemented with either intact isolated rat liver mitochondria or the soluble matrix fraction from isolated mitochondria. In intact hepatocytes, the transhydrogenase precursor was short-lived in the cytosol and was efficiently imported into the membranous fraction. When the cell-free translation mixture was incubated with intact mitochondria, the transhydrogenase precursor was processed to the mature form, to an extent that depended on the amount of added mitochondria. Incubation of the translation mixture with the soluble mitochondria matrix fraction converted the precursor to a mature-sized protein with 75% efficiency, this being blocked by various proteinase inhibitors such as EDTA, 1,10-phenanthroline and leupeptin.
Separate binding sites for antimycin and mucidin in the respiratory chain of the bacterium Paracoccus denitrificans and their occurrence in other denitrificans bacteria
Authors: Kucera I , Hedbávný R , Dadák V
Published: August 2015

By means of the method of fluorimetric titration it has been shown that mucidin does not affect the attachment of antimycin to membranes from anaerobically grown Paracoccus denitrificans. The fluorimetric titration with antimycin can be used in the determination of the amount of the cytochrome bc1 complex in the membrane. In cells inhibited with antimycin, the oxidation of cytochromes c was accompanied by the reduction of cytochrome b; in the presence of mucidin this effect did not take place. The results, which indicated a difference in binding sites, were interpreted in terms of the Q-cycle [Mitchell (1976) J. Theor. Biol. 62, 327-367; Trumpower (1981) Biochim. Biophys. Acta 639, 129-155]. Comparable sensitivity towards antimycin and mucidin was shown by other typical denitrifying bacteria: Pseudomonas stutzeri and Alcaligenes xylosoidans, subspecies denitrificans.
Photoaffinity-labelling of the calcium-channel-associated 1,4-dihydropyridine and phenylalkylamine receptor in guinea-pig hippocampus. A 195 kDa polypeptide carries both drug receptors and has similarities to the α1 subunit of the purified skeletal-muscle calcium channel
Authors: Striessnig J , Knaus H G , Glossmann H
Published: August 2015

This study identifies calcium-antagonist-receptor-carrying polypeptides of calcium channels in guinea-pig hippocampus membranes. The arylazide ligands (-)-[3H]azidopine and [N-methyl-3H]LU49888 [(-)-5-[(3-azidophenethyl) [N-methyl-3H]methylamino]-2-(3,4,5-trimethoxyphenyl-2- isopropylvaleronitrile] were used to selectively label 1,4-dihydropyridine and phenylalkylamine receptors respectively. In the absence of u.v. light, both ligands reversibly bound to a single class of high-affinity receptors with a calcium-channel-typical pharmacological profile. [N-methyl-3H]LU49888 bound to the extent of 849 +/- 188 fmol/mg of protein (mean +/- S.D., n = 3) with a dissociation constant (Kd) of 1.4 +/- 0.3 nM. Under identical assay conditions (-)-[3H]azidopine labelled to the extent of 562 +/- 132 fmol/mg of protein with a Kd of 0.096 +/- 0.024 nM. After u.v. irradiation of the [N-methyl-3H]LU49888- and (-)-[3H]azidopine-labelled membranes, both photo-affinity probes were found to be incorporated specifically into a 190-195 kDa band as shown by SDS/polyacrylamide-gel electrophoresis (SDS/PAGE). Photoincorporation occurred with a protection profile identical with that produced by reversible binding-inhibition. [N-methyl-3H]LU49888, but not (-)-[3H]-azidopine, specifically labelled an additional 265 kDa band. Both photolabelled bands had an identical electrophoretic mobility on SDS/PAGE, irrespective of pretreatment either with 10 mM-N-ethylmaleimide or 10 mM-dithiothreitol. The electrophoretic properties of the 195 kDa polypeptide and the lability of receptor-incorporated (-)-[3H]azidopine to nucleophilic agents resemble those of the previously described drug-receptor-carrying alpha 1 subunit of the purified skeletal-muscle calcium channel. The data suggest that this polypeptide carries both the high-affinity 1,4-dihydropyridine as well as the phenylalkylamine receptor of neuronal calcium channels in guinea-pig hippocampus and is a component of the L-type calcium channel.
Evidence for filaggrin as a component of the cell envelope of the newborn rat
Authors: Richards S , Scott I R , Harding C R , Liddell J E , Powell G M , Curtis C G
Published: August 2015

A substrate of transglutaminase, specific to the epidermis, was identified, by fluorescent and radioactive labelling with the lysine analogues dansylcadaverine and [14C]putrescine respectively, in newborn-rat epidermal homogenates and whole-skin organ cultures. The labelled analogues were preferentially incorporated into the stratum-corneum protein filaggrin in a Ca2+-dependent manner in both ‘in vitro’ systems. When filaggrin was labelled in vivo with [3H]histidine and then incubated with rat epidermal preparations, the label was rendered SDS/thiol-insoluble. Incorporation of [3H]filaggrin into the insoluble envelope fraction was Ca2+-dependent and inhibited by EDTA and exogenous amines. Antisera to newborn-rat filaggrin cross-reacted with purified newborn-rat cell envelopes, and this reaction was blocked by adsorbing the antiserum with purified filaggrin. Quantification of the ‘envelope-bound’ filaggrin showed it to be a significant component, accounting for approx. 10% of the cell-envelope protein.
The biphasic stimulation of insulin secretion by bombesin involves both cytosolic free calcium and protein kinase C
Authors: Swope S L , Schonbrunn A
Published: August 2015

Members of the bombesin family of peptides potently stimulate insulin release by HIT-T15 cells, a clonal pancreatic cell line. The response to bombesin consists of a large burst in secretion during the first 30 s, followed by a smaller elevation of the secretory rate, which persists for 90 min. The aim of this study was to identify the intracellular messengers involved in this biphasic secretory response. Addition of 100 nM-bombesin to cells for 20 s increased the cellular accumulation of [3H]diacylglycerol (DAG) by 40% and that of [3H]inositol monophosphate (InsP), bisphosphate (InsP2) and trisphosphate (InsP3) by 40%, 300%, and 800%, respectively. In contrast, cyclic AMP concentrations were unaffected. Bombesin stimulation of [3H]InsP3 formation was detected at 2 s, before the secretory response, which was not measurable until 5 s. Furthermore, the potency of bombesin to stimulate [3H]InsP3 generation (ED50 = 14 +/- 9 nM) agreed with its potency to stimulate insulin release (ED50 = 6 +/- 2 nM). Consistent with its effects on [3H]InsP3 formation, bombesin raised the intracellular free Ca2+ concentration [(Ca2+]i) from a basal value of 0.28 +/- 0.01 microM to a peak of 1.3 +/- 0.1 microM by 20 s. Chelation of extracellular Ca2+ did not abolish either the secretory response to bombesin or the rise in [Ca2+]i, showing that Ca2+ influx was not required. Although the Ca2+ ionophore ionomycin (100 nM) mimicked the [Ca2+]i response to bombesin, it did not stimulate secretion. However, pretreating cells with ionomycin decreased the effects of bombesin on both [Ca2+]i and insulin release, suggesting that elevation of [Ca2+]i was instrumental in the secretory response to this peptide. To determine the role of the DAG produced upon bombesin stimulation, we examined the effects of another activator of protein kinase C, the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA). TPA did not affect [Ca2+]i, but it increased insulin secretion after a 2 min lag. However, an immediate increase in secretion was observed when ionomycin was added simultaneously with TPA. These data indicate that the initial secretory burst induced by bombesin results from the synergistic action of the high [Ca2+]i produced by InsP3 and DAG-activated protein kinase C. However, activation of protein kinase C alone appears to be sufficient for a sustained secretory response.
High concentrations of exogenous arachidonate inhibit calcium mobilization in platelets by stimulation of adenylate cyclase
Authors: Kowalska M A , Rao A K , Disa J
Published: August 2015

1. Exposure of platelets to exogenous arachidonic acid results in aggregation and secretion, which are inhibited at high arachidonate concentrations. The mechanisms for this have not been elucidated fully. In our studies in platelet suspensions, peak aggregation and secretion occurred at 2-5 microM-sodium arachidonate, with complete inhibition around 25 microM. 2. In platelets loaded with quin2 or fura-2, the cytoplasmic Ca2+ concentration, [Ca2+]i, rose in the presence of 1 mM-CaCl2 from 60-80 nM to 300-500 nM at 2-5 microM-arachidonate, followed by inhibition to basal values at 25-50 microM. Thromboxane production was not inhibited at 25 microM-arachidonate. Cyclic AMP increased in the presence of theophylline, from 3.5 pmol/10(8) platelets in unexposed platelets to 8 pmol/10(8) platelets at 50 microM-arachidonate; all platelet responses were inhibited with doubling of cyclic AMP contents. 3. The adenylate cyclase inhibitor 2′,5′-dideoxyadenosine attenuated the inhibitory effect of arachidonate, suggesting that it is mediated by increased platelet cyclic AMP and that it is unlikely to be due to irreversible damage to platelets. 4. Aspirin or the combined lipoxygenase/cyclo-oxygenase inhibitor BW 755C did not prevent the inhibition by arachidonate of either [Ca2+]i signals or aggregation induced by U46619. 5. Thus high arachidonate concentrations inhibit Ca2+ mobilization in platelets, and this is mediated by stimulation of adenylate cyclase. High arachidonate concentrations influence platelet responses by modulating intracellular concentrations of two key messenger molecules, cyclic AMP and Ca2+.
A slow kinetic transient in RNA synthesis catalysed by wheat-germ RNA polymerase II
Authors: Job C , De Mercoyrol L , Job D
Published: August 2015

Progress curves of U-A-primed RNA synthesis catalysed by wheat-germ RNA polymerase II on a poly[d(A-T)] template exhibit a slow burst of activity. In contrast, the progress curves of single-step addition of UMP to U-A primer in the abortive elongation reaction do not exhibit the slow burst of activity. The correlation between the kinetic transient in the productive pathway of RNA synthesis and the rate of abortive elongation is suggestive of the occurrence of a slow conformational change of the transcription complex during the transition from abortive to productive elongation. The exceptional duration of the transient burst (in the region of 4 min) may suggest a transition of a hysteretic type.
Uroporphyria produced in mice by 20-methylcholanthrene and 5-aminolaevulinic acid
Authors: Urquhart A J , Elder G H , Roberts A G , Lambrecht R W , Sinclair P R , Bement W J , Gorman N , Sinclair J A
Published: August 2015

Iron-loaded male C57BL/6 mice allowed free access to an aqueous solution of 5-aminolaevulinic acid (ALA) (2 mg/ml) as their only drink, develop severe uroporphyria within 9 days of a single intraperitoneal dose of 20-methylcholanthrene (MC) (125 mg/kg). At 21 days, uroporphyrinogen decarboxylase (EC 4.1.1.37) activities are less than 10% of control activities. The porphyria is not dependent on pretreatment with iron and persists for at least 21 days after withdrawal of ALA. The same intraperitoneal dose of MC does not produce porphyria within 21 days when given without ALA. Continuous administration of ALA markedly accelerates the onset of porphyria in iron-loaded male C57BL/6 mice after a single intraperitoneal dose of hexachlorobenzene (200 mg/kg); mice given phenobarbitone and ALA do not become porphyric. MC with ALA does not produce porphyria in iron-loaded male DBA/2 mice. At least two separate events are needed to produce uroporphyria in mammals: induction of a specific form of cytochrome P-450 and stimulation of the formation of intermediates of haem biosynthesis in the liver. These results show that severe, persistent porphyria can be produced in mammals by compounds other than polyhalogenated aromatic hydrocarbons and suggest that a similar mechanism underlies the porphyrogenic action of halogenated and non-halogenated compounds.
Purification of low density lipoprotein receptor from liver and its quantification by anti-receptor monoclonal antibodies
Authors: Gherardi E , Brugni N , Bowyer D E
Published: August 2015

The low density lipoprotein (LDL) receptor has been purified to homogeneity from rabbit liver by a combination of DEAE-Sephacel chromatography, LDL-Sepharose 4B chromatography and preparative SDS/polyacrylamide-gel electrophoresis. The receptor protein had a pI of 4.45 and an Mr of 120 x 10(3)-125 x 10(3) in SDS gels under non-reducing conditions. Incubation of the LDL receptor with neuraminidase decreased its Mr to 105 x 10(3)-110 x 10(3) and increased its pI from 4.45 to 5.25. The purified receptor exhibited all the properties of the membrane-bound receptor including Ca2+-dependent binding of rabbit and human LDL but not of methylated LDL or high density lipoprotein. The amount of LDL receptor present in rabbit liver was measured by a quantitative blotting procedure employing a newly developed rat anti-receptor monoclonal antibody. The affinity and specificity of this monoclonal antibody allowed the quantification of the LDL receptor in detergent extracts of liver homogenate, thus eliminating the loss of receptor associated with the preparation of membrane fractions prior to receptor assay. Livers from adult female New Zealand White rabbits contained 149 +/- 13 ng of LDL receptor/mg of liver protein. Administration of pharmacological doses of 17 alpha-ethinyloestradiol raised the concentration of LDL receptor in liver to 312 +/- 25 ng/mg of liver protein.
Solubilization of functional calcitonin receptors
Authors: Nicholson G C , D'Santos C S , Evans T , Moseley J M , Kemp B E , Martin T J
Published: August 2015

Calcitonin (CT) binding activity has been extracted from a membrane fraction of human placenta using the zwitterionic detergent, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulphonic acid (Chaps). Approximately two-thirds of the available binding sites were extracted using 5 mM-Chaps. The binding characteristics of 125I-labelled salmon CT(125I-sCT) to the solubilized extract were similar to those obtained previously with placental membranes and other targets such as osteoclasts, renal cells and certain human cancer cell lines. 125I-sCT binding was saturable (Bmax. 75 +/- 6 fmol/mg of protein, n = 3) and Scatchard analysis revealed a single class of high-affinity binding sites (Kd 165 +/- 28 pM, n = 3). In competitive-binding studies, various species-specific CTs and CT analogues showed the same rank order of potencies as seen in CT bioassays and several unrelated peptides did not compete at high doses. A biologically active CT analogue, [Arg11,18, Lys14]sCT, derivatized with the photoreactive phenylazide cross-linking agent, N-hydroxysuccinimidyl-4-azidobenzoate, was used to identify receptor components of Mr approximately 88,000 and approximately 71,000 in both particulate placental membranes and the solubilized extract. Receptor components of Mr 85-90,000 have been identified in other CT target cells previously using chemical- and photoaffinity-labelling techniques. These results demonstrate the first successful solubilization of the CT receptor in a form which purification.
Stimulation of surfactant phospholipid biosynthesis in the lungs of rats treated with silica
Authors: Miller B E , Hook G E R
Published: August 2015

The effects of intratracheally instilled silica (10 mg/rat) on the biosynthesis of surfactant phospholipids was investigated in the lungs of rats. The sizes of the intracellular and extracellular pools of surfactant phospholipids were measured 7, 14 and 28 days after silica exposure. The ability of lung slices to incorporate [14C]choline and [3H]palmitate into surfactant phosphatidylcholine (PC) and disaturated phosphatidylcholine (DSPC) was also investigated. Both intra- and extra-cellular pools of surfactant phospholipids were increased by silica treatment. The intracellular pool increased linearly over the 28-day time period, ultimately reaching a size 62-fold greater than controls. The extracellular pool also increased, but showed a pattern different from that of the intracellular pool. The extracellular pool increased non-linearly up to 14 days, and then declined. At its maximum, the extracellular pool was increased 16-fold over the control. The ability of lung slices to incorporate phospholipid precursors into surfactant-associated PC and DSPC was elevated at all time periods. The rate of incorporation of [14C]choline into surfactant PC and DSPC was maximal at 14 days and was nearly 3-fold greater than the rate in controls. The rate of incorporation of [3H]palmitate was also maximal at 14 days, approx. 5-fold above controls for PC and 3-fold for DSPC. At this same time point, the microsomal activity of cholinephosphate cytidylyltransferase was increased 4.5-fold above controls, but cytosolic activity was not significantly affected by silica treatment. These data indicate that biosynthesis of surfactant PC is elevated after treatment of lungs with silica and that this increased biosynthesis probably underlies the expansion of the intra- and extra-cellular pools of surfactant phospholipids.
Synthesis of myo-inositol 1,3,4,5,6-pentakisphosphate from inositol phosphates generated by receptor activation
Authors: Stephens L R , Hawkins P T , Barker C J , Downes C P
Published: August 2015

myo-[3H]Inositol 1,3,4,5,6-pentakisphosphate can be made from myo-[3H]inositol 1,4,5-trisphosphate in a rat brain homogenate or soluble fraction. Although D-myo-inositol 3,4,5,6-tetrakisphosphate can be phosphorylated by a soluble rat brain enzyme to give myo-inositol 1,3,4,5,6-pentakisphosphate, it is not an intermediate in the pathway from myo-inositol 1,4,5-trisphosphate. The intermediates in the above pathway are myo-inositol 1,3,4,5-tetrakisphosphate, myo-inositol 1,3,4-trisphosphate and myo-inositol 1,3,4,6-tetrakisphosphate [Shears, Parry, Tang, Irvine, Michell & Kirk (1987) Biochem. J. 246, 139-147; Balla, Guillemette, Baukal & Catt (1987) J. Biol. Chem. 262, 9952-9955], and it is catalysed by soluble kinase activities of similar anion-exchange mobility and Mr value. Compounds with chromatographic and chemical properties consistent with the structures myo-inositol 1,3,4,5-tetrakisphosphate, myo-inositol 1,3,4,6-tetrakisphosphate and myo-inositol 3,4,5,6-tetrakisphosphate are present in avian erythrocytes, human 1321 N1 astrocytoma cells and primary-cultured murine bone-marrow-derived macrophages. The amounts of these inositol tetrakisphosphates rise upon muscarinic cholinergic stimulation of the astrocytoma cells or stimulation of macrophages with platelet-activating factor.
The design of peptidyldiazomethane inhibitors to distinguish between the cysteine proteinases calpain II, cathepsin L and cathepsin B
Authors: Crawford C , Mason R W , Wikstrom P , Shaw E
Published: August 2015

A series of peptidyldiazomethanes was synthesized and tested as inactivators of the cysteine proteinases calpain II, cathepsin L and cathepsin B. Inactivators that react rapidly and that show a degree of selectivity between the enzymes were identified. Z-Tyr(I)-Ala-CHN2 (where Z represents benzyloxycarbonyl) reacts rapidly with cathepsin L and more slowly with cathepsin B, but does not inhibit calpain II. Z-Leu-Leu-Tyr-CHN2 reacts rapidly with cathepsin L and calpain II but very slowly with cathepsin B. Boc-Val-Lys(epsilon-Z)Leu-Tyr-CHN2 (where Boc represents t-butyloxycarbonyl) reacts more rapidly with calpain II than with cathepsin L or cathepsin B. The discriminating inhibitory effects of these compounds make them potentially useful for investigation of enzyme functions in vivo. The data presented also provide insights into the subsite specificity of calpain.