bluuebunny/crossref_metadata_2025 huggingface dataset:
Isolation and characterization of glyoxylate dehydrogenase from the fungus Sclerotium rolfsii
Authors: Balmforth A J , Thomson A
Published: August 2015

Glyoxylate dehydrogenase (glyoxylate: NAD+ oxidoreductase) was purified 600-fold in three steps from crude extracts of the fungus Sclerotium rolfsii (Corticium rolfsii Curzi). Two of the purification steps involved dye-affinity chromatography. The enzyme is a tetramer of Mr 250 000, with identical subunits of Mr 57 000. Inhibition studies suggest that there is one essential thiol group per active site.
Cleavage of formate from ω,4-dihydroxyacetophenone. An unusual oxygen-requiring reaction in the bacterial catabolism of 4-hydroxyacetophenone
Authors: Hopper D J , Elmorsi E A
Published: August 2015

An enzyme, from a soil bacterium grown on 4-hydroxyacetophenone, cleaved the side chain of omega,4-dihydroxyacetophenone between the keto group and the carbon atom bearing a hydroxy group to give 4-hydroxybenzoate and formate. The reaction was O2-dependent. Partially purified enzyme required no added cofactors for activity.
Extracts of human articular cartilage contain an inhibitor of tissue metalloproteinases
Authors: Dean D D , Woessner J F
Published: August 2015

When human articular cartilage is extracted with 2M-guanidinium hydrochloride at pH 7.5, an inhibitor is obtained that blocks the activity of three metalloproteinases, including collagenase. Molecular-sieve chromatography of the inhibitor gives an Mr value for the inhibitor of 28 500. The inhibitor is stable to heat (60 degrees C, 1h) and acid (pH2, 24 degrees C, 10 min). It is destroyed by trypsin and by reduction and alkylation. It is slowly inactivated by aminophenylmercuric acetate. It binds to concanavalin A-Sepharose and is eluted with alpha-D-1-O-methyl glucopyranoside. Complexes of enzyme and inhibitor are not re-activated by aminophenylmercuric acetate and only partially so by high levels of trypsin. These properties indicate that this inhibitor is a member of the TIMP (tissue inhibitor of metalloproteinases) class. Such an inhibitor, previously found in tissue culture and amniotic fluid, is now shown to be directly extractable from tissue.
The effects of oxygen solubility and high concentrations of salts on photosynthetic electron transport in chloroplast membranes
Authors: Thomasset B , Barbotin J N , Thomas D
Published: August 2015

Chloroplast membranes were isolated in different media containing Hepes [4-(2-hydroxyethyl)-1-piperazine-ethanesulphonic acid] and high concentrations of sorbitol (0.33 M), potassium citrate (0.75 M) or Na2SO4 (1.0 M). Due to the complexity of the media, the oxygen solubility is strongly modified by high concentrations of salts (oxygen solubility for 0.33 M-sorbitol, 0.21 mmol/litre; for 0.75 M-potassium citrate, 0.121 mmol/litre; and for 1.0 M-Na2SO4, 0.112 mmol/litre). The knowledge of these values is necessary to interpret the rate of O2 evolution. For thylakoids isolated in ‘sorbitol buffer’ and then tested in high concentrations of potassium citrate, a slight stimulation of O2 evolution is observed (143-173 mumol of O2/h per mg of chlorophyll a) with potassium ferricyanide as electron acceptor. When we monitor the potassium ferricyanide reduction, no stimulation of electron transport is obtained even if the observed phenomenon is identical with the Photosystem-II oxygen evolution. In the same experiments no stimulation of the photophosphorylation was recorded, but when thylakoids are directly isolated in 0.75 M-potassium citrate, O2 evolution, ferricyanide reduction and photophosphorylation are inhibited by high concentrations of salts. The behaviour of thylakoids seems to be influenced by their initial treatment.
Immunochemical characterization of the androgen-dependent spermine-binding protein of the rat ventral prostate
Authors: Hiipakka R A , Chen C , Schilling K , Oberhauser A , Saltzman A , Liao S
Published: August 2015

A solid-phase radioimmunoassay was developed to measure the level of the androgen-dependent spermine-binding protein (SBP) in the cytosol fraction of the rat ventral prostate during endocrine manipulation. The concentration of SBP and immunologically cross-reacting material (CRM) in the ventral prostate was at least 5000 times higher than the level of CRM detected in rat serum or cytosol from other rat tissues. Cytosol from the ventral prostate of intact rats was separated by DEAE-cellulose chromatography into three major fractions of CRM. One of these fractions corresponded to the elution position of SBP. Cytosol prepared from rats 48 h after castration lacked SBP and one of the two other fractions of CRM. This loss coincided with an increase in CRM in the remaining fraction. No significant difference was detected in the total level of CRM when intact and 48 h-castrated rats were compared. Injection of rats with 5 alpha-dihydrotestosterone (DHT) immediately after castration prevented these changes in the profile of CRM. Several proteins cross-reacting with antibodies to purified SBP were detected in cytosol by using an immunoblot procedure. The highest-Mr band corresponded to SBP. The effect of short- and long-term castration and subsequent DHT treatment on CRM was studied by using the immunoblot technique. Short-term castration (2 days) led to the disappearance of CRM coinciding with SBP (Mr 35 000-38 000) and an increase in smaller forms of CRM (Mr 24 000 and 22 000). Injection of rats with DHT 2 days after castration led to the reappearance of CRM corresponding to SBP, which returned to normal levels within 4 to 5 days of treatment. Long-term castration (up to 14 days) led to a gradual disappearance of all CRM; subsequent DHT treatment led to the reappearance of all forms of CRM and normal levels were attained within 5 days. We have identified SBP and the various forms of CRM as a secretory product of the rat ventral prostate by immunohistochemical staining and by DEAE-cellulose fractionation of prostatic fluid. Prostatic fluid is rich in proteolytic activity and these proteinases may be responsible for processing SBP to small forms of CRM.
The role of calcium in luteinizing hormone-releasing hormone agonist (ICI 118630)-stimulated steroidogenesis in rat Leydig cells
Authors: Sullivan M H F , Cooke B A
Published: August 2015

The luteinizing hormone-releasing hormone (LHRH) agonist ICI 118630 was found to increase testosterone production in purified rat testis Leydig cells in a concentration- and time-dependent manner, but no consistent changes in cyclic AMP levels were detectable. The stimulation of steroidogenesis by LHRH agonist was found to be dependent on the concentration of Ca2+ in the incubation medium; at least 1 mM was required. The calcium ionophore A23187 mimicked the effects of the LHRH agonist on steroidogenesis, and addition of both compounds together did not further increase testosterone production. The calcium ionophore caused a small increase in cyclic AMP which was independent of the concentration of the ionophore and of the calcium concentrations. The evidence obtained in this study indicates that LHRH agonist-stimulated steroidogenesis in rat testis Leydig cells is primarily mediated by calcium and not cyclic AMP.
Dihydrolipoamide dehydrogenase from halophilic archaebacteria
Authors: Danson M J , Eisenthal R , Hall S , Kessell S R , Williams D L
Published: August 2015

Dihydrolipoamide dehydrogenase has been discovered in the halophilic archaebacteria for the first time. The enzyme from both classical and alkaliphilic halobacteria has been investigated. (1) The enzyme specifically catalysed the stoichiometric oxidation of dihydrolipoamide by NAD+. Enzymic activity was optimal at 2 M-NaCl and was remarkably resistant to thermal denaturation. (2) The relative molecular masses (Mr) of the native enzyme from the various species of halobacteria were determined to be within the range 112000-120000. (3) The enzyme exhibited a hyperbolic dependence of catalytic activity on both dihydrolipoamide and NAD+ concentrations. From these steady-state kinetic measurements the dissociation constant (Ks) of dihydrolipoamide was determined to be 57 (+/- 5) microM. (4) The enzyme was only susceptible to inactivation by iodoacetic acid in the presence of its reducing ligands, dihydrolipoamide or NADH. The rate of inactivation followed a hyperbolic dependence on the concentration of dihydrolipoamide, from which the Ks of this substrate was calculated to be 55 (+/- 7) microM. Together with the steady-state kinetic data, the pattern of inactivations is consistent with the involvement in catalysis of a reversibly reducible disulphide bond, as has been found in dihydrolipoamide dehydrogenase from non-archaebacterial species. In eubacterial and eukaryotic organisms, dihydrolipoamide dehydrogenase functions in the 2-oxo acid dehydrogenase complexes. These multienzyme systems have not been detected in the archaebacteria, and, in the context of this apparent absence, the possible function and evolutionary significance of archaebacterial dihydrolipoamide dehydrogenase are discussed.
Calcium- and cyclic AMP-regulated protein kinases of bovine central-nervous-system myelin
Authors: Wu N C , Ahmad F
Published: August 2015

Bovine central-nervous-system myelin was found to contain both Ca2+-activated and cyclic AMP-dependent protein kinases. Each enzyme possesses unique solubility and substrate-specificity characteristics. The Ca2+-activated enzyme, like its substrate (basic protein), is probably deeply embedded in the neural membrane, whereas the cyclic AMP-dependent kinase appears to be much less tightly associated with myelin. Treatment of insoluble myelin fraction housing the Ca2+-activated kinase with phospholipase A2 and phospholipases A2 + C causes a decrease in its ability to become activated by Ca2+. This can be countered by phosphatidylserine and phosphatidylethanolamine. Whereas the activity of the Ca2+-activated membrane-associated kinase is inhibited by chlorpromazine, dibucaine, melittin and Triton X-100, it is activated by certain phorbol diesters (4 beta-phorbol 12-myristate 13-acetate, 4 beta-phorbol 12,13-didecanoate, 4 beta-phorbol 12,13-dibenzoate and 4 beta-phorbol 12,13-diacetate), which appear to exert this effect by lowering the concentration of Ca2+ normally required for the activation of this enzyme. Together these results suggest that the activation of the membrane-associated kinase by Ca2+ most probably requires certain lipids, perhaps those already present in the membrane.
The structure of the inhibitory complex of alloxanthine (1H-pyrazolo[3,4-d]pyrimidine-4,6-diol) with the molybdenum centre of xanthine oxidase from electron-paramagnetic-resonance spectroscopy
Authors: Hawkes T R , George G N , Bray R C
Published: August 2015

Studies were carried out on the inhibitory complex of alloxanthine (1H-pyrazolo[3,4-d]pyrimidine-4,5-diol) with xanthine oxidase, in extension of the work of Williams & Bray [Biochem. J. (1981) 195, 753-760]. By suitable regulation of the reaction conditions, up to 10% of the functional enzyme could be converted into the complex in the Mo(V) oxidation state. The e.p.r. spectrum of the complex was investigated in detail with the help of computer simulation and substitution with stable isotopes. Close structural analogy of the signal-giving species to that of the Very Rapid intermediate in enzyme turnover is shown by g-values (2.0279, 1.9593 and 1.9442) and by coupling to 33S in the cyanide-labile site of the enzyme [A(33S) 0.30, 3.10 and 0.70mT]. However, whereas in the Very Rapid signal there is strong coupling to 17O [Gutteridge & Bray, Biochem. J. (1980) 189, 615-623], instead, in the Alloxanthine signal there is strong coupling to a single nitrogen atom [A(14N) 0.35, 0.35, 0.32 mT]. This is presumed to originate from the 2-position of the heterocyclic ring system. From this work and from earlier kinetic studies it is concluded that alloxanthine, after being bound reversibly at the active centre, reacts slowly with it, in a specific manner, distinct from that in the normal catalytic reaction with substrates. This reaction involves elimination of an oxygen ligand of molybdenum and co-ordination, in this site, of alloxanthine via the N-2 nitrogen atom, to give a complex that is structurally but not chemically closely analogous to that of the Very Rapid species.
Binding of haemoglobin to the red-cell membrane in the presence of copper chloride
Authors: Ribarov S R , Benchev I C , Strashimirov D D
Published: August 2015

Methaemoglobin may be an important factor for initiation and development of lipid peroxidation in Cu(II)-treated red blood cells. It seems likely that the initiation of peroxidation by methaemoglobin is only possible if direct contact between haemoglobin molecule and the cell membrane is realized. In view of this, the binding of haemoglobin to the red-blood-cell membrane in the presence of CuCl2 was studied. It was found that the haemoglobin quenching of the fluorescence of 12-(9-anthroyl)stearic acid-labelled red-blood-cell membranes greatly increases in the presence of CuCl2. This effect is relatively independent of pH and the ionic strength of the medium, indicating that in this case the binding of haemoglobin is not electrostatic in nature. The haemoglobin quenching of the fluorescence of the inside-out and the right-side-out resealed ghosts were almost the same in the presence of CuCl2. This result suggests that, in the presence of ionic copper, both surfaces of the membrane possess approximately equal amounts of sites for the binding of haemoglobin.
Collagen composition of normal and myxomatous human mitral heart valves
Authors: Cole W G , Chan D , Hickey A J , Wilcken D E L
Published: August 2015

The collagens were studied in 13 normal and 19 myxomatous human mitral valves. The collagens of the valve were completely solubilized by using a method consisting of guanidinium chloride extraction, limited pepsin digestions and CNBr cleavage of the residue. The normal valves contained 74% type I, 24% type III and 2% type V collagen. The type I and type III collagens had similar solubility patterns, although only type I collagen was detected in the guanidinium chloride extract. Type V collagen was only detected in the first pepsin extract. The type I and III collagens had higher contents of hydroxylysine than did the same collagens from age-matched dermis. The two-dimensional electrophoretic ‘maps’ of CNBr-cleavage peptides showed low recoveries of the C-terminal alpha 1(I) CB6 and alpha 1(III) CB9 peptides, which are involved in forming intermolecular cross-linkages. Most of the reducible cross-linkages were present in large-Mr peptide complexes, and these complexes were shown by labelling with 125I to include the tyrosine-containing alpha 1(I) CB6 peptide. The myxomatous valves contained 67% type I, 31% type III and 2% type V collagens. There was a significant increase in the concentration of each type of collagen, which consisted of a 9% increase of type I collagen, a 53% increase of type III collagen and a 25% increase of type V collagen. The contents of hydroxylysine in type I and III collagens and the electrophoretic ‘maps’ of the CNBr-cleavage peptides involved in cross-linkages did not differ significantly from the results obtained from the normal valves. The biochemical findings suggest that there is an increased production of collagen, in particular type III collagen, and glycosaminoglycan as well as a proliferation of cells as part of a repair process in the myxomatous valves.
The effect of the iron saturation of transferrin on its binding and uptake by rabbit reticulocytes
Authors: Young S P , Bomford A , Williams R
Published: August 2015

Polyacrylamide-gel electrophoresis in urea was used to prepare the four molecular species of transferrin:diferric transferrin, apotransferrin and the two monoferric transferrins with either the C-terminal or the N-terminal metal-binding site occupied. The interaction of these 125I-labelled proteins with rabbit reticulocytes was investigated. At 4 degrees C the average value for the association constant for the binding of transferrin to reticulocytes was found to increase with increasing iron content of the protein. The association constant for apotransferrin binding was 4.6 × 10(6)M-1, for monoferric (C-terminal iron) 2.5 × 10(7)M-1, for monoferric (N-terminal iron) 2.8 × 10(7)M-1 and for diferric transferrin, 1.1 × 10(8)M-1. These differences in the association constants did not affect the processing of the transferrin species by the cells at 37 degrees C. Accessibility of the proteins to extracellular proteinase indicated that the transferrin was internalized by the cells regardless of the iron content of the protein, since in each case 70% was inaccessible. Cycling of the cellular receptors may also occur in the absence of bound transferrin.
Effects of pH on the interaction of substrates and malonyl-CoA with mitochondrial carnitine palmitoyltransferase I
Authors: Mills S E , Foster D W , McGarry J D
Published: August 2015

The kinetics of carnitine palmitoyltransferase I (CPT I; EC 2.3.1.21) were examined in mitochondria from rat liver, heart and skeletal muscle as a function of pH over the range 6.8-7.6. In all three tissues raising the pH resulted in a fall in the Km for carnitine, no change in the Km for palmitoyl-CoA or Octanoyl-CoA, and a marked decrease in the inhibitory potency of malonyl-CoA. Studies with skeletal-muscle mitochondria established that increasing pH was accompanied by an increase in the Kd of the malonyl-CoA binding site for this ligand, coupled with a decrease in the Kd for fatty acyl-CoA species to compete for malonyl-CoA binding. Three principal conclusions are drawn. (1) The pH-induced shift in malonyl-CoA sensitivity of CPT I is not a phenomenon restricted to liver mitochondria. (2) At any given pH within the range tested, the ability of malonyl-CoA (and closely related compounds) to inhibit enzyme activity is governed by the efficiency of their binding to the malonyl-CoA site. (3) The competitive interaction between fatty acyl-CoA substrates and malonyl-CoA as regards CPT I activity is exerted at the malonyl-CoA binding site. Finally, the possibility is strengthened that the malonyl-CoA binding site is distinct from the active site of CPT I.
Immunoaffinity purification of intact, metabolically active, cholinergic nerve terminals from mammalian brain
Authors: Richardson P J , Siddle K , Luzio J P
Published: August 2015

A method for the immunoaffinity purification of cholinergic nerve terminals from mammalian brain was developed. A sheep antiserum to Torpedo electric-organ synaptic membranes, previously shown to be specific for cholinergic terminals in mammalian brain, was incubated with crude mitochondrial fractions prepared from rat brain. Cholinergic nerve terminals sensitized by this serum were purified from the mitochondrial fractions on a high-capacity cellulose immunoadsorbent bearing a mouse monoclonal anti-(sheep immunoglobulin G) antibody. Adsorption of nerve terminals on to the immunoadsorbent was assessed by using a variety of enzyme markers and gave a maximum yield of 24% of choline acetyltransferase, whereas non-specific binding was less than 1.0% for all of the enzymes measured. Cholinergic terminals were purified 26-fold from rat caudate nucleus, 30-fold from rat hippocampus and 38-fold from rat cerebral cortex. The terminals were shown to be intact, osmotically sensitive and metabolically active.
Identification of two molecular-mass forms of phenylalanine hydroxylase that segregate independently in rats. Specific association of each form with certain rat strains
Authors: Mercer J F B , Grimes A , Jennings I , Cotton R G H
Published: August 2015

The nature of the different molecular-mass forms of phenylalanine hydroxylase in rat livers was examined by immunoprecipitation of the enzyme from crude liver extracts that had been radiolabelled by reductive methylation. The two forms of the enzyme were resolved by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and detected by fluorography. Segregation of the two forms of the enzyme was demonstrated in Sprague-Dawley rats, as would be expected if the two forms were products of allelic genes. In addition, hooded and albino Wistar rat livers contained only the slower-migrating form and Lewis rat livers contained only the faster-migrating form, and hence we suggest that the forms be referred to as W (for Wistar) and L (for Lewis). Peptide mapping showed that the W and L forms are closely related, and the difference between them appears to reside at one or other end of the polypeptide chain. The kidney contained the same forms as the liver in one-tenth the quantity, providing further evidence that the same phenylalanine hydroxylase gene is expressed in liver and kidney.
Dimethyl sulphoxide protects cells against polypeptide toxins and poliovirus
Authors: Sandvig K , Madshus I H , Olsnes S
Published: August 2015

The effect of dimethyl sulphoxide and other cryoprotective compounds on the sensitivity of cells to polypeptide toxins and to poliovirus was tested. In the presence of these compounds, which all affect membrane fluidity, the cells were protected against the toxic proteins and against poliovirus. The large protection obtained was not due to reduced binding and endocytosis of the toxins. Apparently, the cryoprotective compounds interfere with the entry of toxins and of the poliovirus genome across the cell membrane.
Calmodulin activation of cyclic AMP phosphodiesterase in the B16 mouse melanoma
Authors: Walker S W , Mac Neil S , Senior H J , Bleehen S S , Tomlinson S
Published: August 2015

Mouse B16 melanoma extracts of both cultured cells and tumour tissue contain cyclic AMP phosphodiesterase activity, with 95% present in the soluble fraction. Although activation of the enzyme by added calmodulin did not occur, it was found that endogenous calmodulin was present at a level sufficient to activate fully the enzyme. The ability of Ca-calmodulin to stimulate cyclic AMP phosphodiesterase in this tissue was shown by the inhibitory effect of N-(6-aminohexyl)-5-chloronaphthalenesulphonamide (W7), a known calmodulin antagonist; by the activation of the enzyme with exogenous calmodulin observed in supernatants depleted of endogenous calmodulin by passage over fluphenazine-Sepharose 6B in the presence of Ca2+; by the Ca-dependent binding of the enzyme to calmodulin-agarose and its activation by Ca-calmodulin after elution from the column with EGTA-containing buffer. It was calculated that about 50% of the total cyclic AMP phosphodiesterase activity was calmodulin-activated in this tissue.
Characterization of a thyroid-hormone-binding site on nuclear envelopes and nuclear matrices of the male-rat liver
Authors: Lefebvre Y A , Venkatraman J T
Published: August 2015

Nuclear envelopes and nuclear matrices were isolated from the male-rat liver. Incubation of 125I-labelled 3,3′,5-tri-iodothyronine (T3) with the nuclear-envelope fraction resulted in specific binding of T3 to the membranes. Maximum specific binding occurred at 30 degrees C after 2h incubation. Storage for 1 week at -80 degrees C resulted in no loss of binding. Scatchard analysis revealed a class of binding sites with KD 86 nM. 3,3′,5′-Tri-iodothyronine was as effective a competitor of [125I]T3 binding to nuclear envelopes as was L-T3 itself, and tri-iodothyroacetic acid was 70% as potent as T3. L- and D-thyronine did not compete for [125I]T3 binding. Incubation of nuclear envelopes with 0.6 M-NaCl before addition of T3 resulted in the complete loss of specific binding sites, whereas exposure of the membranes to 2.0 M-NaCl after incubation with T3 did not extract binding sites. Nuclear matrices, after incubation with [125I]T3 under the same conditions, were shown to possess a class of binding sites with a similar KD but with approx. 30% of the maximum binding capacity. Nuclear envelopes from hypothyroid animals may possess slightly lower numbers of binding sites compared with nuclear envelopes from the intact animal, whereas nuclear matrices from hypothyroid animals have the same number of binding sites as do nuclear envelopes from the intact animal. In conclusion, nuclear envelopes and nuclear matrices have a class of binding sites with relatively high affinity for T3. It is distinct from nuclear and cytosolic binding sites.
Quasi-elastic light scattering studies on dormant and germinating Bacillus subtilis spores
Authors: Harding S E , Johnson P
Published: August 2015

Spores of Bacillus subtilis in suspension, both dormant and germinating, have been examined by light-scattering methods, both integrated intensity and correlation versions. When intensity of scatter at constant volume was plotted against angle, curves possessing a maximum at about 20 degrees were regularly obtained but without any noticeable features at higher angles. This indicated polydispersity and/or asymmetry among the population. Curves of the intensity correlation function for both dormant and germinating spores at different angles, theta, did not superimpose at the lower angles when plotted appropriately, but did so for theta greater than 35 degrees. This was considered to arise from asymmetry of the spores. By using the high-angle data the apparent diffusion coefficient was determined for both dormant spores and for germinating spores from 1 min after germinant addition. No appreciable difference was observed, from which volume changes greater than 6% during germination could be excluded. The occurrence of germination was confirmed by both absorbance and phase-contrast-microscopy observations.
Ca2+-activated proteinase in the rat. Quantification by immunoassay in the uterus during pregnancy and involution, and in other tissues
Authors: Elce J S , Baenziger J E , Young D C
Published: August 2015

Rat uteri were taken at various stages of pregnancy and involution post partum, and several other tissues were taken from pregnant and non-pregnant animals. Portions of each tissue were homogenized in the presence of proteinase inhibitors, and the amounts of the high-Ca2+-requiring Ca2+-activated proteinase in the supernatants were measured by a two-site immunoradiometric assay using 125I-immunoglobulin G. The proteinase was shown, by protein blotting, to be immunologically identical in all tissues. The amounts in the various tissues, expressed in units of proteinase activity/g wet wt., were: lung, 95; kidney and small intestine, 42; liver, 20; brain, heart and skeletal muscle, 13. Uterine wet weight increased at the end of pregnancy by about 8-fold, but the amounts of proteinase per uterus increased by about 22-fold; alternatively, expressed in units of proteinase activity/g wet wt., the mean uterine values were: non-pregnant, 28.6; term-pregnant, 77.0. As the wet weight of the uterus fell rapidly during involution, the amounts of proteinase activity remained relatively high. The data suggest that the Ca2+-activated proteinase may have some role in tissue resorption during uterine involution, but the high proteinase activity present before parturition must be regulated in ways which are not yet clear.
Rat hepatic uroporphyrinogen III co-synthase. Purification and evidence for a bound folate coenzyme participating in the biosynthesis of uroporphyrinogen III
Authors: Kohashi M , Clement R P , Tse J , Piper W N
Published: August 2015

Rat hepatic uroporphyrinogen III co-synthase was isolated and purified 73-fold with a 13% yield by (NH4)2SO4 fractionation and sequential chromatography on DEAE-Sephacel, Sephadex G-100 (superfine grade) and folate-AH-Sepharose 4B. The purified co-synthase has an Mr of approx. 42 000, and is resolved into two bands, each possessing co-synthase activity, by polyacrylamide-gel electrophoresis. A factor was dissociated from the purified co-synthase. Results of both microbiological and competitive protein-binding assays suggest that it is a pteroylpolyglutamate. The isolated pteroylpolyglutamate factor was co-eluted with authentic N5-methyltetrahydropteroylheptaglutamate on DEAE-Sephacel. Uroporphyrinogen III is formed by cosynthase-free preparations of uroporphyrinogen I synthase in the presence of tetrahydropteroylglutamate. Tetrahydropeteroylheptaglutamate is also able to direct the formation of equivalent amounts of uroporphyrinogen III at a concentration approximately one-hundredth that of tetrahydropteroylmonoglutamate. These results suggest that a reduced pteroylpolyglutamate factor is associated with rat hepatic uroporphyrinogen III co-synthase, and that this may function as a coenzyme for the biosynthesis of uroporphyrinogen III.
Cadmium-binding proteins of rat testes. Characterization of a low-molecular-mass protein that lacks identity with metallothionein
Authors: Waalkes M P , Chernoff S B , Klaassen C D
Published: August 2015

Cadmium-binding proteins in the cytosol of testes from untreated rats were separated by Sephadex G-75 gel filtration. Three major testicular metal-binding proteins (TMBP), or groups of proteins, with relative elution volumes of approx. 1.0 (TMBP-1), 1.7 (TMBP-2) and 2.4 (TMBP-3) were separated. Elution of Zn-binding proteins exhibited a similar pattern. TMBP-3 has previously been thought to be metallothionein (MT), and hence this protein was further characterized and compared with hepatic MT isolated from Cd-treated rats. Estimation of Mr by gel filtration indicated a slight difference between MT (Mr 10000) and TMBP-3 (Mr 8000). Two major forms of MT (MT-I and MT-II) and TMBP-3 (TMBP-3 form I and TMBP-3 form II) were obtained after DEAE-Sephadex A-25 anion-exchange chromatography, with the corresponding subfractions being eluted at similar conductances. Non-denaturing polyacrylamide-gel electrophoresis on 7% acrylamide gels indicated that the subfractions of TMBP-3 had similar mobilities to those of the corresponding subfractions of MT. However, SDS (sodium dodecyl sulphate)/12% (w/v)-polyacrylamide-gel electrophoresis resulted in marked differences in migration of the two corresponding forms of MT and TMBP-3. Co-electrophoresis of MT-II and TMBP-3 form II by SDS/polyacrylamide-gel electrophoresis revealed two distinct proteins. Amino acid analysis indicated much lower content of cysteine in the testicular than in the hepatic proteins. TMBP-3 also contained significant amounts of tyrosine, phenylalanine and histidine, whereas MT did not. U.v.-spectral analysis of TMBP-3 showed a much lower A250/A280 ratio than for MT. Thus this major metal-binding protein in testes, which has been assumed to be MT is, in fact, a quite different protein.
Partial purification and characterization of the soluble phosphatidate phosphohydrolase of rat liver
Authors: Butterwith S C , Hopewell R , Brindley D N
Published: August 2015

A method is described by which the Mg2+-stimulated phosphatidate phosphohydrolase can be purified from the soluble fraction of liver from ethanol-treated rats. The increase in specific activity was about 416-fold. This involved purification by adsorption on calcium phosphate, chromatography on DE-52 DEAE-cellulose, separation on Ultrogel AcA-34 and chromatography on CM-Sepharose 6B. The effects of phosphatidylcholine, phosphatidate and Mg2+, Mn2+ and Zn2+ on the activity are described. Inhibitor studies indicate that the phosphohydrolase contains functional thiol groups and arginine residues.
An additional acidic residue in the membrane portion of the b-subunit of the energy-transducing adenosine triphosphatase of Escherichia coli affects both assembly and function
Authors: Jans D A , Fimmel A L , Hatch L , Gibson F , Cox G B
Published: August 2015

Glycine at position 9 is replaced by aspartic acid in the mutant b-subunit of Escherichia coli F1F0-ATPase coded for by the uncF476 allele. The mutant b-subunit is not assembled into the membrane in haploid strains carrying the uncF476 allele, but, if the mutant allele is incorporated into a multicopy plasmid, then some assembly of the mutant b-subunit occurs. Two revertant strains were characterized, one of which (AN2030) was a full revertant, the other (AN1953) a partial revertant. DNA sequencing indicated that in strain AN2030 the uncF476 mutation had reverted to give the sequence found in the normal uncF gene. The partial-revertant strain AN1953, however, retained the DNA sequence of the uncF476 allele, and complementation analysis indicated that the second mutation may be in the uncA gene. Membranes prepared from the partial-revertant strain carried out oxidative phosphorylation, although the membranes appeared to be impermeable to protons, and the ATPase activity was sensitive to the inhibitor dicyclohexylcarbodi-imide.
Starvation-induced changes of palmitate metabolism and insulin secretion in isolated rat islets stimulated by glucose
Authors: Tamarit-Rodríguez J , Vara E , Tamarit J
Published: August 2015

The influence of 48 h starvation on glucose-induced changes of palmitate metabolism and insulin release in isolated rat islets was investigated. (1) Islet insulin response to 20 mM-glucose was abolished after 48 h starvation, and it was restored by 0.25 mM-2-bromostearate, an inhibitor of fatty acid oxidation. (2) The increase in glucose concentration from 3 to 20 mM was accompanied by a 50% decrease in the oxidation rate of 0.5 mM-[U-14C]palmitate in control (fed) islets, and a concomitant increase (100%) in its incorporation into triacylglycerol and phospholipid fractions. (3) Starvation induced a higher basal (3 mM-glucose) rate of palmitate oxidation, which was resistant to inhibition by 20 mM-glucose. The latter also failed to increase palmitate incorporation into islet triacylglycerols and phospholipids. (4) 2-Bromostearate (0.25 mM) strongly inhibited the high oxidation rate of palmitate in islets of starved rats, and allowed a normal stimulation of its incorporation rate into islet lipids by 20mM-glucose. (5) The results suggest that starvation restricts islet esterification of fatty acids by inducing a higher rate of their oxidative degradation that is insensitive to regulation by glucose.
Drug-binding properties of rat α1-foetoprotein. Binding of warfarin, phenylbutazone, azapropazone, diazepam, digitoxin and cholic acid
Authors: Hervé F , Rajkowski K , Martin M T , Dessen P , Cittanova N
Published: August 2015

As part of an investigation into whether alpha 1-foetoprotein (alpha 1-FP) plays the same transport role in foetal serum as albumin does in the adult, the binding properties of both proteins were compared with respect to the binding of a series of compounds known to be bound by albumin's specific drug-binding sites. The binding of warfarin, phenylbutazone, azapropazone, diazepam, digitoxin and cholic acid by rat alpha 1-FP and serum albumin was studied by equilibrium dialysis at 4 degrees C. Rat alpha 1-FP was shown to have neither albumin's high-affinity site II (diazepam as marker) nor its site III (digitoxin and cholic acid as markers). High-affinity binding by alpha 1-FP was found for the specific markers (warfarin, phenylbutazone, azapropazone) of albumin's drug-binding site I. However, instead of albumin's one high-affinity site/molecule, a mean value of 0.5 site/molecule was obtained with rat alpha 1-FP. Charcoal treatment at neutral pH of rat serum albumin did not affect its measured binding properties, but treatment of the alpha 1-FP led to an increased affinity for warfarin, phenylbutazone and azapropazone without a change in the measured number of sites, indicating competition for binding at this site by (an) endogenous ligand(s). These results are discussed in terms of the structures of the two proteins and with respect to the physiological implications of the differences found.
The Ca2+-activated polyphosphoinositide phosphodiesterase of human and rabbit neutrophil membranes
Authors: Cockcroft S , Baldwin J M , Allan D
Published: August 2015

Addition of Ca2+ to a plasma-membrane fraction derived from human or rabbit neutrophils led to the specific breakdown of polyphosphoinositides. The degradation products were identified as diacylglycerol and inositol bis- and tris-phosphate, thus demonstrating the presence of a Ca2+-activated phospholipase C. The newly generated diacylglycerol resembled the polyphosphoinositides in its fatty acid composition, and in the presence of MgATP2- it was converted into phosphatidate. These results therefore demonstrate the presence in neutrophil plasma membranes not only of polyphosphoinositide phosphodiesterase but also of diacylglycerol kinase.
Properties of rat heart adenosine kinase
Authors: Fisher M N , Newsholme E A
Published: August 2015

Adenosine kinase was purified 870-fold from rat heart by a combination of gel filtration and affinity chromatography. The preparation was free of purine-metabolizing enzymes that could interfere in the assay of the kinase. A study of the properties of the purified enzyme showed that it is activated by Na+ and K+, it possesses a broad pH optimum between 6 and 8, MgATP is the nucleotide substrate, free Mg2+ is an inhibitor with respect to both MgATP and adenosine, and the enzyme is subject to substrate inhibition by adenosine. The severity of this inhibition increases as the concentration of free Mg2+ increase. The Km for MgATP was calculated to be 0.8 mM and that for adenosine, at likely physiological concentrations of MgATP and free MgCl2, was about 0.2 microM. In vivo the enzyme is likely to be saturated with both MgATP and adenosine. Indeed, the adenosine concentration in rat heart in vivo is probably sufficient to cause substrate inhibition, and this would be increased by an increase in free Mg2+ concentration. Changes in the concentrations of adenosine and free Mg2+ may play a role in modifying the activity of the enzyme in vivo.
Activation of Neurospora crassa soluble adenylate cyclase by calmodulin
Authors: Reig J A , Téllez-Iñón M T , Flawiá M M , Torres H N
Published: August 2015

The soluble form of adenylate cyclase was extracted and purified from wild-type Neurospora crassa mycelia. Brain or N. crassa calmodulin significantly enhanced this enzyme activity in assay mixtures containing Mg2+-ATP as substrate. EGTA reverses this calmodulin activation.
Chemical modifications of metallothionein. Preparation and characterization of polymers
Authors: Templeton D M , Cherian M G
Published: August 2015

Reaction of rat liver metallothionein-II with two bifunctional cross-linking reagents, glutaraldehyde and dimethyl suberimidate, produces high yields of polymeric species. It is argued that cross-linking is trapping preformed aggregates of the protein, which therefore represent a stabilized quaternary structure of metallothionein. The two polymeric species differ in a number of respects. With dimethyl suberimidate, the polymer retains all metal-binding sites of the monomer, and has an unaltered isoelectric point. Reaction with glutaraldehyde causes loss of one or two Cd2+/Zn2+-binding sites and elevates the pI. Both species are nearly spherical aggregates, in contrast with the highly asymmetrical metallothionein. Both polymers are linked through lysine residues, and the thiol groups remain reduced. The biological significance of these aggregates is discussed.
Vasoactive-intestinal-polypeptide-stimulated adenosine 3′,5′-cyclic monophosphate accumulation in GH3 pituitary tumour cells. Reversal of desensitization by forskolin
Authors: Guild S , Drummond A H
Published: August 2015

The interaction between forskolin and vasoactive intestinal polypeptide (VIP) in the regulation of cyclic AMP production in GH3 pituitary tumour cells was investigated. Both forskolin (10nM-10 microns) and VIP (10pM-10nM) increased the cyclic AMP content of GH3 cells. Forskolin (50-100nM) was additive with VIP in stimulating cyclic AMP accumulation when low concentrations (less than 1 nM) of the peptide were used, but exhibited a synergistic interaction with higher VIP concentrations (10-100 nM). These effects on cyclic AMP accumulation were reflected in a leftward shift in the concentration-response curve for VIP-stimulated prolactin release from GH3 cells, a process known to be regulated by intracellular cyclic AMP concentrations. The synergy observed did not appear to be related to changes in cyclic nucleotide phosphodiesterase activity, since it was even more marked in the presence of isobutylmethylxanthine, a phosphodiesterase inhibitor. Studies of the time-course of VIP-induced changes in GH3-cell cyclic AMP content revealed that, with high concentrations of VIP, production ceased within 2 min of addition. This attenuation of cyclic AMP synthesis was still observed in the presence of isobutylmethylxanthine, but was markedly inhibited by low concentrations of forskolin (50-100nM). The results suggest that VIP-induced cyclic AMP production rapidly becomes desensitized. This process, which is prevented by forskolin, may be related to changes in the ability of the guanine nucleotide regulatory protein to couple receptor occupancy to activation of adenylate cyclase.
The capacity of basic peptides to trigger exocytosis from mast cells correlates with their capacity to immobilize band 3 proteins in erythrocyte membranes
Authors: Dufton M J , Cherry R J , Coleman J W , Stanworth D R
Published: August 2015

The effect of mast-cell-triggering peptides on the rotational properties of band 3, a protein component of the human erythrocyte membrane, was measured by observing flash-induced transient dichroism of the triplet probe eosin maleimide. In the presence of melittin, polylysine and five synthetic peptides, varying degrees of retardation in the rotational motion of band 3 were produced. When placed in order of band 3 immobilizing activity, the peptides formed a series identical with their order of efficacy in releasing 5-hydroxytryptamine from rat peritoneal mast cells. The correspondence in the abilities to immobilize band 3 in the erythrocyte and trigger mast cells is significant because structure-activity analyses of the peptides show both processes to have the same cationic, hydrophobic and stereochemical requirements. Probably, the immobilization of band 3 proteins reflects an ability of the basic peptides to aggregate anionic surface moieties, and therefore a similar mechanism is implied in mast-cell triggering.
The effect of low-density lipoproteins on the synthesis of cyclic nucleotides induced by prostacyclin in isolated platelets
Authors: Bruckdorfer K R , Buckley S , Hassall D G
Published: August 2015

Isolated platelets are strongly sensitized by the presence of low-density lipoproteins (LDL) so that they aggregate with very low concentrations of other agonists or exhibit spontaneous aggregation. Prostacyclin (PGI2) is a potent inhibitor of aggregation, but its action was reversed by LDL. This effect of LDL was accompanied by a decrease in the synthesis of cyclic AMP induced by PGI2, but its efficacy depended on the relative concentrations of LDL and PGI2. PGI2 also enhanced the synthesis of cyclic GMP, but this was completely reversed by the presence of LDL. LDL did not remove inhibitory prostaglandins, e.g. E1, from their receptor sites. The lipoproteins also decreased cyclic AMP synthesis induced by forskolin, which has its effect on the GTP-sensitive protein or the catalytic unit of the adenylate cyclase enzyme complex. It is proposed that LDL may act on the enzyme catalytic unit via an inhibitory GTP-sensitive protein or by a separate mechanism which indirectly impedes the production of cyclic AMP.
Kinetics of protein modification reactions. Plot of fractional enzyme activity versus extent of protein modification in cases where all modifiable groups are essential for enzyme activity
Authors: Rakitzis E T
Published: August 2015

The plot of fractional enzyme activity versus extent of protein modification, for cases where all enzyme modifiable groups of a certain kind are essential for activity, is found to be nearly independent of the number, per enzyme active site, of modifiable groups involved. Such plots usually, by a fallacious extension of the initial portion of the plot on the extent-of-modification axis, are interpreted to mean the modification of one single group per enzyme active site (or per enzyme molecule). The possible relevance of these findings to cases in the literature is discussed.
The active site of the P99 β-lactamase from Enterobacter cloacae
Authors: Joris B , Dusart J , Frere J M , van Beeumen J , Emanuel E L , Petursson S , Gagnon J , Waley S G
Published: August 2015

Labelling the beta-lactamase of Enterobacter cloacae P99 with a poor substrate or a mechanism-based inactivator points to an active-site serine residue in a sequence closely resembling that of the ampC beta-lactamase. These results establish the P99 enzyme as a class-C beta-lactamase, and the concurrence of the two approaches helps to confirm the reliability of determining active-site sequences with the aid of mechanism-based inactivators.
Radioimmunochemical evidence for a role of carbohydrate in antigenic determinant(s) on human liver α-L-fucosidase
Authors: Alhadeff J A , Andrews-Smith G L
Published: August 2015

A competitive-binding radioimmunoassay method was employed to investigate the role of carbohydrate in antigenic determinant(s) of human liver alpha-L-fucosidase. Competition curves were used to quantify the concentrations of competitors needed to cause 30% inhibition of the precipitation of 125I-labelled alpha-L-fucosidase. The isoelectric forms of alpha-L-fucosidase, which are related by sialic acid residues, were separated preparatively and used as competitors in the radioimmunoassay. A pattern of increasing effectiveness as competitors with increasing acidity of the forms was found, suggesting that sialic acid may be involved in the antigenic determinant(s) of alpha-L-fucosidase. Specificity was exhibited when sugar and sugar derivatives were used as competitors in the radioimmunoassay: a 51-fold range of competitive ability was found, and sialic acids (N-acetylneuraminic acid and N-glycollylneuraminic acid) and colominic acid (a polymer of N-acetylneuraminic acid) were the best competitors. The results of our studies suggest that carbohydrate contributes to antigenic determinant(s) of alpha-L-fucosidase and that sialic acid is probably the major sugar involved.
A comparison of the low-density-lipoprotein receptor from bovine adrenal cortex, rabbit and rat liver and adrenal glands by lipoprotein blotting
Authors: Kroon P A , Thompson G M , Chao Y S
Published: August 2015

This paper describes the use of lipoprotein blotting to detect low-density-lipoprotein (LDL) receptors in rat and rabbit liver and adrenal glands and in bovine adrenal glands. Using this technique we show that the rabbit and rat liver LDL receptors have Mr values of 128000 and 145000 respectively. Mr values for the rabbit, rat and bovine adrenal receptors are 131000, 142000 and 132000 respectively. Differences between the bovine adrenal and rat liver receptors are not due to differences in the degree of sialylation. Lipoprotein blotting can be used to detect dietary- and drug-induced changes in the concentrations of LDL receptors. When rabbits are fed on a cholesterol-rich diet, liver LDL receptors cannot be detected, consistent with the suppression of hepatic LDL receptors by cholesterol feeding. Pharmacological doses of 17 alpha-ethinyloestradiol cause a marked increase in hepatic LDL-receptor activity in the rat. This is accompanied by a corresponding increase in the number of LDL receptors detected by lipoprotein blotting. The Mr of the induced receptor is identical with that of the receptor from control rats, which suggests that the induced receptors are produced by the same gene as LDL receptors normally present in the liver.