bluuebunny/crossref_metadata_2025 huggingface dataset:
A two-state thermodynamic and kinetic analysis of the allosteric functioning of the haemoglobin of an extreme poikilotherm
Authors: Brittain T
Published: August 2015

The blood of the extreme poikilotherm Trematomus borchgrevinki contains one major haemoglobin component, which may be separated from the minor species by ion-exchange chromatography. This haemoglobin shows co-operative CO-binding isotherms at pH 8.2. An analysis of the temperature-dependence of the binding curves has allowed the thermodynamic constants associated with the two-state allosteric parameters L, KR and KT to be measured. The binding of CO at lower pH (6.2) is characterized by the maintenance of the T-state to relatively high degrees of saturation. Kinetic investigations with the use of flash photolysis of the haemoglobin-CO complex under various conditions has allowed the determination of the thermodynamic parameters association with the T-state and R-state rate constants. An amalgamation of these data has allowed the mathematical simulation of predicted time courses for CO binding to this haemoglobin, by using the two-state model, which very closely represents those obtained experimentally. The overall findings show that this haemoglobin closely follows the two-state model of co-operative interaction.
Cadmium metabolism by rat liver endothelial and Kupffer cells
Authors: Caperna T J , Failla M L
Published: August 2015

The metabolism of cadmium was investigated in Wistar-rat liver non-parenchymal cells. Kupffer and endothelial cells, the major cell populations lining the sinusoidal tracts, were isolated by collagenase dispersion and purified by centrifugal elutriation. At 20 h after subcutaneous injection of the metal salt (1.5 mg of Cd/kg body weight), endothelial cells accumulated 2-fold higher concentrations of Cd than did Kupffer or parenchymal cells. Most of the Cd in non-parenchymal cells was associated with cytosolic metallothionein (MT), the low-Mr heavy-metal-binding protein(s). When MT was quantified in cytosols from cells isolated from control rats by a 203Hg competitive-binding assay, low levels were found to be present in Kupffer, endothelial and parenchymal cells. Cd injection significantly increased MT levels in all three cell types. The induction of MT synthesis was investigated in vitro by using primary monolayer cultures. The incorporation of [35S]cysteine into MT increased 47% over constitutive levels in endothelial-cell cultures after the addition of 0.8 microM-Cd2+ to the medium for 10 h. MT synthesis in Kupffer cells was not observed. The lack of MT synthesis by monolayer cultures of Kupffer cells in vitro was associated with a decreased capacity of these cells to accumulate heavy metals from the extracellular medium. This apparent decreased ability to transport metals did not reflect a general defect in either cellular function or metabolic activity, since isolated Kupffer cells incorporated [3H]leucine into protein at rates comparable with those shown by liver parenchymal cells and readily phagocytosed particles.
Integrated rate equations for enzyme-catalysed first-order and second-order reactions
Authors: Boeker E A
Published: August 2015

Generalized rate equations covering all mechanisms giving hyperbolic initial-rate kinetics with stoichiometry A in equilibrium P, A in equilibrium P + Q, A + B in equilibrium P and A + B in equilibrium P + Q were integrated. The results are regular and reasonably economical.
Analysis of the mechanism of chloramphenicol acetyltransferase by steady-state kinetics. Evidence for a ternary-complex mechanism
Authors: Kleanthous C , Shaw W V
Published: August 2015

The mechanism of the enzymic reaction responsible for chloramphenicol resistance in bacteria was examined by steady-state kinetic methods. The forward reaction catalysed by chloramphenicol acetyltransferase leads to inactivation of the antibiotic. Use of alternative acyl donors and acceptors, as well as the natural substrates, has yielded data that favour the view that the reaction proceeds to the formation of a ternary complex by a rapid-equilibrium mechanism wherein the addition of substrates may be random but a preference for acetyl-CoA as the leading substrate can be detected. Chloramphenicol and acetyl-CoA bind independently, but the correlation between directly determined and kinetically derived dissociation constants is imperfect because of an unreliable slope term in the rate equation. The reverse reaction, yielding acetyl-CoA and chloramphenicol, was studied in a coupled assay involving citrate synthase and malate dehydrogenase, and is best described by a rapid-equilibrium mechanism with random addition of substrates. The directly determined dissociation constant for CoA is in agreement with that derived from kinetic measurements under the assumption of an independent-sites model.
Evidence for the reversibility of the acyl-CoA:lysophosphatidylcholine acyltransferase in microsomal preparations from developing safflower (Carthamus tinctorius L.) cotyledons and rat liver
Authors: Stymne S , Stobart A K
Published: August 2015

Acyl exchange between acyl-CoA and position 2 of sn-phosphatidylcholine occurs in the microsomal preparations of developing safflower cotyledons. Evidence is presented to show that the acyl exchange is catalysed by the combined back and forward reactions of an acyl-CoA:lysophosphatidylcholine acyltransferase (EC 2.3.1.23). The back reaction of the enzyme was demonstrated by the stimulation of the acyl exchange with free CoA and by the observation that the added CoA was acylated with acyl groups from position 2 of sn-phosphatidylcholine. Re-acylation of the, endogenously produced, lysophosphatidylcholine with added acyl-CoA occurred with the same specificity as that observed with added palmitoyl lysophosphatidylcholine. A similar acyl exchange, catalysed by an acyl-CoA:lysophosphatidylcholine acyltransferase, occurred in microsomal preparations of rat liver. The enzyme from safflower had a high specificity for oleate and linoleate, whereas arachidonate was the preferred acyl group in the rat liver microsomal preparations. The rate of the back reaction was 3-5% and 0.2-0.4% of the forward reaction in the microsomal preparations of safflower and rat liver respectively. Previous observations, that the acyl exchange in safflower microsomal preparations was stimulated by bovine serum albumin and sn-glycerol 3-phosphate, can now be explained by the lowered acyl-CoA concentrations in the incubation mixture with albumin and in the increase in free CoA in the presence of sn-glycerol 3-phosphate (by rapid acylation of sn-glycerol 3-phosphate with acyl groups from acyl-CoA to yield phosphatidic acid). Bovine serum albumin and sn-glycerol 3-phosphate, therefore, shift the equilibrium in acyl-CoA:lysophosphatidylcholine acyltransferase-catalysed reactions towards the rate-limiting step in the acyl exchange process, namely the removal of acyl groups from phosphatidylcholine. The possible role of the acyl exchange in the transfer of acyl groups between complex lipids is discussed.
The computerized derivation of steady-state rate equations for enzyme kinetics
Authors: Herries D G
Published: August 2015

A FORTRAN 77 program is described for the derivation of steady-state rate equations for enzyme kinetics. Input is very simple and consists of the two enzyme forms and the two rate constants for each step in the mechanism. The program may be run interactively or off-line. The results are produced after collecting together the algebraic coefficients of like concentration terms, taking account of sign. A fully interactive BASIC version running on a BBC Microcomputer is also available. Details of the programs have been deposited as Supplementary Publication SUP 50126 (45 pages) with the British Library Lending Division, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies may be obtained as indicated in Biochem. J. (1984) 217, 5.
Control of gene expression by glucocorticoid hormones
Authors: Rousseau G G
Published: August 2015

Glucocorticoids control the expression of a small number of transcriptionally active genes by increasing or decreasing mRNA concentration. Either effect can result from a transcriptional or a post-transcriptional mechanism. Induction of mouse mammary tumour virus RNA results from a stimulation of transcription initiation and depends on the presence of defined regions in proviral DNA. These regions bind the glucocorticoid receptor and behave functionally as proto-enhancers. Glucocorticoid-inducible genes can retain their sensitivity to the hormone after transfer to a heterologous cell by transfection techniques. Non-inducible genes can become inducible when linked to the promoter region of an inducible gene. The mechanisms by which the receptor-steroid complex stimulates or inhibits transcription or influences mRNA stability are unknown. Receptor binding to nucleic acids appears to be a necessary but not sufficient condition. It is likely that the receptor also interacts with chromatin proteins. This might lead to a catalytic modification of these proteins, resulting in a modulation of gene expression. Development of glucocorticoid-sensitive, biochemically defined, cell-free transcription systems should provide a tool to delineate the molecular determinants of this essential regulatory mechanism.
The removal of cytosolic-type aldehyde dehydrogenase from preparations of sheep liver mitochondrial aldehyde dehydrogenase and the unusual properties of the purified mitochondrial enzyme in assays
Authors: Allanson S , Dickinson F M
Published: August 2015

The pI approximately 5.2 isoenzymes of mitochondrial aldehyde dehydrogenase were separated from the other isoenzymes by pH-gradient chromatography on DEAE-Sephacel. The pI approximately 5.2 material is immunologically identical with cytosolic aldehyde dehydrogenase. It also shows sensitivity to 20 microM-disulfiram and insensitivity to 4M-urea in assays. These and other criteria seem to establish that the material is identical with the cytosolic enzyme. Mitochondrial enzyme that had been purified to remove pI approximately 5.2 isoenzymes shows concentration-dependent lag phases in assays. These effects are possibly due to the slow establishment of equilibrium between tetramer and either dimers or monomers, with the dissociated species being intrinsically more active than the tetramer.
Azotobacter chroococcum 7Fe ferredoxin. Two pH-dependent forms of the reduced 3Fe clusters and its conversion to a 4Fe cluster
Authors: George S J , Richards A J M , Thomson A J , Yates M G
Published: August 2015

Ferredoxin from Azotobacter chroococcum has been studied by low-temperature magnetic-circular-dichroism and electron-paramagnetic-resonance spectroscopy. When aerobically isolated ferredoxin contains a [3Fe-4S] and [4Fe-4S] cluster. Anaerobic treatment with dithionite in the presence of ethanediol reduces the [3Fe-4S] cluster to give two spectroscopically distinct forms RI and RII which are reversibly interconvertible with a pKa approximately 7.5. The higher-pH form, RII, has a high affinity for ferrous ion and converts readily to a [4Fe-4S]1+ cluster, scavenging iron from the medium. The presence of the iron chelator EDTA inhibits this conversion.
Reversal of dietary-induced insulin resistance in muscle of the rat by adenosine deaminase and an adenosine-receptor antagonist
Authors: Budohoski L , Challiss R A J , Cooney G J , McManus B , Newsholme E A
Published: August 2015

Transfer of young rats from a maintenance diet to a breeding diet plus 10% sucrose in the drinking water for 4 weeks caused the development of insulin resistance. Inclusion of the enzyme adenosine deaminase or the adenosine-receptor antagonist 8-phenyltheophylline caused a marked increase in the sensitivity of the soleus-muscle strips isolated from the diet-induced insulin-resistant rats: the concentration of insulin giving 50% of maximum response of glycolysis shifted from 500 to less than 20 microunits/ml.
Localization of the complement-component-C3b-binding site and the cofactor activity for factor I in the 38kDa tryptic fragment of factor H
Authors: Alsenz J , Lambris J D , Schulz T F , Dierich M P
Published: August 2015

Trypsin treatment of human factor H (H160) [enzyme/substrate ratio 1:100 (w/w), 30 min, 37 degrees C] generated a 38 kDa (H38) and a 142 kDa (H142) fragment linked by disulphide bonds (H38/142). The fragments were purified by reduction with 2-mercapto-ethanol, gel filtration on a Sephadex G-200 column and affinity chromatography with monoclonal anti-(factor H) antibody coupled to Sepharose 4B. This monoclonal antibody bound to a site in the 38 kDa fragment. To localize the C3b binding site in factor H we used two enzyme-linked immunosorbent assays (e.l.i.s.a.). For the first test, e.l.i.s.a. plates were coated with C3b; H160, H38/142, H38 and H142 were added, and their binding was monitored by goat anti-(factor H) and peroxidase-labelled rabbit anti-goat antibodies. Only intact factor H bound to the C3b-coated plates. For the second test, e.l.i.s.a. plates were coated with comparable amounts of factor H or its fragments, and C3b was offered at several dilutions. In contrast with the results from the first assay, C3b bound to intact factor H, H38/142 and H38 but not to H142, thus characterizing H38 as the fragment carrying the C3b-binding site. To identify the fragment responsible for the cofactor activity of factor H (cleavage of fluid-phase C3b by factor I), 125I-C3b was incubated with either H38 or H142 and factor I. H142 had no cofactor activity, whereas H38 had the same cofactor function as intact H. To further investigate the relationship between the C3b-binding site and the site of factor H essential for its cofactor activity, we made use of monoclonal antibodies directed against the H38. Those antibodies inhibiting the binding of C3b to H160 also inhibited the cofactor function, whereas those without effect on the C3b binding also did not interfere with the cofactor activity. This suggests that the C3b-binding site and the site essential for the cofactor activity of factor H are both localized in the 38 kDa tryptic fragment of factor H in close proximity or are identical.
Calmodulin activation of adenylate cyclase in the mouse B16 melanoma
Authors: Mac Neil S , Walker S W , Senior H J , Pollock A , Brown B L , Bleehen S S , Munro D S , Tomlinson S
Published: August 2015

Calmodulin antagonists inhibited hormone-stimulated cyclic AMP accumulation in both cultured cells and cell lysates of mouse B16 melanoma. Particulate preparations of B16 melanoma contained 34-45% of total cell calmodulin, which could not be dissociated by extensive washing irrespective of the presence of EGTA in the buffer. The adenylate cyclase activity in such preparations was unaffected by the addition of exogenous calmodulin. However, the rare-earth-metal ion La3+, which can mimic or replace Ca2+ in many systems, produced an immediate inhibition of agonist-stimulated adenylate cyclase activity and preincubation of particulate preparations was La3+ followed by washing with La3+-free buffer dissociated calmodulin (96% loss) from particulate preparations. The loss of calmodulin from particulate preparations was associated with a decrease in agonist responsiveness (74%) and a marked change in the Ca2+-sensitivity of the enzyme, low concentrations of calcium (approx. 10 nM) now failing to stimulate enzyme activity, high concentrations of calcium (greater than or equal to 100 nM) producing greater-than-normal inhibition of enzyme activity. Direct activation of adenylate cyclase by the addition of pure calmodulin was now demonstrable in such calmodulin-depleted particulate preparations. Half-maximal stimulation of agonist-responsive adenylate cyclase occurred at 80 nM-calmodulin in the presence of 10 microM free Ca2+. Maximal stimulation by calmodulin (at 300-600 nM) restored enzyme activity to 89 +/- 5% (mean +/- S.E.M., n = 7) of the activity in untreated, calmodulin-intact, preparations.
Mucin release and calcium fluxes in isolated rat submandibular acini
Authors: McPherson M A , Dormer R L
Published: August 2015

A method is described for preparing isolated rat submandibular acini by collagenase digestion followed by mechanical dispersion. As assessed by Trypan Blue exclusion, phase contrast microscopy, ATP content and release of mucins and lactate dehydrogenase, the acini are morphologically and functionally intact. Secretory function of isolated acini was similar to that of intact tissue in terms of time-course, dose dependence and degree of stimulation of mucin release by adrenergic secretagogues. Mucin release was increased to the same extent (approx. 3-4-fold) by either isoproterenol or noradrenaline at a maximally effective concentration (10 microM). Stimulation of mucin release by isoproterenol (10 microM), noradrenaline (10 microM) or adrenaline (10 microM) was inhibited by propranolol (30 microM) but not by phentolamine (30 microM). Isoproterenol (10 microM) increased both 45Ca2+ uptake and efflux from the acini, which was shown to represent a net release of calcium. However, there was a delay (approx. 10 min) in onset of stimulation of 45Ca2+ mobilization which was not apparent in isoproterenol stimulation of mucin release. Our results indicate that increases in intracellular calcium mobilization in response to a beta-adrenergic secretagogue do not trigger mucin secretion from rat submandibular acini.
Activities of glucokinase and hexokinase in mammalian and avian livers
Authors: Stanley J C , Dohm G L , McManus B S , Newsholme E A
Published: August 2015

A radiochemical assay for glucokinase activity was developed for use in high-speed supernatants of liver. The maximum activities of glucokinase ranged from 0.4 to 3.8 mumol/min per g fresh wt. at 30 degrees C in some avian and mammalian livers, including pigeon, guinea pig and man, in which previous reports indicated zero activities. The reported maximum rates of hepatic glycogen synthesis in livers of rat and man in vivo are similar to the calculated glucokinase activities at 10mM-glucose; therefore glucokinase activity should not limit glycogen synthesis from glucose.
Protein synthesis in rat tissues during lactation. No effect of diethyl ether anaesthesia
Authors: Sampson D A , Masor M , Jansen G R
Published: August 2015

Rates of protein synthesis were measured in mammary gland, liver, intestinal mucosa and muscle of lactating rats by using a flooding dose of [3H]phenylalanine that was injected into diethyl ether-anaesthetized dams via a lateral vein or into undisturbed dams via a jugular cannula. Ether anaesthesia did not alter rates of protein synthesis significantly in any tissue.
Difference in glucose sensitivity of liver glycolysis and glycogen synthesis. Relationship between lactate production and fructose 2,6-bisphosphate concentration
Authors: Hue L , Sobrino F , Bosca L
Published: August 2015

Incubation of isolated rat hepatocytes from fasted rats with 0-6 mM-glucose caused an increase in [fructose 2,6-bisphosphate] (0.2 to about 5 nmol/g) without net lactate production. A release of 3H2O from [3-3H]glucose was, however, detectable, indicating that phosphofructokinase was active and that cycling occurred between fructose 6-phosphate and fructose 1,6-bisphosphate. A relationship between [fructose 2,6-bisphosphate] and lactate production was observed when hepatocytes were incubated with [glucose] greater than 6 mM. Incubation with glucose caused a dose-dependent increase in [hexose 6-phosphates]. The maximal capacity of liver cytosolic proteins to bind fructose 2,6-bisphosphate was 15 nmol/g, with affinity constants of 5 × 10(6) and 0.5 × 10(6) M-1. One can calculate that, at 5 microM, more than 90% of fructose 2,6-bisphosphate is bound to cytosolic proteins. In livers of non-anaesthetized fasted mice, the activation of glycogen synthase was more sensitive to glucose injection than was the increase in [fructose 2,6-bisphosphate], whereas the opposite situation was observed in livers of fed mice. Glucose injection caused no change in the activity of liver phosphofructokinase-2 and decreased the [hexose 6-phosphates] in livers of fed mice.
The mechanism of Klebsiella pneumoniae nitrogenase action. Pre-steady-state kinetics of an enzyme-bound intermediate in N2 reduction and of NH3 formation
Authors: Thorneley R N F , Lowe D J
Published: August 2015

The reduction of N2 to 2NH3 by Klebsiella pneumoniae nitrogenase was studied by a rapid-quench technique. The pre-steady-state time course for N2H4, formed on quenching by the acid-induced hydrolysis of an enzyme-bound intermediate in N2 reduction, showed a 230 ms lag followed by a damped oscillatory approach to a constant concentration in the steady state. The pre-steady-state time course for NH3 formation exhibited a lag of 500 ms and a burst phase that was essentially complete at 1.5s, before a steady-state rate was achieved. These time courses have been simulated by using a previously described kinetic model for the mechanism of nitrogenase action [Lowe & Thorneley (1984) Biochem. J. 224, 877-886]. A hydrazido(2-) structure (=N-NH2) is favoured for the intermediate that yields N2H4 on quenching. The NH3-formation data indicate enzyme-bound metallo-nitrido (identical to N) or -imido (=NH) intermediates formed after N-N bond cleavage to produce the first molecule of NH3 and which subsequently give the second molecule of NH3 by hydrolysis on quenching. The simulations require stoichiometric reduction of one N2 molecule at each Mo and the displacement of one H2 when N2 binds to the MoFe protein. Inhibition by H2 of N2-reduction activity occurs before the formation of the proposed hydrazido(2-) species, and is explained by H2 displacement of N2 at the active site.
The mechanism of Klebsiella pneumoniae nitrogenase action. Simulation of the dependences of H2-evolution rate on component-protein concentration and ratio and sodium dithionite concentration
Authors: Thorneley R N F , Lowe D J
Published: August 2015

The rate constants from Table 1 and Scheme 2 of Lowe & Thorneley [(1984) Biochem. J. 224, 877-886] were used to simulate the rate of H2 evolution, under various conditions, from nitrogenase isolated from Klebsiella pneumoniae. These rates depend on both the ratio and concentrations of the MoFe protein and Fe protein that comprise nitrogenase. The simulations explain the shapes of ‘protein titration’ and ‘dilution effect’ curves. The concept of an apparent Km for the reductant Na2S2O4 is shown to be invalid, since the dependence of H2-evolution rate on the square root of S2O4(2-) concentration is not hyperbolic and depends on the ratio and absolute concentrations of the MoFe protein and Fe protein.
Inositol lipids, phosphatidate and diacylglycerol share stearoylarachidonoylglycerol as a common backbone in thrombin-stimulated human platelets
Authors: Mauco G , Dangelmaier C A , Smith J B
Published: August 2015

Gel-filtered human platelets were stimulated with 5i.u. of thrombin/ml for times up to 1 min. The fatty acid composition of inositol-containing phospholipids, phosphatidic acid and diacylglycerol was determined by g.l.c. in control and thrombin-stimulated platelet suspensions. Inositol phospholipids were found to have similar proportions of stearic and arachidonic acids, the sum of these representing 86.6% of the total fatty acids in phosphatidylinositol (PtdIns), 76.9% in phosphatidylinositol 4-phosphate (PtdIns4P) and 85.4% in phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2]. However, arachidonic and stearic acids were less abundant in phosphatidic acid (PtdA) and diacylglycerols in non-stimulated platelets. A transient decrease in the mass of PtdIns(4,5)P2 was observed after 5-10s of thrombin stimulation, followed by an increase after 30s. The amounts of PtdIns4P and PtdIns decreased throughout the experiment. A transient accumulation of stearoylarachidonoylglycerol was observed at 5s, whereas stearoylarachidonoylglycerol 3-phosphate (PtdA) was produced in increasing amounts throughout the experiment. The decrease in inositol-containing phospholipids was not fully compensated for by the production of diacylglycerol or PtdA [or PtdIns(4,5)P2] at 1 min. All the changes in inositol phospholipids, as well as those observed in diacylglycerols and PtdA, were due to a parallel reduction or increase in the contents of stearic and arachidonic acids, with a stoichiometry equal to 1. Taken together, this suggests an interconversion of all these lipids with the utilization of a common backbone, stearoylarachidonoylglycerol. The deacylation of this diacylglycerol could account for up to 4-5nmol of arachidonate/10(9) platelets after 1 min stimulation by thrombin.
Localization and synthesis of the acetylcholine-binding site in the α-chain of the Torpedo californica acetylcholine receptor
Authors: McCormick D J , Atassi M Z
Published: August 2015

The sequence of the alpha-chain of the acetylcholine receptor of T. californica has been determined by recent cloning studies. The integrity of the disulphide bond between Cys-128 and cys-142 has been shown to be important for the maintenance of the binding activity of the receptor, thus implicating the regions around the disulphide bridge in binding with acetylcholine. In the present work, a synthetic peptide containing this loop region (residues 125-147) was synthesized. Solid-phase radiometric binding assays demonstrated a high binding of 125I-labelled alpha-bungarotoxin to the synthetic peptide. It was further shown that the free peptide bound well to [3H]acetylcholine. Additional experiments demonstrated that pretreatment of peptide 125-147 with 2-mercaptoethanol destroyed its binding activity, clearly showing that the integrity of the disulphide structure was essential for binding. Unlabelled acetylcholine also inhibited the binding of labelled acetylcholine to the synthetic peptide. The region 125-147, therefore, contains essential elements of the acetylcholine binding site of the Torpedo receptor.
Extracellular and cellular proteins of rat cells with O-glycosidically linked fucose
Authors: Morton P A , Steiner S M
Published: August 2015

Fucose-labelled proteins were examined for the release of low-Mr O-linked fucose substituents after mild alkaline-borohydride treatment. A component tentatively identified as glucosylfucitol (DS) and an apparently higher-Mr component (TS), which also contained fucitol, were observed to be released over a broad molecular-size range of proteins. Approx. 90% of the DS-releasing proteins were in the particulate fraction, whereas only approx. 66% of the TS-releasing proteins were in that fraction. In addition to cell-associated proteins, a substantial proportion of DS-containing proteins were shed into the medium. For example, after 96 h of labelling there was 6-fold more of these components in the growth medium than were cell-associated. Moreover, the incorporation of labelled fucose into both the DS and TS appeared to be cell-population-density-dependent. Despite the apparent wide distribution of these novel fucose substituents in cellular proteins, it seems reasonable to suggest that they have not been routinely observed largely because each represents less than 0.5% of the fucose bound to protein.
Functional receptors for epidermal growth factor in an epithelial-cell line derived from the rat small intestine
Authors: Blay J , Brown K D
Published: August 2015

Epidermal growth factor (EGF) regulates the proliferation of cells of a rat intestinal epithelial-cell line (RIE-1) in culture. Confluent RIE-1 cells were stimulated to proliferate by EGF with a half-maximal effect at 1-2 ng/ml. In contrast, the growth of sparse RIE-1 cells was inhibited by the growth factor. Binding studies at 4 degrees C with 125I-EGF identified two classes of binding sites for EGF on RIE-1 cells, one of high affinity (KD = 1.8 × 10(-10)M; 1.8 × 10(4) receptors/cell) and one of lower affinity (KD = 5.2 × 10(-9)M; 6.3 × 10(4) receptors/cell). After binding to the cells at 37 degrees C, 125I-EGF was rapidly internalized and subsequently degraded. Degradation products were released into the medium after a lag of 15-30 min. The degradation of 125I-EGF did not occur at 4 degrees C and was inhibited at 37 degrees C by chloroquine, methylamine or NH4Cl, but not by colchicine. Exposure of RIE-1 cells to EGF caused a time- and dose-dependent loss of EGF receptors from the cell surface. The recovery of receptors after the removal of EGF was retarded in the absence of serum and prevented by the presence of cycloheximide or actinomycin D. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis separation of the 125I-EGF-receptor complex from RIE-1 cells after covalently cross-linking with disuccinimidyl suberate indicated a receptor of Mr congruent to 160 000. The demonstration of functional EGF receptors in this cell line provides further evidence that EGF may regulate intestinal-epithelial-cell physiology.
Dye-sensitized photo-oxidation of enzymes
Authors: Tsai C S , Godin J R P , Wand A J
Published: August 2015

Heart lipoamide dehydrogenase, liver alcohol dehydrogenase and egg-white lysozyme are photo-oxidized in the presence of various dye sensitizers. The photodynamic process is preceded by the binding between the enzyme and the sensitizers. Among the commonly used dyes, halogenated xanthines and thiazine are effective sensitizers for the photo-inactivation of these three enzymes. Histidine residues are the primary target for the sensitized photo-oxidation that inactivates lipoamide dehydrogenase and alcohol dehydrogenase. However, the destruction of tryptophan residues is responsible for the photo-inactivation of lysozyme. The deuterium medium effect and the quenching effect by various scavengers of the potential photo-oxidative intermediates implicate the participation of the mixed type I-type II mechanism, with the involvement of singlet oxygen being of greater importance, in the photo-inactivation of the enzymes.
Phospholipid association with the bovine cardiac mitochondrial adenosine triphosphatase
Authors: Brown R E , Montgomery R I , Spach P I , Cunningham C C
Published: August 2015

The association of different phospholipids with a lipid-depleted oligomycin-sensitive ATPase from bovine cardiac mitochondria [Serrano, Kanner & Racker (1976) J. Biol. Chem. 251, 2453-2461] has been examined using three approaches. First, reconstitution of the ATPase with different synthetic diacyl phospholipids resulted in a 2-10-fold stimulation of ATPase specific activity depending upon the particular phospholipid employed. The phospholipid headgroup region displayed the following order of ATPase reactivation potential: dioleoylphosphatidylglycerol greater than dioleoylphosphatidic acid greater than dioleoylphosphatidylcholine. Furthermore, the ATPase showed higher levels of specific activity when reconstituted with dioleoyl phospholipid derivatives compared with dimyristoyl derivatives. Second, examination of the phospholipid remaining associated with the lipid-depleted ATPase upon purification showed that phosphatidylcholine, phosphatidylethanolamine, and diphosphatidylglycerol were present. No relative enrichment of any of these phospholipids (compared with their distribution in submitochondrial particles) was noted. Therefore, no preferential association between the ATPase and any one phospholipid could be found in the mitochondrial ATPase. Third, the sodium cholate-mediated phospholipid exchange procedure was employed for studying the phospholipid requirements of the ATPase. Replacement of about 50% of the mitochondrial phospholipid remaining with the lipid-depleted ATPase could be achieved utilizing either synthetic phosphatidic acid or phosphatidylcholine. Examination of the displaced mitochondrial phospholipid showed that phosphatidylcholine, phosphatidylethanolamine, and diphosphatidylglycerol were replaced with equal facility.
Quantitative analysis of intermediary metabolism in hepatocytes incubated in the presence and absence of glucagon with a substrate mixture containing glucose, ribose, fructose, alanine and acetate
Authors: Rabkin M , Blum J J
Published: August 2015

Hepatocytes were isolated from the livers of fed rats and incubated, in the presence and absence of 100 nM-glucagon, with a substrate mixture containing glucose (10 mM), fructose (4 mM), alanine (3.5 mM), acetate (1.25 mM), and ribose (1 mM). In any given incubation one substrate was labelled with 14C. Incorporation of 14C into glucose, glycogen, CO2, lactate, alanine, glutamate, lipid glycerol and fatty acids was measured after 20 and 40 min of incubation under quasi-steady-state conditions [Borowitz, Stein & Blum (1977) J. Biol. Chem. 252, 1589-1605]. These data and the measured O2 consumption were analysed with the aid of a structural metabolic model incorporating all reactions of the glycolytic, gluconeogenic, and pentose phosphate pathways, and associated mitochondrial and cytosolic reactions. A considerable excess of experimental measurements over independent flux parameters and a number of independent measurements of changes in metabolite concentrations allowed for a stringent test of the model. A satisfactory fit to the data was obtained for each condition. Significant findings included: control cells were glycogenic and glucagon-treated cells glycogenolytic during the second interval; an ordered (last in, first out) model of glycogen degradation [Devos & Hers (1979) Eur. J. Biochem. 99, 161-167] was required in order to fit the experimental data; the pentose shunt contributed approx. 15% of the carbon for gluconeogenesis in both control and glucagon-treated cells; net flux through the lower Embden-Meyerhof pathway was in the glycolytic direction except during the 20-40 min interval in glucagon-treated cells; the increased gluconeogenesis in response to glucagon was correlated with a decreased pyruvate kinase flux and lactate output; fluxes through pyruvate kinase, pyruvate carboxylase, and phosphoenolpyruvate carboxykinase were not coordinately controlled; Krebs cycle activity did not change with glucagon treatment; flux through the malic enzyme was towards pyruvate formation except for control cells during interval II; and ‘futile’ cycling at each of the five substrate cycles examined (including a previously undescribed cycle at acetate/acetyl-CoA) consumed about 26% of cellular ATP production in control hepatocytes and 21% in glucagon-treated cells.
Integrated rate equations for irreversible enzyme-catalysed first-order and second-order reactions
Authors: Boeker E A
Published: August 2015

Integrated rate equations are presented that describe irreversible enzyme-catalysed first-order and second-order reactions. The equations are independent of the detailed mechanism of the reaction, requiring only that it be hyperbolic and unbranched. The results should be directly applicable in the laboratory.
Kinetic study of the photochemical changes of (ZZ)-bilirubin IX α bound to human serum albumin. Demonstration of (EZ)-bilirubin IX α as an intermediate in photochemical changes from (ZZ)-bilirubin IX α to (EZ)-cyclobilirubin IX α
Authors: Itoh S , Onishi S
Published: August 2015

The present study was performed to elucidate why the photochemical reaction of (ZZ)-bilirubin bound to human serum albumin is singularly selective, and only one of the two (EZ)- and (ZE)-bilirubins, the (ZE)-isomer, is produced. In a kinetic study of the photochemical reaction in vitro, the sum of the relative rate constants of photochemical transformation of (EZ)-bilirubin into both (EZ)-cyclobilirubin and (ZZ)-bilirubin, with a significant preference for the former, was proved to be considerably larger than that of the transformation of (ZZ)-bilirubin into (EZ)-bilirubin. Therefore only one of the geometrical isomers, namely (ZE)-bilirubin, is apparently formed. It was concluded that (EZ)-bilirubin photochemically undergoes (EZ)-cyclization, i.e. structural photoisomerization, while bound to its high-affinity site on human serum albumin, and is an intermediate in the transformation of (ZZ)-bilirubin into (EZ)-cyclobilirubin.
Effect of sucrose starvation on sycamore (Acer pseudoplatanus) cell carbohydrate and Pi status
Authors: Rébeillé F , Bligny R , Martin J B , Douce R
Published: August 2015

The mobilization of stored carbohydrates during sucrose starvation was studied with sycamore (Acer pseudoplatanus) cells. When sucrose was omitted from the nutrient medium, the intracellular sucrose pool decreased rapidly during the first hours of the experiment, whereas the starch content remained practically unchanged. After 10h of sucrose starvation, starch hydrolysis replaced sucrose breakdown. From this moment, the phosphate-ester pool and respiration rate decreased with time. Conversely, the intracellular Pi concentration increased. 31P n.m.r. of intact sycamore cells indicated that, under these conditions, most of the Pi accumulated in the vacuole. These results strongly suggest that starch breakdown, in contrast with sucrose hydrolysis, is not rapid enough to maintain a high cellular metabolism.
Effect of glucose on 7-hydroxycoumarin glucuronide production in periportal and pericentral regions of the liver lobule
Authors: Conway J G , Kauffman F C , Thurman R G
Published: August 2015

The effect of starvation and glucose addition on glucuronidation was assessed in sublobular regions of the lobule in perfused livers from phenobarbital-treated rats. Fibre-optic micro-light guides were placed on periportal and pericentral areas on the surface of livers to monitor the fluorescence (excitation 366 nm, emission 450 nm) of free 7-hydroxycoumarin from the tissue surface. After infusion of 7-hydroxycoumarin (80 microM) under normoxic conditions, steady-state increases in fluorescence were reached in 6-8 min in both regions. Subsequently, the formation of non-fluorescent 7-hydroxycoumarin glucuronide was inhibited completely by perfusion with N2-saturated perfusate containing 20 mM-ethanol. The difference in fluorescence between anoxic and normoxic perfusions was due to glucuronidation under these conditions. In livers from fed rats, rates of glucuronidation in periportal and pericentral regions of the liver lobule were 8 and 19 mumol/h per g, respectively. In contrast, rates of glucuronidation were 3 and 9 mumol/h per g, respectively, in periportal and pericentral regions of livers from starved rats. Infusion of glucose (20 mM) had no effect on rates of glucuronidation in livers from fed rats; however, glucose increased rates of glucuronidation rapidly (half-time, t0.5 = 1.5 min) in periportal and pericentral regions to 7 and 17 mumol/h per g, respectively in livers from starved rats. These results indicate that the rapid synthesis of the cofactor UDP-glucuronic acid derived from glucose is an important rate-determinant for glucuronidation of 7-hydroxycoumarin in both periportal and pericentral regions of livers from starved rats.
Quantification of tissue fibronectin from terminal villi of placenta
Authors: Bray B A
Published: August 2015

Tissue fibronectin (TFn) was solubilized from the terminal villi of perfused human placentas by sequential chemical extractions and plasmin digestion. Alternatively, plasmin digestion of intact tissue solubilized all the TFn, which amounted to 1.8-2.9% of the dry weight of the villi. Concomitantly, 69% of the tissue was solubilized. The non-equilibrium competitive e.l.i.s.a. (enzyme-linked immunoabsorbent assay), in which the TFn was immunologically identical with plasma fibronectin (PFn), was used for the quantification of TFn. This study demonstrates that the bulk of TFn can be obtained in a form that can be quantified by e.l.i.s.a. and that TFn is immunologically identical with PFn. Thus the fibronectin molecule is not significantly altered as it is incorporated into the connective-tissue matrix and could exchange with PFn.
Intercellular pathway of leucine catabolism in rat spermatogenic epithelium
Authors: Grootegoed J A , Jansen R , van der Molen H J
Published: August 2015

A unique intercellular pathway of leucine catabolism was observed in vitro in rat spermatogenic epithelium. Sertoli cells convert leucine via transmination into 4-methyl-2-oxovalerate, and spermatocytes and spermatids reduce exogenous 4-methyl-2-oxovalerate to 2-hydroxy-4-methylvalerate, which is then released by the spermatogenic cells. The NADH-dependent reduction of 4-methyl-2-oxovalerate could be catalysed by the male-germ-cell-specific lactate dehydrogenase isoenzyme LDH-C4 in the cytosol of the spermatogenic cells, concomitant with the NAD+-dependent conversion of exogenous lactate into pyruvate.
Role of antizyme in degradation of ornithine decarboxylase in HTC cells
Authors: Murakami Y , Hayashi S
Published: August 2015

A good correlation was observed between the reciprocal of the half-life of ornithine decarboxylase (ODC) activity in the presence of cycloheximide and the relative amount of ODC-antizyme complex to total ODC (free ODC plus complexed ODC) activity in HTC cells examined at various times after cell dilution or change of medium. Pretreatment of cells with putrescine increased the relative amount of ODC-antizyme complex and decreased the half-life of ODC decay. These results suggested that antizyme plays a key role in ODC degradation.
Structure and interactions of cartilage proteoglycan binding region and link protein
Authors: Bonnet F , Dunham D G , Hardingham T E
Published: August 2015

Binding region and link protein were prepared from pig laryngeal cartilage proteoglycans after chondroitinase ABC and trypsin digestion. Experiments on gel chromatography showed the purified binding region to interact reversibly with hyaluronate (HA), and this binding was also shown to be stabilized by native link protein. The trypsin-prepared link protein showed properties of self-association in solution that were partially inhibited by oligosaccharides (HA10-16) and abolished by modification of free amino groups (lysine residues) with 2-methylmaleic anhydride. The Mr (sedimentation equilibrium) of the modified link protein was 41 700. Analysis of binding region showed it to contain 25% (w/w) carbohydrate, mainly in galactose, glucosamine, mannose and galactosamine. It contained some keratan sulphate, as digestion with endo-beta-D-galactosidase (keratanase) removed 28% galactose and 25% glucosamine and the Mr (sedimentation equilibrium) decreased from 66 500 to 60 800. After keratanase digestion the interaction with polyclonal antibodies specific for binding region was unaffected, but the response in a radioimmunoassay with a monoclonal antibody to keratan sulphate was decreased by 47%. Preparation of a complex between binding region, link protein and HA approximately 34 showed a single component (5.5S) of Mr (sedimentation equilibrium) 133 500. In this complex the antigenic determinants of link protein appeared masked, as previously found with proteoglycan aggregates. The isolated binding region and link protein were thus shown to retain properties comparable with those involved in the structure and organization of proteoglycan aggregates.
Lysosomal glycogen storage induced by Acarbose, a 1,4-α-glucosidase inhibitor
Authors: Geddes R , Taylor J A
Published: August 2015

The 1,4-alpha-glucosidase inhibitor. Acarbose, when injected intraperitoneally disturbs liver lysosome metabolism, causing distinct and persistent inhibition of the enzymes and acute disturbances of lysosomal glycogen metabolism. A feedback control mechanism appears to operate, affecting cytosolic carbohydrate metabolism. A model is suggested for the adult form of lysosomal storage disease. The biochemical effects closely resemble those occurring in glycogenosis type II (Pompe's disease), and these have been confirmed by electron microscopy.
Nucleotide sequence of the Rhodospirillum rubrum atp operon
Authors: Falk G , Hampe A , Walker J E
Published: August 2015

The nucleotide sequence was determined of a 8775-base-pair region of DNA cloned from the photosynthetic non-sulphur bacterium Rhodospirillum rubrum. It contains a cluster of five genes encoding F1-ATPase subunits. The genes are arranged in the same order as F1 genes in the Escherichia coli unc operon. However, as in the related organism Rhodopseudomonas blastica, neither genes for components of F0, the membrane sector of ATP synthase, nor a homologue of the E. coli uncI gene are associated with this locus, as they are in E. coli.
Studies in vivo and in vitro on the uptake and degradation of soluble collagen α1(I) chains in rat liver endothelial and Kupffer cells
Authors: Smedsrød B , Johansson S , Pertoft H
Published: August 2015

Intravenously administered 125I-labelled monomeric alpha 1 chains (125I-alpha 1) of collagen type I were rapidly cleared and degraded by the liver of rats. Isolation of the liver cells after injection of the label revealed that the uptake per liver endothelial cell equalled the uptake per Kupffer cell, whereas the amount taken up per hepatocyte was negligible. The uptake of 125I-alpha 1 in cultured cells was 10 times higher per liver endothelial cell than per Kupffer cell. The ligand was efficiently degraded by cultures of both cell types. However, spent medium from cultures of Kupffer cells, unlike that from cultures of other cells, contained gelatinolytic activity which degraded 125I-alpha 1. The presence of hyaluronic acid, chondroitin sulphate or mannose/N-acetylglucosamine-terminal glycoproteins, which are endocytosed by the liver endothelial cells via specific receptors, did not interfere with binding, uptake or degradation of 125I-alpha 1 by these cells. Unlabelled alpha 1 and heat-denatured collagen inhibited the binding to a much greater extent than did native collagen. The presence of fibronectin or F(ab')2 fragments of anti-fibronectin antibodies did not affect the interaction of the liver endothelial cells, or of other types of liver cells, with 125I-alpha 1. The accumulation of fluorescein-labelled heat-denatured collagen in vesicles of cultured liver endothelial cells is evidence that the protein is internalized. Moreover, chloroquine, 5-dimethylaminonaphthalene-1-sulphonylcadaverine (dansylcadaverine), monensin and cytochalasin B, which impede one or more steps of the endocytic process, inhibited the uptake of 125I-alpha 1 by the liver endothelial cells. Leupeptin, an inhibitor of cathepsin B and ‘collagenolytic cathepsins’, inhibited the intralysosomal degradation of 125I-alpha 1, but had no effect on the rate of uptake of the ligand. The current data are interpreted as follows. (1) The ability of the liver endothelial cells and the Kupffer cells to sequester circulating 125I-alpha 1 efficiently may indicate a physiological pathway for the breakdown of connective-tissue collagen. (2) The liver endothelial cells express receptors that specifically recognize and mediate the endocytosis of collagen alpha 1(I) monomers. (3) The receptors also recognize denatured collagen (gelatin). (4) Fibronectin is not involved in the binding of alpha 1 to the receptors. (5) Degradation occurs intralysosomally by leupeptin-inhibitable cathepsins.