bluuebunny/crossref_metadata_2025 huggingface dataset:
Oxidation and enzyme-activated irreversible inhibition of rat liver monoamine oxidase-B by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)
Authors: Tipton K F , McCrodden J M , Youdim M B
Published: August 2015

The compound 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), which produces symptoms resembling Parkinson's disease in humans, acts both as a substrate and an enzyme-activated irreversible inhibitor of the B-form of monoamine oxidase from rat liver. Analysis of the inhibitory process showed the compound to be considerably more efficient as a substrate than as an irreversible inhibitor, with about 17000 mol of product being formed per mol of enzyme inactivated. The half-time of the inhibitory process was about 22 min. With the A-form of the enzyme, the compound had a lower Km value and a considerably lower maximum velocity than the corresponding values obtained with the B-form. Under the conditions used in the present work the inhibition of the A-form of the enzyme was largely reversible.
Synthesis and degradation rates of collagens in vivo in whole skin of rats, studied with 18O2 labelling
Authors: Molnar J A , Alpert N , Burke J F , Young V R
Published: August 2015

Rats of synthesis and degradation in vivo of collagens in 0.5 M-acetic acid-soluble and -insoluble extracts from skins of three growing rats were determined by using a labelling procedure involving exposure of the animals to an atmosphere of 18O2 for 36 h. For comparison, rats also received injections of [2H]proline. Serial skin biopsies were taken at frequent intervals over 392 days. Enrichment of 18O and 2H in the hydroxyproline of the collagen fractions was determined by gas chromatography-mass spectrometry. Changes in size of the soluble and insoluble collagen pools were considered in the evaluation of isotope kinetic data. The insoluble collagen fraction showed no degradation. The efflux (mean +/- S.D., expressed as mumol of hydroxyproline) from the soluble collagen pool was estimated to be 59.9 +/- 1.9 per day from the 18O data, and 25.5 +/- 7.5 per day from the 2H results. The finding indicates significant reutilization of 2H-radiolabelled proline for hydroxyproline synthesis. From these isotope data and estimates of size of the collagen pools it was determined that 55% of the collagen disappearing from the soluble pool was due to maturation into insoluble collagens and 45% of the disappearance was a result of actual degradation of soluble collagen. These results confirm the utility of 18O2 as a non-reutilizable label for studies of collagen turnover in vivo.
Cathepsin S The cysteine proteinase from bovine lymphoid tissue is distinct from cathepsin L (EC 3.4.22.15)
Authors: Kirschke H , Schmidt I , Wiederanders B
Published: August 2015

Cathepsin S was purified from bovine spleen by acid autolysis, (NH4)2SO4 fractionation and chromatography on CM-Sephadex C-50, CM-cellulose and activated-thiol-Sepharose. Cathepsin L was isolated from lysosomal fractions of rat liver, rat kidney and bovine liver. Generally, cathepsin L was bound tightly to CM-Sephadex C-50. Preparations of cathepsin L from rat liver, rat kidney and bovine liver were shown to have kinetic constants for the substrate benzyloxycarbonyl-Phe-Arg-7-(4-methyl)coumarylamide in the same range (Km 2-3 microM). Benzyloxycarbonyl-Phe-Phe-diazomethane proved to be a sensitive irreversible inhibitor of cathepsin L from different species. Cathepsin S differed in all these characteristics from cathepsin L. A polyclonal antibody to cathepsin L from rat reacted with bovine cathepsin L but not with bovine cathepsin S.
NADPH: cytochrome P-450 reductase in olfactory epithelium Relevance to cytochrome P-450-dependent reactions
Authors: Reed C J , Lock E A , De Matteis F
Published: August 2015

The presence of a very active cytochrome P-450-dependent drug-metabolizing system in the olfactory epithelium has been confirmed by using 7-ethoxycoumarin, 7-ethoxyresorufin, hexobarbitone and aniline as substrates, and the reasons for the marked activity of the cytochrome P-450 in this tissue have been investigated. The spectral interaction of hexobarbitone and aniline with hepatic and olfactory microsomes has been examined. By this criterion there was no evidence for marked differences in the spin state of the cytochromes of the two tissues, or for the olfactory epithelium containing a greater amount of cytochrome capable of binding hexobarbitone, a very actively metabolized substrate. Rates of NADPH and NADH: cytochrome c reductase activity were found to be higher in the olfactory epithelium than in the liver, and direct evidence was obtained for a greater amount of the NADPH-dependent flavoprotein in the olfactory microsomes. Investigation of male rats and male and female mice, as well as male hamsters, demonstrated that, in all cases, the cytochrome P-450 levels of the olfactory epithelium were lower than those of the liver, while the 7-ethoxycoumarin de-ethylase and NADPH:cytochrome c reductase activities were higher. A correlation was found between 7-ethoxycoumarin de-ethylase and NADPH:cytochrome c reductase activities for both tissues in all species examined. The ratio of reductase to cytochrome P-450 was found to be considerably higher in the olfactory epithelium (1:2-1:3) than in the liver (1:11-1:15), regardless of the species examined, suggesting that facilitated electron flow may contribute significantly to the cytochrome P-450 catalytic turnover in the olfactory tissue.
The characterization of an acidic calmodulin-binding protein in brain cytoskeleton and membrane fractions
Authors: Strocchi P , Gilbert J M
Published: August 2015

One of the most abundant acidic proteins in rat brain has an Mr of 68,000 and a pI of 5.6 (68K 5.6 protein) when analysed by two-dimensional gel electrophoresis. The 68K 5.6 protein was found in large relative amounts in brain cytoskeleton preparations and in membrane and supernatant fractions. High-salt washing and proteolytic digestion did not remove this protein from the membrane elements. The 68K 5.6 protein was also found in the microtubule-associated protein fraction of purified microtubules and was present in large relative amounts in preparations of intermediate-filament proteins. The 68K 5.6 protein binds to calmodulin in the presence of Ca2+ ions, and we found it to be an abundant acidic calmodulin-binding protein in brain tissue.
Active-site directed inactivation of rat ovarian 20 α-hydroxysteroid dehydrogenase
Authors: Ricigliano J W , Penning T M
Published: August 2015

Rat ovarian 20 alpha-hydroxysteroid dehydrogenase plays a pivotal role in leuteolysis and parturition by catalysing the reduction of progesterone to give the progestationally inactive steroid 20 alpha-hydroxyprogesterone. Putative mechanism based inhibitors of this enzyme were synthesized as potential progestational maintaining agents, including the epimeric allylic alcohol pair 3 beta-hydroxy-alpha-vinyl-5 alpha-androstane-17 beta-methanol and the related vinyl ketone 1-(3 beta-hydroxy-5 alpha-androstan-17 beta-yl)-2-propen-1-one. The vinyl ketone inactivates rat ovarian 20 alpha-hydroxysteroid dehydrogenase, semi-purified by poly(L-lysine)-agarose column chromatography, in a rapid time-dependent manner. Analysis of the pseudo-first-order inactivation plots gave a Ki of 2.0 microM for the inhibitor and a t1/2 for the enzyme of 20 s at saturation. These data indicate that the vinyl ketone is a potent and efficient inactivator of the ovarian dehydrogenase. Neither dialysis in the presence or absence of a competing nucleophile nor gel filtration reserves the inactivation, suggesting that a stable covalent bond is formed between the enzyme and steroid ligand. Both substrates (20 alpha-hydroxyprogesterone and NADP+) protect the enzyme from inactivation; moreover, initial velocity measurements in the presence of saturating concentrations of both substrates indicate that the vinyl ketone can behave as a competitive inhibitor, yielding a Ki value identical with that obtained in the inactivation experiments. Our results imply that the vinyl ketone is an active-site directed alkylating agent. By contrast the allylic alcohol pair 3 beta-hydroxy-alpha-vinyl-5 alpha-androstane-17 beta-methanol are neither substrates nor inhibitors of the ovarian enzyme and appear to be excluded from the catalytic site. The rapid inactivation observed with the vinyl ketone suggests that this compound may be useful as a progestational maintaining agent.
Influence of temperature on the enzymic semisynthesis of human insulin by coupling and transpeptidation methods
Authors: Morihara K , Ueno Y , Sakina K
Published: August 2015

The influence of temperature of enzymic semisynthesis of human insulin ester was determined by using coupling and transpeptidation methods with trypsin and Achromobacter lyticus proteinase I as catalysts. The optimal reaction conditions were studied at the selected temperatures of 25, 12 and 4 degrees C. The results showed that the synthesis rates by both methods with trypsin increased as the temperature increased, but the final product yield correspondingly decreased. Therefore the reaction with trypsin should be done below 12 degrees C, preferably at 4 degrees C. This agrees well with the stability of trypsin at these temperatures. When the catalyst was Achromobacter lyticus proteinase I, no such complex temperature effects were observed, and the findings indicated that the reactions should be conducted below 37 degrees C for enzyme stability.
Micro-injection of inositol 1,3,4,5-tetrakisphosphate activates sea urchin eggs by a mechanism dependent on external Ca2+
Authors: Irvine R F , Moor R M
Published: August 2015

Micro-injection of submicromolar concentrations of inositol 1,3,4,5-tetrakisphosphate caused a raising of the fertilization envelope in eggs of the sea urchin Lytechinus variegatus. This effect was dependent both on the presence of extracellular Ca2+ and on co-injection with a Ca2+-mobilizing compound, inositol 2,4,5-trisphosphate. Inositol 1,3,4,5-tetrakisphosphate was the most potent compound tested in this assay; removal of the 3- or 5-phosphates or randomization of the phosphates in the inositol ring decreased its potency. These results show that inositol 1,3,4,5-tetrakisphosphate is an intracellular second messenger, and suggest that its function is to control cellular Ca2+ homoeostasis at the plasma membrane.
The propeptide Asn1–Tyr126 is the storage form of rat atrial natriuretic factor
Authors: Thibault G , Garcia R , Gutkowska J , Bilodeau J , Lazure C , Seidah N G , Chrétien M , Genest J , Cantin M
Published: August 2015

Granules from rat atria were isolated by differential centrifugation and by a 53% (v/v) Percoll gradient after tissue homogenization in 0.25 M-sucrose/50 mM-Na2EDTA. About 40% of the immunoreactive ANF (atrial natriuretic factor) sedimented with the atrial granules during differential centrifugations. On the Percoll gradient, two distinct bands were observed. Cell debris, mitochondria, lysosomes, myofilaments and microsomes were mostly contained in the lightest-density (rho) (1.03-1.07 g/ml) fraction, as demonstrated by electron microscopy and by enzymic markers such as lactate dehydrogenase, monoamine oxidase, cytochrome c reductase, beta-glucuronidase and acid phosphatase. Atrial granules were mostly contained in the denser (rho 1.11-1.15 g/ml) band and were only slightly contaminated by lysosomes, as shown by beta-glucuronidase activity. Analysis of the ANF content in these isolated granules by h.p.l.c., amino acid composition and sequencing demonstrated that it was only the pro-ANF [ANF-(Asn1-Tyr126)-peptide]. The precursor was present in all granules, as demonstrated by immunocytochemistry. Since hormonal propeptides usually undergo intracellular processing, and the matured peptides are subsequently stored in the secretory granules, these results indicate that the processing pathway of ANF may be different from that of other hormonal peptides.
Biosynthesis and maturation of lactase-phlorizin hydrolase in the human small intestinal epithelial cells
Authors: Naim H Y , Sterchi E E , Lentze M J
Published: August 2015

The biosynthesis and maturation of the human intestinal lactase-phlorizin hydrolase (LPH; EC 3.2.1.23-3.2.1.62) has been studied in cultured intestinal biopsies and mucosal explants. Short time pulse labelling revealed on high mannose intermediate of Mr 215,000 which was converted upon endo-beta-N-acetylglucosaminidase H (endo-H) digestion to a polypeptide of Mr 200,000. The brush border form of LPH was revealed after longer pulse periods and has Mr 160,000. It possesses mainly complex oligosaccharide chains and, owing to its partial endo-H sensitivity, at least one chain of the high mannose type. Leupeptin partially inhibited the appearance of the Mr-160,000 polypeptide. Monensin treatment of biopsies resulted in the modification of the Mr-160,000 species to the Mr-140,000 molecule, which was endo-H sensitive. Pulse-chase analysis indicated a slow post-translational processing of the high mannose precursor (Mr 215,000) to yield the mature brush-border form (Mr 160,000) of LPH. Our results further indicate that LPH is synthesized as a single polypeptide precursor which is intracellularly cleaved to yield the mature brush border of LPH. The data presented suggest that this cleavage occurs during the translocation of the molecule across the Golgi complex.
Co-operativity in seminal ribonuclease function: binding studies
Authors: Di Donato A , Piccoli R , D'Alessio G
Published: August 2015

Binding of nucleotides to bovine seminal RNAase was studied by differential spectrophotometry and equilibrium dialysis. Cytidine 3′-phosphate, the reaction product of the hydrolytic, rate-limiting step of the reaction, was found to be capable, in contrast to related nucleotides, of discriminating between the two structurally identical active sites of the enzyme. Negative co-operativity, with a ‘half-of-sites’ reactivity, was found at lower concentrations of ligand, whereas at higher concentrations positive co-operativity was detected. These findings exclude that the non-hyperbolic kinetics previously reported for the hydrolytic step of the reaction are due to hysteretic effect. A model of mixed-type co-operativity is proposed for interpreting the binding data.
Characterization of a low-relative-molecular-mass prolyl 4-hydroxylase from the green alga Chlamydomonas reinhardii
Authors: Kaska D D , Günzler V , Kivirikko K I , Myllylä R
Published: August 2015

Prolyl 4-hydroxylase was partially purified and characterized from the unicellular green alga, Chlamydomonas reinhardii. This enzyme differed from all the animal and plant prolyl 4-hydroxylases studied so far in that its Mr was only about 40,000 by gel filtration, being thus less than one-sixth of those determined for the vertebrate and higher-plant enzymes. The algal enzyme did not hydroxylate to any significant extent chick-embryo protocollagen or triple-helical (Pro-Pro-Gly)10, whereas a low hydroxylation rate was found with denatured (Pro-Pro-Gly)10. Poly(L-proline), which is an effective inhibitor of the vertebrate enzymes but acts as a substrate for some higher-plant enzymes, was a good substrate. In the absence of poly(L-proline) the enzyme catalysed an uncoupled decarboxylation of 2-oxoglutarate. Studies of the Km values for the co-substrates and cofactors and the specificity of the 2-oxoglutarate requirement, as well as inhibition studies with selected 2-oxoglutarate analogues, suggested that the catalytic site of the algal enzyme is similar to, but not identical with, those of the vertebrate enzymes. The existence of distinct similarities was further demonstrated by an inhibition of the algal enzyme activity with a monoclonal antibody to the beta-subunit of human prolyl 4-hydroxylase. The amount of prolyl 4-hydroxylase activity in the algal cells was not altered by signals which recognize the presence or absence of the cell wall, as determined in studies on experimental cell-wall regeneration and wall-less mutants.
Role of avidin and other biotin-binding proteins in the deposition and distribution of biotin in chicken eggs. Discovery of a new biotin-binding protein
Authors: White H B , Whitehead C C
Published: August 2015

In addition to the previously characterized egg-yolk biotin-binding protein (BBP-I), we have discovered another BBP (BBP-II) in the plasma and yolk from laying hens. BBP-I is stable to 65 degrees C, whereas BBP-II is stable to 45 degrees C. Both proteins are normally saturated with biotin and together they account for most, if not all, of the biotin in hen plasma and yolk, except in hens fed excessive amounts of biotin (greater than 1 mg of biotin/kg of feed). The maximal production of BBP-I is attained at lower levels of dietary biotin (approximately 50 micrograms/kg) than for BBP-II (approximately 250 micrograms/kg); however, the maximal production of BBP-II is severalfold greater than for BBP-I. Consequently, as dietary biotin increases, the ratio of BBP-II to BBP-I increases and becomes constant at dietary intakes of biotin above 250 micrograms/kg. The observation that the amounts of these proteins are limited by biotin in the normal dietary range (less than 250 micrograms/kg) suggests that biotin is required for the synthesis, secretion or stability of these proteins. Although both plasma vitamin-protein complexes are transported to the oocyte and concentrated in the yolk, BBP-II is transferred more efficiently. Thus biotin deposition in the yolk is a function of the amounts and relative concentrations of the two proteins. Dietary biotin above 250 micrograms/kg exceeds the transport capacity of BBP-I and BBP-II in the plasma; however, unbound biotin does not accumulate. Rather it is efficiently scavenged by avidin in the oviduct and transferred to the egg albumen. Only when avidin becomes saturated at high dietary intake does free or weakly bound biotin accumulate in plasma and yolk. The synthesis of avidin is independent of dietary biotin. Small amounts of BBPs with the heat-stability of avidin or BBP-I respectively are present in the plasma of adult males or immature chickens. BBP-II, the major BBP in the plasma and yolk of laying hens, was not detected in the plasma of non-laying chickens.
Chlorpromazine and carnitine-dependency of rat liver peroxisomal β-oxidation of long-chain fatty acids
Authors: Vamecq J
Published: August 2015

The enzyme targets for chlorpromazine inhibition of rat liver peroxisomal and mitochondrial oxidations of fatty acids were studied. Effects of chlorpromazine on total fatty acyl-CoA synthetase activity, on both the first and the third steps of peroxisomal beta-oxidation, on the entry of fatty acyl-CoA esters into the peroxisome and on catalase activity, which allows breakdown of the H2O2 generated during the acyl-CoA oxidase step, were analysed. On all these metabolic processes, chlorpromazine was found to have no inhibitory action. Conversely, peroxisomal carnitine octanoyltransferase activity was depressed by 0.2-1 mM-chlorpromazine, which also inhibits mitochondrial carnitine palmitoyltransferase activity in all conditions in which these enzyme reactions are assayed. Different patterns of inhibition by the drug were, however, demonstrated for both these enzyme activities. Inhibitory effects of chlorpromazine on mitochondrial cytochrome c oxidase activity were also described. Inhibitions of both cytochrome c oxidase and carnitine palmitoyltransferase are proposed to explain the decreased mitochondrial fatty acid oxidation with 0.4-1.0 mM-chlorpromazine reported by Leighton, Persico & Necochea [(1984) Biochem. Biophys. Res. Commun. 120, 505-511], whereas depression by the drug of carnitine octanoyltransferase activity is presented as the factor responsible for the decreased peroxisomal beta-oxidizing activity described by the above workers.
A putative protein-sequestration site involving intermediate filaments for protein degradation by autophagy. Studies with microinjected purified glycolytic enzymes in 3T3-L1 cells
Authors: Doherty F J , Wassell J A , Mayer R J
Published: August 2015

Several glycolytic enzymes (lactate dehydrogenase, pyruvate kinase, glyceraldehyde-3-phosphate dehydrogenase) were radiolabelled by [125I]iodination, conjugation with 125I-labelled Bolton & Hunter reagent and reductive [3H]methylation, and their degradative rates after microinjection into 3T3-L1 cells compared with that of the extracellular protein bovine serum albumin. Although the albumin remains largely cytosolic in recipient cells, the glycolytic enzymes rapidly (less than 30 min) become insoluble, as measured by detergent and salt extractions. The microinjected glycolytic enzymes appear to form disulphide-linked aggregates, are found in a cell fraction rich in vimentin-containing intermediate filaments and histones (nuclear-intermediate-filament fraction), and are degraded slowly by a lysosomal mechanism, as judged by the effects of inhibitors (NH4Cl, leupeptin, 3-methyladenine). 125I-labelled bovine serum albumin appears to be degraded rapidly and non-lysosomally. Prolonged treatment (96 h) of cultured cells with leupeptin results in the accumulation of pulse-labelled ([35S]methionine for 24 h) endogenous cell proteins in the detergent-and salt-non-extractable residue, but NH4Cl and 3-methyladenine do not have this effect. The findings are in terms of the interpretation of experiments involving microinjection of proteins to study intracellular protein protein degradation by autophagy.
Stimulation of adipocyte phospholipid methyltransferase activity by phorbol 12-myristate 13-acetate. Differential regulation of phospholipid methyltransferase and lipolysis
Authors: Kelly K L
Published: August 2015

The present studies demonstrate that treatment of rat adipocytes with the phorbol ester phorbol 12-myristate 13-acetate (PMA) causes a dose-dependent stimulation of phospholipid methyltransferase (PLMT) activity. The stimulatory effect of PMA was not additive with that of isoprenaline or forskolin. The sensitivity of stimulated PLMT activity to inhibition by insulin, however, was decreased in the presence of PMA. The inhibitory effect of a maximal concentration of insulin on PLMT was unchanged in the presence of PMA. In contrast with the effects on PLMT, the lipolytic response of adipocytes to isoprenaline and the anti-lipolytic response to insulin were unaffected by PMA. These data suggest that PLMT is, whereas hormone-sensitive lipase is not, an intracellular target for the action of PMA. The lack of effect of PMA on lipolysis suggests that PLMT and hormone-sensitive lipase can be regulated by separate mechanisms. Furthermore, phorbol esters do not interfere in the regulatory pathway whereby insulin inhibits PMLT or lipolysis.
Oxidized polyamines and the growth of human vascular endothelial cells. Prevention of cytotoxic effects by selective acetylation
Authors: Morgan D M L
Published: August 2015

The responses of human umbilical-vein vascular endothelial cells in culture to the naturally occurring polyamines spermine, spermidine and putrescine, their acetyl derivatives and oxidation products were examined. In the absence of human polyamine oxidase, exposure of cells to polyamines (up to 160 microM) had no adverse effects. In the presence of polyamine oxidase, spermine and spermidine were cytotoxic, but putrescine was not. Acetylation of the aminopropyl group of spermidine or both aminopropyl groups of spermine prevented this cytotoxicity. The amino acids corresponding to the polyamines, representing a further stage of oxidation, were also without effect. The cytotoxic effects were irreversible. Use of bovine serum amine oxidase in place of the human enzyme gave qualitatively similar results.
Separation of multiple isomers of inositol phosphates formed in GH3 cells
Authors: Dean N M , Moyer J D
Published: August 2015

A high-performance-liquid-chromatography (h.p.l.c.) separation was developed, which resolves isomers of inositol monophosphate (IP), inositol bisphosphate (IP2), and inositol trisphosphate (IP3) in a single run. In GH3 cells labelled with [3H]inositol, treated with Li+ and thyrotropin-releasing hormone (TRH), radiolabelled components identified as inositol 1-phosphate (I1P), inositol 2-phosphate (I2P), inositol 4-phosphate (I4P), inositol 1,4-bisphosphate [I(1,4)P2], inositol 1,3,4-trisphosphate [I(1,3,4)P3] and inositol 1,4,5-trisphosphate [I(1,4,5)P3] are present, as are multiple unidentified IP2 peaks. After TRH stimulation, both I1P and I4P increase, the increase in I4P preceding that of I1P; I(1,4)P2 and an unknown IP2 increase; and both I(1,3,4)P3 and I(1,4,5)P3 increase, the increase in I(1,4,5)P3 being rapid and transient, whereas the increase in I(1,3,4)P3 is slower and more sustained. The most rapidly appearing inositol phosphates produced after TRH stimulation are I(1,4)P2 and I(1,4,5)P3.
Properties of the canalicular bile acid transport system in rat liver
Authors: Meier P J , Meier-Abt A S , Boyer J L
Published: August 2015

4,4-Di-isothiocyanostilbene-2,2′-disulphonic acid inhibition of taurocholate efflux from canalicular vesicles was used to demonstrate that potential driven and ‘carrier’-mediated canalicular excretion of taurocholate occur via a common, rather than two separate, pathways. This electrogenic canalicular bile acid ‘carrier ’ preferentially transports trihydroxylated and conjugated dihydroxylated bile acids, but not the unphysiological oxo bile acids, and possibly extends its substrate specificity to other amphipathic molecules such as sulphobromophthalein.
Effect of α-difluoromethylornithine on DNA methylation in murine erythroleukaemic cells. Relationship to stimulation of induced differentiation
Authors: Papazafiri P , Osborne H B
Published: August 2015

Murine erythroleukaemic (MEL) cells cultured with alpha-difluoromethylornithine (DFMO) accumulated decarboxylated S-adenosylmethionine(decarboxylated AdoMet). In the absence of the inducer hexamethylenebisacetamide (HMBA), this accumulation of decarboxylated AdoMet was associated with a concomitant and proportional increase in DNA hypomethylation. In the presence of HMBA, DFMO, which stimulates the erythrodifferentiation of MEL cells, enhanced the differentiation-associated DNA hypomethylation. However, this differentiation-associated DNA hypomethylation was neither temporally nor quantitatively correlated with the accumulation of decarboxylated AdoMet in these cells. Therefore DFMO probably stimulates the HMBA-induced differentiation of MEL cells and the associated DNA hypomethylation via the effect of this drug on polyamine biosynthesis.
Inactivation of horse liver mitochondrial aldehyde dehydrogenase by disulfiram. Evidence that disulfiram is not an active-site-directed reagent
Authors: Sanny C G , Weiner H
Published: August 2015

The inhibition of mitochondrial (pI 5) horse liver aldehyde dehydrogenase by disulfiram (tetraethylthiuram disulphide) was investigated to determine if the drug was an active-site-directed inhibitor. Stoichiometry of inhibition was determined by using an analogue, [35S]tetramethylthiuram disulphide. A 50% loss of the dehydrogenase activity was observed when only one site per tetrameric enzyme was modified, and complete inactivation was not obtained even after seven sites per tetramer were modified. Modification of only two sites accounted for a loss of 75% of the initial catalytic activity. The number of functioning active sites per tetrameric enzyme, as determined by the magnitude of the pre-steady-state burst of NADH formation, did not decrease until approx. 75% of the catalytic activity was lost. These data indicate that disulfiram does not modify the essential nucleophilic amino acid at the active site of the enzyme. The data support an inactivation mechanism involving the formation of a mixed disulphide with a non-essential cysteine residue, resulting in a lowered specific activity of the enzyme.
Modulation of monocyte complement synthesis by interferons
Authors: Hamilton A O , Jones L , Morrison L , Whaley K
Published: August 2015

Recombinant Escherichia coli-derived gamma-interferon has been shown to stimulate synthesis of the second component of complement (C2), factor B and C1 inhibitor, but to inhibit synthesis of the third component (C3). alpha- and beta-interferons stimulate synthesis of factor B and C3 inhibitor, inhibit C5 synthesis but do not alter synthesis of C2. alpha- and beta-interferons act synergistically with gamma-interferon to enhance both factor B and C1-inhibitor synthesis.
Characterization of primary amino acid sequence of human complement control protein factor I from an analysis of cDNA clones
Authors: Catterall C F , Lyons A , Sim R B , Day A J , Harris T J R
Published: August 2015

A cDNA clone of the mRNA coding for the human complement system control protein Factor I has been isolated. The predicted amino acid sequence obtained from the DNA sequence demonstrates a protein consisting of a heavy chain (Mr 35,400) linked to a light chain (Mr 27,600), both of which contain three sites for N-linked glycosylation. The light chain has clear homology with other serine proteinases, most notably in the region of the catalytically active and structurally important amino acids and shares some of the features characteristic of the plasminogen activators. The heavy chain has a clear ‘mosaic’ nature typical of the plasma serine proteinases; in particular it contains class A and class B LDL (low-density lipoprotein) receptor repeats with conserved cysteine residues similar to those found in other complement proteins.
Ca2+ uptake by corpus-luteum plasma membranes. Evidence for the presence of both a Ca2+-pumping ATPase and a Ca2+-dependent nucleoside triphosphatase
Authors: Minami J , Penniston J T
Published: August 2015

Plasma-membrane vesicles from rat corpus luteum showed an ATP-dependent uptake of Ca2+. Ca2+ was accumulated with a K1/2 (concn. giving half-maximal activity) of 0.2 microM and was released by the bivalent-cation ionophore A23187. A Ca2+-dependent phosphorylated intermediate (Mr 100,000) was detected which showed a low decomposition rate, consistent with it being the phosphorylated intermediate of the transport ATPase responsible for Ca2+ uptake. The Ca2+ uptake and the phosphorylated intermediate (E approximately P) displayed several properties that were different from those of the high-affinity Ca2+-ATPase previously observed in these membranes. Both Ca2+ uptake and E approximately P discriminated against ribonucleoside triphosphates other than ATP, whereas the ATPase split all the ribonucleoside triphosphates equally. Both Ca2+ uptake and E approximately P were sensitive to three different Hg-containing inhibitors, whereas the ATPase was inhibited much less. Ca2+ uptake required added Mg2+ (Km = 2.2 mM), whereas the ATPase required no added Mg2+. The maximum rate of Ca2+ uptake was about 400-fold less than that of ATP splitting; under different conditions, the decomposition rate of E approximately P was 1,000 times too slow to account for the ATPase activity observed. All of these features suggested that Ca2+ uptake was due to an enzyme of low activity, whose ATPase activity was not detected in the presence of the higher-specific-activity Ca2+-dependent ATPase.
Hexokinase isoenzymes from the Novikoff hepatoma. Purification, kinetic and structural characterization, with emphasis on hexokinase C
Authors: Radojković J , Ureta T
Published: August 2015

The purification to homogeneity of hexokinases B and C from the cytosol of rat Novikoff hepatoma was achieved by a protocol using an initial chromatography on Blue 2-agarose to separate the isoenzymes from each other. After that step each hexokinase was subjected to chromatography on DEAE-cellulose, hydroxyapatite and Sephacryl S-300, followed by re-chromatography on hydroxyapatite. The final preparations of hexokinases B and C had specific activities of 86 and 23.5 units/mg of protein respectively, and gave single bands on electrophoresis under non-denaturing conditions or in SDS/polyacrylamide gels. Mr values of about 100,000 were found for both isoenzymes either by Sephacryl S-300 chromatography or by SDS/polyacrylamide-gel electrophoresis. Values of apparent Km for glucose and ATP of pure hexokinase B were similar to those reported for the enzyme from other sources. The apparent Km value for glucose of hexokinase C was 0.025 mM. Marked inhibition of hexokinase C by glucose concentrations above 0.2 mM was found. The effect was partially relieved by ATP concentrations above 1 mM and was independent of pH. Glucose 6-phosphate was inhibitory, but the Ki value (0.18 mM) is higher than those reported for other animal hexokinases. The amino acid composition of hexokinase C was found to be similar to those reported for hexokinases B and D. Also, an immune serum directed against hexokinase A was able, at low dilutions, to bind hexokinases B and C. An immune serum directed against hexokinase C was able, at low dilutions, to bind hexokinase B and also, but weakly, hexokinase A.
Quantitative tissue isolation from Drosophila freeze-dried in acetone
Authors: Fujita S C , Inoue H , Yoshioka T , Hotta Y
Published: August 2015

Freeze-drying procedures were developed to enable collection of tissues from Drosophila flies. The flies were frozen in acetone at -86 or -94 degrees C, and dehydrated therein. After drying, many tissues could be easily taken in entirety and free of neighbouring tissues without action of degradative enzymes. Seven polypeptide species specific to retina, and nine specific to cornea, were identified on two-dimensional electrophoretograms. Phospholipids of the dried tissues could be studied by t.l.c., and phosphatidic acid of the fly head was found to occur predominantly in the retina. Activity of three enzymes in the dried tissues could be assayed. The results of protein, phospholipid and enzyme analyses were corroborated by analyses by ‘genetic dissection’ using an eyeless mutant line.
Glutathione S-transferases of mouse lung. Selective binding of benzo[a]pyrene metabolites by the subunits which are preferentially induced by t-butylated hydroxyanisole
Authors: Singh S V , Creadon G , Das M , Mukhtar H , Awasthi Y C
Published: August 2015

Six isoenzymes of glutathione S-transferase (GST) present in mouse lung have been purified and characterized. GST I (pI 9.8) is a dimer of Mr-26,500 subunits and GST II is a heterodimer of Mr-26,500 and -22,000 subunits, and GST III (pI 7.9) and IV (pI 6.4) are dimers of Mr-24,500 subunits. GST V (pI 5.7) is a heterodimer of Mr-24,500 and -23,000 subunits, whereas GST VI (pI 4.9) is a dimer of Mr-23,000 subunits. Immunological studies indicate that the Mr-24,500 subunits present in GST III (pI 7.9) are distinct from those present in GST IV (pI 6.4) and V (pI 5.7). Structural and immunological studies provide evidence that at least five distinct types of subunits in their different binary combinations give rise to various GST isoenzymes of mouse lung. These isoenzymes express varying degrees of catalytic activities towards a wide range of electrophilic substrates including benzo[a]pyrene 7,8-oxide and benzo[a]pyrene 4,5-oxide. The dietary antioxidant t-butylated hydroxyanisole (BHA) preferentially induces GST II and III. Also, these two isoenzymes selectively bind benzo[a]pyrene (B[a]P) metabolites, indicating that they play an important physiological role in the detoxification of B[a]P metabolites. The preferential induction of the GST isoenzymes involved in the detoxification of activated B[a]P metabolites indicates that the anti-neoplastic activity of BHA against B[a]P-induced neoplasia in mouse lung [Wattenberg (1973) J. Natl. Cancer Inst. 50, 1541-1544] may be due to the enhanced detoxification of B[a]P metabolites.
Heterogeneity of growth-hormone receptors detected with monoclonal antibodies to human growth hormone
Authors: Thomas H , Green I C , Wallis M , Aston R
Published: August 2015

The specificity of hormone-receptor interactions has been examined with the aid of monoclonal antibodies (MABs) (EB1, EB2, QA68 and NA71) defining four non-overlapping antigenic determinants on human growth hormone (hGH). The results indicate that growth-hormone receptors in liver obtained from different sources differ with regard to their affinities and relative numbers; they may also differ with respect to the region of the growth-hormone molecule to which they bind. Antibody NA71 effectively inhibited hormone binding to all receptor preparations tested, although with various degrees of potency. Monoclonal antibody EB1 demonstrated a graded inhibition with respect to its ability to block 125I-hGH binding to receptors from various sources, the maximum inhibition being seen in receptor preparations from mouse and ovine liver and the minimum in rat liver. MABs EB2 and QA68 also showed various abilities to inhibit hormone-receptor interaction, depending on the origin of the receptor preparation. Furthermore, the receptor-binding characteristics of hormone-antibody complexes were dependent on whether the binding-site preparation was derived from pregnant, lactating or ‘normal’ animals. A particularly striking difference between the ability of hormone-MAB complexes to bind to receptors from different sources was seen for microsomes (microsomal fractions) derived from livers of animals of the ‘Little’ mouse strain. These animals become progressively obese, and it was shown that MABs were considerably more effective in inhibiting 125I-labelled hGH binding to microsomes from phenotypically obese mice than to those derived from their non-obese littermates. The results can be explained by the presence of multiple receptor types for GH, the relative proportions of which vary according to the physiological state of the animal, and possibly between species.
Activation of phospholipase C associated with isolated rabbit platelet membranes by guanosine 5′-[γ-thio]triphosphate and by thrombin in the presence of GTP
Authors: Hrbolich J K , Culty M , Haslam R J
Published: August 2015

Rabbit platelets were labelled with [3H]inositol and a membrane fraction was isolated in the presence of ATP, MgCl2 and EGTA. Incubation of samples for 10 min with 0.1 microM-Ca2+free released [3H]inositol phosphates equivalent to about 2.0% of the membrane [3H]phosphoinositides. Addition of 10 microM-guanosine 5′-[gamma-thio]triphosphate (GTP[S]) caused an additional formation of [3H]inositol phosphates equivalent to 6.6% of the [3H]phosphoinositides. A half-maximal effect was observed with 0.4 microM-GTP[S]. The [3H]inositol phosphates that accumulated consisted of 10% [3H]inositol monophosphate, 88% [3H]inositol bisphosphate ([3H]IP2) and 2% [3H]inositol trisphosphate ([3H]IP3). Omission of ATP and MgCl2 led to depletion of membrane [3H]polyphosphoinositides and marked decreases in the formation of [3H]inositol phosphates. Thrombin (2 units/ml) or GTP (4-100 microM) alone weakly stimulated [3H]IP2 formation, but together they acted synergistically to exert an effect comparable with that of 10 microM-GTP[S]. The action of thrombin was also potentiated by 0.1 microM-GTP[S]. Guanosine 5′-[beta-thio]diphosphate not only inhibited the effects of GTP[S], GTP and GTP with thrombin, but also blocked the action of thrombin alone, suggesting that this depended on residual GTP. Incubation with either GTP[S] or thrombin and GTP decreased membrane [3H]phosphatidylinositol 4-phosphate ([H]PIP) and prevented an increase in [3H]phosphatidylinositol 4,5-bisphosphate ([3H]PIP2) observed in controls. Addition of unlabelled IP3 to trap [3H]IP3 before it was degraded to [3H]IP2 showed that only about 20% of the additional [3H]inositol phosphates that accumulated with GTP[S] or thrombin and GTP were derived from the action of phospholipase C on [3H]PIP2. The results provide further evidence that guanine-nucleotide-binding protein mediates signal transduction between the thrombin receptor and phospholipase C, and suggest that PIP may be a major substrate of this enzyme in the platelet.
Regulation of hepatic very-low-density lipoprotein secretion in rats fed on a diet high in unsaturated fat
Authors: Gibbons G F , Pullinger C R
Published: August 2015

Rats were fed ad libitum on either a standard, high-carbohydrate, chow diet or a similar diet supplemented with 15% unsaturated fat (corn oil). Hepatocytes were prepared either during the dark phase (D6-hepatocytes) or during the light phase (L2-hepatocytes) of the diurnal cycle. In hepatocytes from rats fed on the unsaturated-fat-containing diet, secretion of very-low-density lipoprotein (VLDL) triacylglycerol was inhibited to a greater extent in the D6- than in the L2-hepatocytes. Plasma non-esterified fatty acid concentrations were elevated to the same extent at both D6 and L2 in the unsaturated-fat-fed animals. The secretion of VLDL esterified and non-esterified cholesterol was relatively insensitive to changes in the unsaturated-fat content of the diet. This resulted in proportionate increases in the content of these lipid constituents compared with that of triacylglycerol in the nascent VLDL. There was also an increase in the ratio of esterified to non-esterified cholesterol in the nascent VLDL produced by hepatocytes of the unsaturated-fat-fed animals. In the D6-hepatocytes from the unsaturated-fat-fed animals, the decrease in the secretion of VLDL triacylglycerol could not be reversed by addition of exogenous oleate (0.7 mM) to the incubation medium. In contrast, addition of a mixture of lactate (10 mM) and pyruvate (1 mM) stimulated both fatty acid synthesis de novo and the rate of VLDL triacylglycerol secretion. Secretion of esterified and non-esterified cholesterol also increased under these conditions. Insulin suppressed the secretion of VLDL triacylglycerol and cholesteryl ester under a wide range of conditions in all types of hepatocyte preparations. Non-esterified cholesterol secretion was unaffected. In hepatocytes prepared from the fat-fed animals, these effects of insulin were more pronounced at D6 than at L2. Glucagon also inhibited VLDL lipid secretion in all types of hepatocyte preparations. The decrease in cholesterol secretion was due equally to decreases in the rates of secretion of both esterified and non-esterified cholesterol.
Identification and characterization of a Ca2+-calmodulin-sensitive cyclic nucleotide phosphodiesterase in a human lymphoblastoid cell line
Authors: Epstein P M , Moraski S , Hachisu R
Published: August 2015

This study examines the pattern and regulatory properties of cyclic nucleotide phosphodiesterases in a human lymphoblastoid B-cell line (RPMI 8392) established from a patient with acute lymphocytic leukaemia. In this cell line, phosphodiesterase activity measured at 0.25 microM-cyclic AMP is approx. 7-fold greater than that in isolated human peripheral-blood lymphocytes, and 16% of the phosphodiesterase activity in RPMI 8392 cells is associated with particulate fractions. Phosphodiesterase activity in crude fractions of this cell line is reproducibly stimulated by about 60-80% by Ca2+-calmodulin. In the presence of 20 nM-calmodulin, half-maximal stimulation occurs at 0.7 microM-Ca2+. The cytosolic phosphodiesterase activity of RPMI 8392 cells is separated into two forms by DEAE-Sephacel chromatography. The first form is eluted at approx. 0.2 M-sodium acetate, catalyses the hydrolysis of both cyclic AMP and cyclic GMP, and is stimulated 3-fold by Ca2+-calmodulin. This form exhibits non-linear kinetics for cyclic AMP in the absence of calmodulin, with extrapolated Km values of 0.8 and 4 microM, and non-linear kinetics in the presence of calmodulin, with extrapolated Km values of 0.5 and 1 microM. The Vmax. values are increased approx. 3-fold by calmodulin. The second form is eluted at approx. 0.6 M-sodium acetate, is specific for cyclic AMP, and insensitive to stimulation by Ca2+-calmodulin. The Ca2+-calmodulin-sensitive phosphodiesterase from the DEAE-Sephacel column can be adsorbed to a calmodulin-Sepharose affinity column and eluted with EGTA. This enzymic activity can also be immunoprecipitated by a monoclonal antibody directed against a calmodulin-bovine heart phosphodiesterase complex. This study documents the existence of Ca2+-calmodulin-sensitive phosphodiesterase in a cultured lymphoblastoid cell line derived from a leukaemic patient.
The synthesis of fluorescent chlorotriazinylaminofluorescein-concanavalin A and its use as a glycoprotein stain on sodium dodecyl sulphate/polyacrylamide gels
Authors: Mahoney C W , Azzi A
Published: August 2015

Concanavalin A, a specific glycoprotein probe, was optimally labelled to a maximum stoichiometry of 0.4 mol of chlorotriazinylaminofluorescein (CTAF)/mol of concanavalin A monomer under mild reaction conditions (pH 8.0, 6 h), and under these conditions the CTAF concanavalin A preparation retains its carbohydrate-binding ability and is able to penetrate SDS/7.5-15%-polyacrylamide gradient gels. CTAF-concanavalin A gives fluorescent bands for the glycoproteins transferrin, fetuin and deoxyribonuclease and shows no fluorescent response for the non-glycoproteins bovine serum albumin and soya-bean trypsin inhibitor. The detection limit of sensitivity for CTAF-concanavalin A, which is similar to that of fluorescein isothiocyanate-concanavalin A, is in the range 5-25 micrograms of glycoprotein. CTAF-concanavalin A is a suitable probe for the detection of glycoproteins in higher-percentage (greater than or equal to 10%) SDS/polyacrylamide gels, and will probably have other applications in, for example, fluorescent energy transfer and other structure-function studies.
Pathways of reducing equivalents in hepatocytes from rats. Estimation of cytosolic fluxes by means of 3H-labelled substrates for either A- or B-specific dehydrogenases
Authors: Vind C , Hunding A , Grunnet N
Published: August 2015

The metabolism of [2-3H]lactate and [2-3H]glycerol was studied in isolated hepatocytes from fed rats. In order to estimate the rate of equilibrium between the 4A and 4B hydrogen atoms of NADH, we compared the flow of 3H from [2-3H]lactate and [2-3H]glycerol, the oxidations of which are catalysed by A- and B-type dehydrogenases, respectively. Hepatocytes were incubated with lactate, glycerol and ethanol and tracer amounts of [2-3H]lactate or [2-3H]glycerol and the labelling rates of lactate, ethanol, glucose and glycerol phosphate were determined. The data were used to calculate the oxidation rate of NADH catalysed by lactate dehydrogenase, alcohol dehydrogenase, triosephosphate dehydrogenase and glycerol phosphate dehydrogenase. The rates were calculated by obtaining the best fit of a model to the experimental data by using a least-squares procedure. The results support our model and suggest that the fluxes through various dehydrogenases are sufficient to equilibrate the 4A and 4B hydrogen atoms of cytosolic NADH. The validity of the metabolic models used was evaluated by comparison of rates of NADH oxidation catalysed by cytosolic dehydrogenases as calculated by two different models.