bluuebunny/crossref_metadata_2025 huggingface dataset:
Subcellular distribution of lysophospholipase of rat intestinal mucosa
Authors: Charbonnier Monique , Grataroli Renée , Portugal Henri , Lafont Huguette , Nalbone Gilles
Published: December 2009

We have studied the subcellular localization of rat intestinal lysophospholipase activity and some of the biochemical properties of this enzyme. After subcellular fractionation, an enriched activity was found in the high-speed pellet fraction containing the microsomes and the brush border membranes. Subsequently, these organelles were isolated. Using the classical calcium-precipitation method to isolate brush border membranes, we failed to demonstrate any significant recovery of lysophospholipase activity associated with this fraction. The microsomal fraction was further isolated after density gradient centrifugation, and most of the lysophospholipase activity was recovered with this fraction. Because further purification of the enzyme was unsuccessful, some of the biochemical properties of the enzyme were determined on the partially purified microsomal fraction. The optimum pH of the activity was centered at 7.0, and the enzyme did not require bivalent cations. By using double reciprocal plots, we determined the [Formula: see text] to be 0.4 mM; the [Formula: see text], 23 μmol∙h−1∙mg protein−1. The enzyme was strongly inhibited by detergents having a low critical micellar concentration and less inhibited by those having a higher critical micellar concentration.
Three types of stereospecificity and the kinetic deuterium isotope effect in the oxidative deamination of dopamine as catalyzed by different amine oxidases
Authors: Yu Peter H.
Published: December 2009

When the stereospecifically deuterated dopamine enantiomers, (R)- and (S)-[α-2H1]dopamine, are incubated with amine oxidases, the deuterium atom may be either retained to form monodeuterated 3,4-dihydroxyphenylacetaldehyde, or eliminated to produce the nondeuterated or protio-aldehyde product. These two aldehydes can be separated from one another and identified by high-performance liquid chromatography with electrochemical detection. Three types of stereospecific abstraction of a hydrogen from the α-carbon of dopamine during deamination have been observed. In the first type, the pro-R hydrogen is removed from the α-carbon. Enzymes in this category are mitochondrial monoamine oxidases A and B, as isolated from different tissues and species. The second type of deamination involves the abstraction of pro-S hydrogen from the α-carbon of dopamine. Soluble enzymes, such as rat aorta benzylamine oxidase or diamine oxidase from hog kidney and pea seedling, have been found to belong to this group. Bovine plasma amine oxidase exhibits the third type of deamination where no absolute stereospecificity is required. This enzyme catalyzes the oxidation of either (S)- or (R)-[α-2H1]dopamine, preferably breaking the C—H bond rather than the C—2H bond in both cases. The kinetic deuterium isotope effect during the deamination of dopamine catalyzed by the different amine oxidases varies greatly; VH/VD ranges from 1.5 to 5.5. The high magnitude of the isotope effect suggests that hydrogen abstraction may be the rate-limiting step (i.e., in reactions catalyzed by benzylamine oxidase and monoamine oxidase). When the isotope effect is low (i.e., for diamine oxidases from hog kidney or pea seedling), it is uncertain if the breaking of the bond is rate limiting.
β-1,4-mannosyl-glycoprotein β-1,4-N-acetylglucosaminyltransferase III activity in human B and T lymphocyte lines and in tonsillar B and T lymphocytes
Authors: Narasimhan S. , Lee J. W. W. , Cheung R. K. , Gelfand E. W. , Schachter H.
Published: December 2009

β-1,4-mannosyl-glycoprotein β-1,4-N-acetylglucosaminyltransferase III (GlcNAc-T III) catalyzes the incorporation of a "bisecting" N-acetylglucosamine (GlcNAc) residue in β1—4 linkage to the β-linked mannose of the core of asparagine linked–protein bound oligosaccharides (N-glycans). The activity of GlcNAc-T III was determined in Triton X-100 extracts of four human Epstein-Barr virus (EBV)-infected B-cell lines, in four T-cell lines originally established from lymphocytes of patients with acute lymphatic leukemia, and in human tonsillar B and T lymphocytes. The four EBV-transformed B-cell lines showed appreciable GlcNAc-T III activities (ranging from 3.4 to 19.0 nmol∙h−1∙mg protein−1), while the tonsillar resting B lymphocytes had much less activity (0.68 nmol∙h−1∙mg protein−1). The four T-cell lines and the tonsillar T lymphocytes had negligible GlcNAc-T III activities (ranging from 0.02 to 0.25 nmol∙h−1∙mg protein−1). Enzyme product was identified by high resolution proton nuclear magnetic resonance spectroscopy and methylation analysis. This is the first demonstration of GlcNAc-T III activity in human lymphocytes. The presence of GlcNAc-T III in B-cell lines correlates with the reported occurrence of bisecting GlcNAc residues in the oligosaccharides of human immunoglobulins G, A1, M, and D, tonsillar class II antigens, and membrane glycoproteins from B lymphocytes. The negligible GlcNAc-T III activity of the four human T-cell lines and of tonsillar T lymphocytes agrees with the reported absence of bisected structures in N-glycans from human T lymphocyte membrane glycoproteins.
Phosphorylation of liver plasma membrane-bound calmodulin
Authors: Ghosh Shobha , Church Jon G. , Roufogalis Basil D. , Villalobo Antonio
Published: December 2009

In highly purified rat liver plasma membrane preparations, membrane-bound calmodulin was phosphorylated by a membrane-bound protein kinase using [γ-32P] ATP as phosphate donor. Maximum phosphorylation of calmodulin occurred in the absence of calcium ion, but was significantly decreased in its presence. Plasma membrane-bound calmodulin was identified by the following criteria: (i) extraction from the membrane by EGTA, (ii) stimulation of the activity of the Ca2+-calmodulin-dependent enzyme, (3′:5′ AMP)-phosphodiesterase, by the EGTA extract, and (iii) electrophoretic comigration of EGTA-extracted protein with standard bovine brain calmodulin, both in the presence and the absence of Ca2+. Phosphorylation of the plasma membrane-bound calmodulin was shown by electrophoretic comigration of the 32P-labelled molecule with bovine brain calmodulin, the absence of phosphorylation of this protein band in calmodulin-depleted membranes, and a Western blot of the phosphorylated band using a calmodulin antibody. Treatment of plasma membrane preparations with sheep anticalmodulin serum prevented the phosphorylation of the calmodulin band. Phosphocalmodulin, which could be partially extracted from the membrane by EGTA, comigrated with bovine brain calmodulin in polyacrylamide gel electrophoresis.
Regulation of uptake of inositol by glucose in cultured retinal pigment epithelial cells
Authors: Khatami Mahin , Rockey John H.
Published: December 2009

Long term and acute effects of glucose on myo-inositol (MI) uptake were studied in primary cultures of bovine retinal pigment epithelial (RPE) cells. RPE cells were grown under low (5 mM) or high (20, 40, or 50 mM) glucose levels in the growth medium for up to 18 days. The concentrative capacity of confluent RPE cells to accululate [3H]MI (10 μM) was reduced up to 41% as the glucose concentration in the growth medium increased. When the growth medium glucose was switched from 5 to 40 mM, or vice versa, the capacity of cells to accumulate MI was reversed. Treatment of cells grown in 40 or 50 mM glucose with 0.1 mM Sorbinil (an aldose reductase inhibitor) minimally reversed the ability of cells to accumulate MI. RPE cells, grown in 5 mM glucose, were incubated with 10–60 mM D-glucose or its nonmetabolizable analogues (acute effect). Kinetics of MI uptake inhibition by α-methyl glucose according to Dixon plots were characteristic of competitive inhibition (Ki = 28 mM). MI uptake was strongly inhibited by phlorizin. The ability of RPE cells to bind 5 μM [3H]phlorizin also was reduced by increased glucose levels in the growth medium. These studies indicated that MI and glucose shared the same transporter system. Glucose in the incubation medium directly interfered with MI binding to the transporter. High glucose feeding of the cells also down-regulated the transporter density. The uptake and function of solutes such as MI in tissues that operate on the glucose carrier system may be severely impaired in diabetes.
Binding of a 30-kDa protein to the pyruvate kinase gene of Neurospora crassa
Authors: Devchand Medhavinee R. , Kapoor M.
Published: December 2009

Extracts of a wild-type strain of Neurospora crassa, electrophoresed on SDS–polyacrylamide gels and electroblotted onto nitrocellulose sheets, were hybridized to an end-labelled pyruvate kinase (PK) gene fragment containing the 5′ noncoding sequence and a large part of the coding region. A 30-kDa protein was found to bind strongly to the PK gene DNA, while binding weakly to plasmid pUC12 DNA and to total N. crassa DNA. Probing of blots with individual restriction fragments derived from the PK gene showed that the protein binding occurred primarily to the 5′ noncoding region. Nonspecific DNA from pUC12, PK gene DNA from the recombinant plasmid pNP460 (pUC12 containing a 1.8-kilobase EcoRI insert of the PK gene DNA), along with a 0.7-kilobase EcoRI-AccI restriction fragment containing the 5′ flanking region, were used in filter-binding experiments to analyze the kinetics of binding. Formation of protein–DNA complexes was demonstrated by monitoring the electrophoretic mobility of this fragment on nondenaturing gels.
The mechanism of the nonenzymatic iodination of tyrosine by molecular iodine
Authors: Dunford H. Brian , Adeniran Adejare J.
Published: December 2009

Over the pH range 7–10, at very low buffer concentration, the nonenzymatic iodination of tyrosine obeys the rate law[Formula: see text]where kapp is the measured second order rate constant based upon the total initial concentrations of molecular iodine and tyrosine and K2 (units M) is the equilibrium constant for [Formula: see text]. The value of k′ is 3.5 × 10−8 M∙s−1. There are three plausible mechanisms that fit the experimental data. One, the simplest, is a concerted process in which hypoiodous acid attacks tyrosine with its phenolic group unionized. The other two involve the formation of an iodinated quinoid reactive intermediate species in a rapid pre-equilibrium between unionized tyrosine and either hypoiodous acid or molecular iodine. The pre-equilibrium, if it occurs, favors the initial reactants. It is followed by a slow step in which the quinoid is converted to mono-iodinated tyrosine. Positive deviations from the rate law for pH dependence indicate that some specific acid catalysis (H3O+) is occurring in the pH range 5–7. In the presence of sufficient buffer, general acid–base catalysis is observed with acetic acid acting as a general acid catalyst in the vicinity of pH 5 and carbonate acting as a general base at pH ~ 9.5. The nonenzymatic iodination of tyrosine occurs more rapidly as the pH is increased, in marked contrast to the peroxidase-catalyzed iodination, which has its optimum at low pH.
Structural studies of the capsular polysaccharide from Actinobacillus (Haemophilus) pleuropneumoniae serotype 4
Authors: Altman Eleonora , Brisson Jean-Robert , Perry Malcolm B.
Published: December 2009

The capsular polysaccharide of Actinobacillus (Haemophilus pleuropneumoniae serotype 4 (ATCC 33378) is composed of D-glucose (one part), 2-acetamido-2-deoxy-D-galactose (one part), and phosphate (one part). From hydrolysis, dephosphorylation, methylation, and 1H and 13C nuclear magnetic resonance studies, the polysaccharide was found to be a high molecular weight polymer of a repeating disaccharide unit, joined through monophosphate diester linkages and having the following structure:[Formula: see text]
Monoacylglycerol lipase activity in cardiac myocytes
Authors: Severson David L. , Hee-Cheong Mariette
Published: December 2009

Monoacylglycerol lipase activity in homogenates of isolated myocardial cells (myocytes) from rat hearts was recovered in both particulate and soluble subcellular fractions. The activity present in the microsomal (100 000 × g pellet) fraction was solubilized by treatment with Triton X-100 and combined with the 100 000 × g supernatant fraction; the properties of monoacylglycerol lipase were investigated with this soluble enzyme preparation. The Km for the hydrolysis of a 2-monoolein substrate was 16 μM. The rates of hydrolysis of 1-monoolein and 2-monoolein were identical, and 1-monoolein was a competitive inhibitor (Ki = 20 μM) of the hydrolysis of 2-monoolein. Monoacylglycerol lipase activity was regulated by product inhibition according to the following order of potency: fatty acyl CoA > free fatty acids > fatty acyl carnitine.
Evidence that hatching enzyme of the sea urchin Strongylocentrotus purpuratus is a chymotrypsin-like protease
Authors: Post Laura L. , Schuel Regina , Schuel Herbert
Published: December 2009

The sea urchin blastula secretes a hatching enzyme (HE) that dissolves the fertilization envelope. HE was collected from the supernatant seawater of cultures of hatched Strongylocentrotus purpuratus blastulae, and concentrated 20 times by ultrafiltration. The proteolytic activity of HE using casein as substrate was inhibited by the chymotrypsin inhibitors, chymostatin and N-tosyl-L-phenylalanine chloromethyl ketone. The activity was not inhibited by inhibitors (antipain, elastatinal, pepstatin, phosphoramidon, soybean trypsin inhibitor, and Nα-p-tosyl-L-lysine chloromethyl ketone) of other types of proteases. HE did not hydrolyze the synthetic trypsin substrate, α-N-benzoyl-L-arginine ethyl ester, but did hydrolyze the synthetic substrate of chymotrypsin, N-benzoyl-L-tyrosine ethyl ester (BTEE). The BTEEase activity of HE was completely inhibited by the chymotrypsin inhibitors chymostatin and 2-nitro-4-carboxyphenyl N,N-diphenylcarbamate (NCDC). Chymostatin inhibited the natural hatching of sea urchin blastulae. Application of HE to freshly fertilized sea urchin eggs, 2 h after insemination, caused premature dispersal of the hardened fertilization envelope. Chymostatin and NCDC inhibited HE-induced lysis of the fertilization envelope, while inhibitors of other types of proteases were ineffective. These data suggest that sea urchin HE is a chymotrypsin-like protease we call "chymostrypsin."
Tissue-specific expression of phosphoenolpyruvate carboxylase in sorghum
Authors: Abergel Elisabeth A. , Glick Bernard R.
Published: December 2009

The synthesis and expression of phosphoenolpyruvate carboxylase (PEP-C) mRNA in sorghum tissue (i.e., leaf, shoot, and root) were monitored for several days after seed germination by Northern hybridization and measurement of de novo protein synthesis. In 2- to 8-day-old leaves and shoots, PEP-C accounted for approximately 8.9 and 2.5%, respectively, of the newly synthesized proteins. In addition, mRNA from both leaves and shoots, detected by hybridization with a heterologous PEP-C probe, is present in relatively constant amounts from 2 to 9 days after seed germination. In roots, on the contrary, a peak of RNA that hybridizes to the probe was observed approximately 6 days after seed germination, when de novo synthesis of the enzyme was barely detectable in this tissue. Northern hybridization experiments indicate the presence of putative mRNA species of approximately 3.1 and 5.9 kilobases. The data are rationalized in terms of the postulated existence of a stable but translationally inactive precursor of PEP-C mRNA in sorghum roots.
Gene expression in Brassica tissues and species following heat shock
Authors: Baszczynski Chris L.
Published: December 2009

Several tissues from the three oilseed Brassica species have been analyzed for polypeptides synthesized in vivo and in vitro under control (23 °C) and heat-shock conditions. The various tissues have characteristically unique patterns of polypeptide synthesis, the general pattern being maintained following heat shock. In addition, all tissues exhibit synthesis of a set of qualitatively similar heat shock protein (HSP) that differ, however, in relative intensity among tissues. Synthesis of control polypeptides as well as high molecular-weight HSP is similar in hypocotyls of the three genomically interrelated species B. napus, B. campestris, and B. juncea; however, the three species exhibit variation in in vivo and in vitro synthesized polypeptides in the 18 000–27 000 molecular-weight region, with several prominent differences in the number and distribution of the major class of low molecular-weight (20 000) HSP. Several of the HSP appear to be unique to B. juncea and one is unique to B. napus. Immunochemical studies of in vitro synthesized Brassica polypeptides using antisera to maize HSP of molecular weight 18 000 substantiate the observed differences in synthesis of various members of the 20 000 molecular-weight HSP family among the three species, and indicate that the Brassica low molecular-weight HSP are immunologically similar to the small HSPs of other plants including maize, soybean, pea, and Arabidopsis.
Purine metabolism in cultured aortic and coronary endothelial cells
Authors: Rosiers Christine Des , Nees Stephan , Gerlach Eckehart
Published: December 2009

Purine salvage pathways in cultured endothelial cells of macrovascular (pig aorta) and microvascular (guinea pig coronary system) origin were investigated by measuring the incorporation of radioactive purine bases (adenine or hypoxanthine) or nucleosides (adenosine or inosine) into purine nucleotides. These precursors were used at initial extracellular concentrations of 0.1, 5, and 500 μM. In both types of endothelial cells, purine nucleotide synthesis occurred with all four substrates. Aortic endothelial cells salvaged adenine best among purines and nucleosides when applied at 0.1 μM. At 5 and 500 μM, adenosine was the best precursor. In contrast, microvascular endothelial cells from the coronary system used adenosine most efficiently at all concentrations studied. The synthetic capacity of salvage pathways was greater than that of the de novo pathway. As measured using radioactive formate or glycine, de novo synthesis of purine nucleotides was barely detectable in aortic endothelial cells, whereas it readily occurred in coronary endothelial cells. Purine de novo synthesis in coronary endothelial cells was inhibited by physiological concentrations of purine bases and nucleosides, and by ribose or isoproterenol. The isoproterenol-induced inhibition was prevented by the β-adrenergic receptor antagonist propranolol. The end product of purine catabolism in aortic endothelial cells was found to be hypoxanthine, whereas coronary endothelial cells degraded hypoxanthine further to xanthine and uric acid, a reaction catalyzed by the enzyme xanthine dehydrogenase.Key words: purine metabolism, aortic endothelial cells, coronary endothelial cells, β-adrenergic receptor.
Reversible ATP-dependent inactivation of glycerophosphate acyltransferase from rat adipose tissue
Authors: Walsh M. , Durocher V. , Rodriguez A.
Published: December 2009

Microsomal glycerophosphate acyltransferase from rat adipose tissue is shown to be inactivated with time upon incubation with ATP. The inactivation can be observed in postmitochondrial supernatant as well as in washed microsomes. However, the effect is more pronounced upon addition of the cytosolic fraction. This activity is specific for ATP, is dependent on the nucleotide concentration, and is prevented when ATP is substituted by β,γ-methylene-ATP. Some protection is provided by amiloride but not by EGTA or cAMP-protein kinase inhibitor. Also, the level of enzyme inactivation is not modified by addition of cAMP-dependent protein kinase and its substrates. Inactivated glycerol-phosphate acyltransferase from ATP-treated microsomes can be reactivated by incubation with partially purified protein phosphatase from rat liver. These results suggest the existence in adipose tissue of a protein kinase (cAMP independent) that may be involved in the regulation of glycerophosphate acyltransferase.Key words: glycerophosphate acyltransferase, lipid synthesis regulation, adipose tissue, protein kinase.
Cytosol components from human placenta and rat liver in iodothyronine 5- and 5′-deiodination
Authors: Iwase Katsumi , Hummel Brian C. W. , Walfish Paul G.
Published: December 2009

Using either human placental microsomal 5-deiodinase as enzyme (5-DI) and thyroxine as substrate or rat liver (RL) microsomal 5′-deiodinase (5′ DI) as enzyme and reverse [(3′- or 5′-)-125I]triiodo-L-thyronine ([125I]rT3) as substrate, activation of 5′-DI in the presence of NADPH was observed using either human placental or rat liver cytosolic components, but there was no activation of 5-DI. Both could be activated by DTT, with higher concentrations being required for 5-DI than for 5′-DI. Iopanoic acid, dicumarol, and sodium arsenite inhibited 5′-DI and 5-DI activated by DTT. In the presence of DTT, 1 mM 6-propyl-2-thiouracil had no effect on 5-DI but inhibited 5′ DI. Thus, human placental and rat liver cytosolic components are interchangeable in activating hepatic 5′-DI in the presence of NADPH. However, if an endogenous cofactor system involved in the activation of human placental 5-DI exists, it probably differs from the activator of liver 5′-DI.Key words: iodothyronines, deiodination cofactors, placenta, liver, cytosol.
Regulation of phosphatidylcholine metabolism in mammalian hearts
Authors: Hatch Grant M. , O Karmin , Choy Patrick C.
Published: December 2009

Phosphatidylcholine is the major phospholipid in the mammalian heart. Over 90% of the cardiac phosphatidylcholine is synthesized via the CDP-choline pathway. The rate-limiting step of this pathway is catalyzed by CTP:phosphocholine cytidylyltransferase. Current evidence suggests that phosphatidylcholine biosynthesis in the heart is regulated by the availability of CTP and the modulation of cytidylyltransferase activity. Phosphatidylcholine is degraded mainly by the actions of phospholipase A1 and A2, with the formation of lysophosphatidylcholine. Lysophosphatidylcholine may be further deacylated by lysophospholipase or reacylated back into the parent phospholipid by the action of acyltransferase. The accumulation of lysophosphatidylcholine in the heart may be one of the biochemical factors for the production of cardiac arrhythmias.Key words: phosphatidylcholine, phospholipids, biosynthesis, metabolism, heart.
Mutation of amino acids thought to polarize the oxaloacetate carbonyl in citrate synthase severely reduces but does not abolish activity of the enzyme
Authors: Anderson Deborah H. , Duckworth Harry W.
Published: December 2009

Oligonucleotide-directed mutagenesis has been used to alter two active site residues of Escherichia coli citrate synthase, histidine-305 and arginine-314. Both residues are thought to be involved in the polarization of the carbonyl group of oxaloacetate and thus facilitate attack at the carbonyl carbon by acetyl-CoA. In one mutant, designated CS305H→A, His-305 was mutated to alanine and in the other, designated CS314R→L, Arg-314 was changed to leucine. Both mutants have greatly reduced turnover numbers, less than 0.1% of the wild-type value. The dissociation constant for formation of the binary enzyme–oxaloacetate complex, Ki, OAA, is at least 950 μM for CS305H→A, and about 500 μM for CS314R→L, 28 and 15 times the wild-type value, respectively. The Michaelis constants for the two substrates, KOAA and KAcCoA, which measure the affinity of the enzyme for the catalytically significant ternary complex, are less radically altered: values of KAcCoA are actually 3.5-fold and 4.6-fold lower for CS305H→A and CS314R→L, respectively. These kinetic effects are taken to mean that both His-305 and Arg-314 are important for the successful formation of an efficient transition state, very likely by polarizing the carbonyl group of oxaloacetate as has been suggested, and that the residual kinetic activity, in both mutants, occurs by a mechanism which benefits from only part of this polarization. Allosteric properties of the mutant enzymes, as measured by NADH inhibition and binding, and KCl activation, are normal. Inhibition of the enzyme by α-ketoglutarate, an oxaloacetate analogue, is only slightly weakened by the mutations, a finding which suggests that this analogue is not a substrate itself because it cannot bind in the active site in such a way as to undergo carbonyl polarization.Key words: citrate synthase, mutagenesis, active site, enzyme mechanism, oxaloacetate.
Effect of hydrocortisone on the maturation of human foetal kidney explants in serum-free organ culture
Authors: Bertrand Lyne , Brière Normand
Published: December 2009

The effect of hydrocortisone on the in vitro maturation of human foetal kidney was investigated. Following legal therapeutic abortions, explants of renal cortex from foetuses aged 13–18 weeks were cultured for 5 days in serum-free Leibovitz's L-15 medium at 37 °C in a mixture of 95% air – 5% CO2, without hormone (controls) or with hydrocortisone at concentrations of 12.5, 25, or 50 ng/mL, which are the levels representative of different gestational periods. During the studied period of culture, the overall architecture of the renal structures was preserved without any evident signs of nephrogenesis induced by hydrocortisone. DNA synthesis was measured by incorporation of [3H]thymidine and was stimulated on day 5 by 80% with the addition of hydrocortisone at 12.5 ng/mL, and by 131% with 50 ng/mL. In autoradiograms, the sites of [3H]thymidine incorporation were the same after hydrocortisone addition, but the number of labelled nuclei was higher in 5-day explants supplemented with hydrocortisone at 50 ng/mL. The activities of some brush border enzymes (leucylnaphthylamidase, maltase, and alkaline phosphatase) were not influenced by hydrocortisone when compared with controls. Trehalase activity was decreased on day 5 with 12.5 and 50 ng/mL. A concentration of 12.5 ng/mL diminished γ-glutamyltransferase activity by 29% on day 5. The incorporation of [3H]leucine into proteins was not influenced by any concentration of the glucocorticoid hormone. This study indicates that hydrocortisone directly influences cell proliferation and certain brush border enzymic activities in human developing kidney maintained in organ culture.Key words: hydrocortisone, organ culture, human foetus, kidney, development.
Covalent reaction of cerulenin at the active site of acyl-CoA reductase of Photobacterium phosphoreum
Authors: Wall Lee , Meighen Edward
Published: December 2009

Inhibition of bioluminescence in Photobacterium phosphoreum by cerulenin has been demonstrated to be due to a specific inactivation of the acyl-CoA reductase subunit of the fatty acid reductase complex required for synthesis of the aldehyde substrate for the luminescent reaction. In contrast, the activities of the other luminescence-related enzymes, acyl-protein synthetase, acyl-transferase, and luciferase, were unaffected by cerulenin. Myristoyl-CoA, but not NADPH, protected the acyl-CoA reductase against cerulenin inhibition. Cerulenin blocked the acylation of the reductase with myristoyl-CoA and the reaction with N-ethylmaleimide. A shift in mobility of the reductase polypeptide on sodium dodecyl sulfate – polyacrylamide gel electrophoresis occurred after reaction with cerulenin, a shift which could be blocked by reaction with N-ethylmaleimide. These results demonstrate that cerulenin blocks aldehyde synthesis by covalent reaction with the acyl-CoA reductase and indicate that the reaction may occur at a cysteine residue involved in the formation of the acyl–reductase intermediate.Key words: bioluminescence, cerulenin, acyl-CoA reductase.
Features of the cell-free translation of a spider fibroin mRNA
Authors: Candelas Graciela C. , Ortiz Anselmo , Ortiz Nayda
Published: December 2009

The massive production of fibroin by the large ampullate glands of the spider, Nephila clavipes, serves as a model system in which to study the synthesis and control of a large secretory protein. Their tissue-specific product, fibroin, produced during the entire adult life of the female, is approximately 320 kilodaltons, and rich in glycine and alanine. Highly purified fibroin mRNA from the glands has been translated in a rabbit reticulocyte cell-free system with variable supplements. The translational products analyzed by SDS–PAGE display two features, tRNA modulation and discontinuous pauses during elongation. tRNA complements exert their effects both in the translational efficiency and in the size of the peptides generated. The pauses observed during translation generate subsets of smaller discrete peptides, visualized in the gels as ladders of variable relative intensities, appearing exclusively and concomitantly with the fibroin. Definitive linkage between the discrete peptides and fibroin synthesis process has been established by their selective labeling with specific radioactive amino acids.Key words: translation, tRNA modulation, fibroin, protein synthesis, cell-free synthesis.
The purification and properties of monoacylglycerol kinase from bovine brain
Authors: Shim Yhong-Hee , Lin C.-H. , Strickland K. P.
Published: December 2009

Monoacylglycerol kinase (MGK) has been purified from bovine brain by six steps: isolation of cytosol, DEAE–cellulose chromatography, ammonium sulfate fractionation (0–40%), Bio-Gel A-1.5m, hydroxylapatite, and ATP–agarose column chromatography. The overall purification was 938 times with a 4.8% yield. The column separations (particularly Bio-Gel A-1.5m) and SDS- and nondenaturing-polyacrylamide gel electrophoresis of enzyme purified from ATP–agarose indicated that MGK exists as a complex (~ 350 kilodaltons) that is stabilized by 0.5 M NaCl and, on complete dissociation, yields a major protein of 72 kilodaltons. Dithiothreitol, EDTA, and ATP helped to stabilize MGK during purification. The protein peak eluted from hydroxylapatite by 25 mM phosphate activated and stabilized MGK activity. Phosphatidylcholine, phosphatidylethanolamine, and sphingomyelin inhibited MGK. These phospholipids and others activated MGK synergistically with the above protein peak. MGK copurified with diacylglycerol kinase (DGK) throughout giving MGK to DGK ratios of 0.05–0.36. Optimal activity required 0.5 mM 2-monoolein and 10 mM MgCl2. Strong inhibition by p-chloromercuriphenyl sulfonic acid, N-ethyl-maleimide, and 5,5′-dithio-bis(2-nitrobenzoic acid), and prevention of this inhibition by dithiothreitol indicated the involvement of intact SH groups in the action of MGK. Purified MGK showed preference for substrates with unsaturated fatty acids except for 1- or 2-monostearin. Overall the preference favored the selective generation of 1-stearoyl- and 2-arachidonoyl-lysophosphatidic acid.Key words: monoacylglycerol kinase, diacylglycerol kinase, bovine brain cytosol, purification.
Diethylene glycol distearate as an embedding medium for immunofluorescence microscopy
Authors: Valdimarsson Gunnar , Huebner Erwin
Published: December 2009

Diethylene glycol distearate was tested as an embedding medium for indirect immunofluorescence microscopy. Rhodnius ovarioles were embedded in diethylene glycol distearate, sectioned at 1–2 μm, mounted onto coverlips, and stained with anti-tubulin antibodies followed by fluorescein-conjugated secondary antibodies. Flat, brightly stained sections with low background fluorescence were routinely obtained, suggesting that diethylene glycol diesterate may be generally applicable for immunofluorescence localization of cytoskeletal proteins in tissues.Key words: diethylene glycol distearate, immunofluorescence, cytoskeleton, insect ovariole.
Assembly of Escherichia coli fumarate reductase holoenzyme
Authors: Latour David J. , Weiner Joel H.
Published: December 2009

The production and assembly of the four fumarate reductase polypeptides into holoenzyme was studied in vivo in a T7-promoter-conditional expression system. No posttranslational modification of any of the subunits was detected, although the ratio of polypeptides produced varied with the temperature at which expression occurred. FrdC and FrdD, the membrane anchor polypeptides, assembled rapidly into the membrane and then were capped with FrdA and FrdB in separate events. Truncation of the C-terminal domain of FrdD by insertion of transposon Tn5 into the frdD cistron interfered with membrane insertion of the anchor polypeptides and assembly of the holoenzyme. Proteolytic degradation of truncated FrdD was implicated in the production of a soluble FrdABC trimer.Key words: fumarate reductase, membrane protein assembly, T7 expression-promoter system, membrane anchor polypeptides, electron transport.
Mannose toxicity in Ehrlich ascites tumor cells
Authors: Hernández D. , Fuente M. De la
Published: December 2009

Mannosephosphate isomerase (MPI) showed a higher activity than hexokinase (HKM) in its ability to phosphorylate mannose in the spleen, thymus, brain, liver, striated muscles, kidneys, and testes from BALB/c mice. This led to a HKM/MPI ratio of less than 1 in all the organs and tissues mentioned. In contrast, Ehrlich ascites tumor cells obtained from the peritoneum of BALB/c mice had low MPI activity (half of the HKM activity and, therefore, a ratio of 2). Mannose, which is nontoxic to nontumor cells at a concentration of 0.1 M, induced marked in vitro mortality of the tumor cells. Incubation of Ehrlich ascites tumor cells with mannose resulted in a high accumulation of mannose-6-phosphate and a marked depletion of ATP which did not appear when the cells were incubated with glucose. These facts may explain the selective mortality caused by mannose in the tumor cells studied.Key words: mannose, mannosephosphate isomerase, tumor cells.
Soybean mitochondrial transcripts capped in vitro with guanylyltransferase
Authors: Auchincloss Andrea H. , Brown Gregory G.
Published: December 2009

A method was devised which permitted the efficient labelling of soybean mitochondrial transcripts carrying initiator 5′ di- or tri-phosphate termini with [α-32P]GTP and the capping enzyme guanylyltransferase. The RNAs labelled in this manner appear to represent several different transcription initiation sites and to be present at only low to moderate abundance in soybean mtRNA preparations.Key words: transcription initiation, promoter, mitochondrial DNA, RNA processing, gene expression.
Evidence for two mechanisms of thrombin-induced platelet activation: one proteolytic, one receptor mediated
Authors: McNicol Archibald , Gerrard Jon M. , MacIntyre D. Euan
Published: December 2009

The possibility that thrombin-induced platelet reactivity could occur via both a receptor-related and a proteolytic process was examined. Thrombin elicited the formation of considerably more [32P)phosphatidic acid (an index of phospholipase C catalysed phosphoinositide metabolism) than did platelet activating factor, 5-hydroxytryptamine, ADP, and the thromboxane A2 analogue EP171, when these agents were added either alone or in combination. Co-addition of thrombin and EP171 did not evoke significantly more [32P]phosphatidic acid than did thrombin alone. The protease inhibitor leupeptin, decreased but did not abolish [32P]phosphatidic acid formation elicited by either thrombin alone or thrombin in combination with EP171. The serine protease, trypsin, stimulated an increase in [32P]phosphatidic acid and this effect was additive with that of EP171. This augmentation by trypsin of EP171-induced [32P]phosphatidic acid formation was inhibited by leupeptin. These results are consistent with the concept that thrombin-induced activation of phospholipase C occurs by two distinct mechanisms: one via proteolysis, which is sensitive to leupeptin, and the other via receptor activation, a process shared by EP171. The individual components of this dual mechanism can be mimicked by the co-addition of a receptor-directed agonist (EP171) and a proteolytic agent (trypsin).Key words: platelet, thrombin, proteolysis, phosphoinositide.
Relationships between sensitivity to hydroxyurea and 4-methyl-5-amino-1-formylisoquinoline thiosemicarbazone (MAIQ) and ribonucleotide reductase RNR2 mRNA levels in strains of Saccharomyces cerevisiae
Authors: Rittberg Dawn A. H. , Wright Jim A.
Published: December 2009

Ribonucleotide reductase is responsible for providing the deoxyribonucleotide precursors for DNA synthesis. In most species the enzyme consists of a large and a small subunit, both of which are required for activity. In mammalian cells, the small subunit is the site of action of several antitumor agents, including hydroxyurea and 4-methyl-5-amino-1-formylisoquinoline thiosemicarbazone (MAIQ). The mRNA levels for the small subunit of ribonucleotide reductase (RNR2) and sensitivity to hydroxyurea and MAIQ were determined in four strains of the yeast, Saccharomyces cerevisiae. Two strains exhibited significantly different sensitivities to both hydroxyurea and MAIQ, which closely correlated with differences in the levels of RNR2 mRNA. These results are consistent with recent observations with mammalian cells in culture, and indicate that a common mechanism of resistance to hydroxyurea and related drugs occurs through the elevation in ribonucleotide reductase message levels. A transplason mutagenized strain with marked structural modifications in RNR2 DNA and mRNA showed an extreme hypersensitivity to hydroxyurea but not to MAIQ, providing evidence that the two drugs do not inhibit the RNR2 subunit by the same mechanism. In addition, a yeast strain isolated for low but reproducible resistance to MAIQ exhibited a sensitivity to hydroxyurea similar to the parental wild-type strain, supporting the idea that the two drugs inhibit the activity of RNR2 by unique mechanisms. These yeast strains provide a useful approach for further studies into the regulation of eucaryotic ribonucleotide reduction and drug resistance mechanisms involving a key rate-limiting step in DNA synthesis.Key words: Saccharomyces cerevisiae, reductase, messenger RNA, hydroxyurea, thiosemicarbazone.
Effects of serum amyloid A protein on lymphocytes, HeLa, and MRC5 cells in culture
Authors: Peristeris Platon , Gaspar Alexandre , Gros Pascal , Laurent Philippe , Bernon Hélène , Bienvenu Jacques
Published: December 2009

A major human acute phase protein, the serum amyloid A protein, has been tested in vitro for its effect on lymphocyte proliferation, the formation of E-stable rosettes, as well as the growth of HeLa and MRC5 cell cultures. Serum amyloid A protein has been found to be markedly inhibitory at 30, 100, 200, and 300 μg/mL, and is a very potent inhibitor of in vitro biological functions.Key words: serum amyloid A protein, lymphocytes, HeLa cells, MRC5 cells.
Immunochemical relatedness of two peroxidase isozymes from peanut cell culture
Authors: Hu Chunfang , Huystee Robert B. van
Published: December 2009

The immunological relationships between a cationic and an anionic peroxidase from peanut cell suspension culture were probed with polyclonal antibodies raised against each and with a monoclonal antibody raised against the cationic isozyme. Partial identity of the two isozymes was established by cross-reactivities of the polyclonal antibodies directed to one peroxidase with the alternate isozyme. Some peptides from Western blots of the partially hydrolysed peroxidases were recognized by both antisera, even though the peptide maps of the two isozymes differed. In contrast, the monoclonal antibody reacted more specifically to the fragments derived from the cationic isozyme. The anti-anionic peroxidase serum was passed over a cationic peroxidase (C.PRX) – Sepharose affinity column to separate antibodies cross-reacting with C.PRX from those specific to the anionic isozyme. Deglycosylation of the cationic and the anionic peroxidases eliminated binding of the antibody fraction eluted from the C.PRX–Sepharose column to the deglycosylated forms of peroxidases. These data suggest (i) that complex structural relationships exist between the cationic and the anionic peanut peroxidases, (ii) that the carbohydrate moieties of the two isozymes are antigenically similar to each other, and (iii) that epitope mapping will help in characterizing further each isoperoxidase.Key words: peroxidase, isozymes, ELISA, immunoblotting, peptide mapping.
Biosynthesis of glycosylated dolichol intermediates and asparagine-linked glycoproteins during the germination of soybean seed embryos
Authors: Rip Jack W. , Williams Joyce A. , Crick Dean C. , Rymerson Robert T. , Carroll Kenneth K.
Published: December 2009

Soybean embryos germinated in the presence of [14C]glucose or [14C]mannose incorporated considerable radioactivity into chloroform–methanol (2:1) (CM) soluble and chloroform–methanol–water (10:10:3) (CMW) soluble and insoluble components. The amount of [14C]mannosyl dolichyl phosphate in CM extracts increased over the 24 h of germination, while their [14C]glucosyl dolichyl phosphate content increased up to about 7 h. Incorporation of glucose exceeded incorporation of mannose by about fivefold. The amount of mannose- and glucose-labeled oligosaccharyl dolichyl pyrophosphates in CMW-soluble extracts increased during germination and, based on Concanavalin A Sepharose binding, never exceeded 2% of the total label incorporated into CM-soluble and CMW-soluble, and CMW-insoluble components combined. Nearly equal amounts of [14C]glucose and [14C]mannose appear in oligosaccharyl dolichyl pyrophosphates in the CMW-soluble fraction. Although the CMW-insoluble component contained the largest single amount of radioactivity from either sugar, much of it was not in protein. The protein content of embryos increased during germination, as did the incorporation of [14C]mannose into concanavalin A binding and nonbinding proteins. Microsomes isolated from nongerminated and germinated embryos contained (i) a dolichol kinase, (ii) glycosyl transferases required for the formation of oligosaccharyl dolichyl pyrophosphates, and (iii) a phosphatase which acts on dolichyl phosphate. Kinase activity doubled in 20 h, while phosphatase activity remained at the relatively high initial level. Activity of all three glycosyl transferases was highest after 2 h of germination, but decreased back to zero-time levels by 20 h. The N-acetylglucosaminyl, mannosyl, and glucosyl transferase activities were initially present in a ratio of about 1:10:40 and this ratio did not change appreciably over the period of observation.Key words: dolichol, dolichyl phosphate, glycosyl transferases, oligosaccharyl dolichyl pyrophosphate, soybean seed embryo germination.
Studies on the ATP requirements of in vitro chromatin assembly
Authors: Kmiec Eric B. , Sekiguchi JoAnn M. , Cole Allyson D.
Published: December 2009

To gain a more complete understanding of the process of in vitro chromatin assembly, an examination of the energy requirements of nucleosome formation must be undertaken. The experiments outlined in this manuscript address this issue by making use of the Xenopus laevis S-150 cell-free extract. The S-150 catalyzes chromatin assembly on circular DNA templates dependent either on the exogenous addition of ATP or regeneration of endogenous ATP. We define two distinct, but temporally ordered, phases of the overall process. The first, nucleosome formation, occurs in the presence of endogenous levels of ATP, while the second phase, chromatin assembly, which we define as the development of properly spaced nucleosomes, requires a higher level of ATP. Both phases lead to a distribution of molecules with similar superhelical densities. Taken together, these data suggest that chromatin assembly may consist of two distinct steps differing in their strategy cofactor requirement. The experiments presented in this manuscript support the concept that nucleosomes first assemble, perhaps randomly, on the DNA and are gradually matured into a canonical chromatin structure with periodic spacing.Key words: DNA topoisomerase, chromatin assembly, energy requirements.
Effect of deglycosylation of N-linked sugar chains on glucose oxidase from Aspergillus niger
Authors: Takegawa Kaoru , Fujiwara Kentaro , Iwahara Shojiro , Yamamoto Kenji , Tochikura Tatsurokuro
Published: December 2009

Endo-β-N-acetylglucosaminidase from Flavobacterium sp. released about 30% of the N-linked sugar chains from the glucose oxidase of Aspergillus niger. To elucidate the role of the carbohydrate moiety, the enzymatic properties of native and carbohydrate-depleted glucose oxidases were compared. It was found that their catalytic activities and thermal and pH stabilities were identical. However, the carbohydrate-depleted glucose oxidase was more rapidly precipitated by the addition of trichloroacetic acid and ammonium sulfate than the native enzyme. These results show that the N-linked sugar chains of the glucose oxidase contributed to the high solubility of the enzyme in water.Key words: glucose oxidase, Aspergillus niger, carbohydrate depletion, endo-β-N-acetylglucosaminidase, enzyme stability.
Functional heterogeneity of virgin and memory B murine lymphocytes revealed by the utilization of cyclosporin A: an overview
Authors: Le Moal Mikaël A. , Truffa-Bachi Paolo
Published: December 2009

The effects of cyclosporin A on the generation and revelation of B memory cells by thymus-independent (TI) antigens was investigated. A class 1 (TNP-LPS) and a class 2 (TNP-Ficoll) TI antigens were used for priming and elicitation. Evidence is presented that cyclosporin A does not interfere with the generation of hapten-specific (TNP) B memory cells by TNP-LPS or DNP-Ficoll. Cyclosporin A does not affect the revelation of B memory cells by TNP-LPS, but inhibits their revelation by TNP-Ficoll. These findings are discussed in terms of two distinct B cell lineages leading to antibody-forming cells and memory cells precursors, and in terms of heterogeneity of B memory cells.Key words: cyclosporin A, memory cells, thymus-independent antigens.
Association of acetylated microtubules, vimentin intermediate filaments, and MAP 2 during early neural differentiation in EC cell culture
Authors: Falconer Marcia M. , Vielkind Ursula , Brown David L.
Published: December 2009

Pluripotent P19 embryonal carcinoma cell cultures can be induced to differentiate into neurons and glial cells by the addition of 10−6 M retinoic acid. During early neural differentiation, a bundle of colchicine-stable, acetylated microtubules is formed. This acetylated microtubule array apparently extends to form neurites during neurogenesis. In this paper, we analyze changes in vimentin and MAP 2 distributions during neural differentiation with respect to the changes in the acetylated microtubule array. During a brief period early in differentiation, indirect immunofluorescence staining shows the colocalization of colchicine-stable acetylated microtubules, vimentin, and MAP 2. Using acrylamide to disrupt the organization of vimentin intermediate filaments and estramustine to disrupt the binding of MAP 2 to microtubules, we show that acetylated microtubules, MAP 2, and vimentin intermediate filaments are arranged in an interdependent cytoskeletal array. We suggest this array may serve to stabilize processes in neural stem cells, before the final decision to differentiate into neurons or glia is made.Key words: embryonal carcinoma culture, MAP 2, microtubules, neural differentiation, vimentin.