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The Journal of Biological Chemistry Dec 1983The mechanism of the metabolic inactivation of leukotrienes C4, D4, and E4 by human polymorphonuclear leukocytes activated by phorbol myristate acetate has been analyzed...
The myeloperoxidase-dependent metabolism of leukotrienes C4, D4, and E4 to 6-trans-leukotriene B4 diastereoisomers and the subclass-specific S-diastereoisomeric sulfoxides.
The mechanism of the metabolic inactivation of leukotrienes C4, D4, and E4 by human polymorphonuclear leukocytes activated by phorbol myristate acetate has been analyzed with the use of activated cells, their supernatants, the isolated components of the myeloperoxidase system, and chemically synthesized hypochlorous acid (HOC1). The metabolic products were resolved by reverse-phase high performance liquid chromatography in one or more solvent systems, and each product was characterized relative to chemically synthesized standards by its reverse-phase high performance liquid chromatography retention time, UV absorption spectrum, nonvascular smooth muscle spasmogenic activity, and immunoreactivity. The phorbol myristate acetate-activated human polymorphonuclear leukocytes converted each of the sulfidopeptide leukotrienes to products identical with synthetic diastereoisomers of 6-trans-leukotriene B4 by the following criteria: retention times in two solvent systems, lambda max of 269 nm with shoulders at 259 and 279 nm, and mass spectral analysis. Each of the sulfidopeptide leukotrienes was simultaneously converted to subclass-specific S-diastereoisomeric sulfoxides which co-chromatographed with synthetic standards, exhibited a bathochromic shift to a lambda max at 284 nm, were fully immunoreactive, and possessed less than 5% spasmogenic activity of the corresponding sulfidopeptide leukotrienes. When the sulfidopeptide leukotrienes were incubated with supernatants of phorbol myristate acetate-activated cells in the presence of 0.1 mM H2O2, with a mixture of 100 milliunits of myeloperoxidase, 0.1 mM H2O2, and 10 mM Cl-, or with chemically prepared HOCl, the metabolic products formed were the same as those obtained with the activated cells, indicating that extracellular inactivation was due to HOCl produced by the action of released myeloperoxidase.
Topics: Cell-Free System; Chromatography, High Pressure Liquid; Humans; Leukotriene B4; Leukotriene E4; Neutrophils; Peroxidase; Peroxidases; SRS-A; Stereoisomerism; Sulfoxides; Tetradecanoylphorbol Acetate
PubMed: 6317683
DOI: No ID Found -
The Journal of Biological Chemistry Nov 1981To determine the relative contributions of glucose, insulin, dexamethasone, and triiodothyronine to the induction of hepatic glucose-6-phosphate dehydrogenase,...
To determine the relative contributions of glucose, insulin, dexamethasone, and triiodothyronine to the induction of hepatic glucose-6-phosphate dehydrogenase, hepatocytes isolated from normal or adrenalectomized rats, either fasted or fed, were examined in culture. Addition of insulin (42 milliunits/ml, 0.9 microM) and dexamethasone (1 microM) to hepatocytes obtained from 3-day-fasted rats and cultured for 48 h in serum-free Dulbecco's medium resulted in a 7- to 11-fold increase in Glc-6-P dehydrogenase specific activity compared with a 2- to 3-fold increase in activity in control cultures incubated without added hormones. The effects of insulin and dexamethasone were independent of DNA synthesis, dose-dependent, and additive; each contributing about one-half of the total response. Medium glucose was neither sufficient nor necessary for the insulin- or dexamethasone-stimulated increase in Glc-6-P dehydrogenase specific activity. Addition of triiodothyronine (10 microM) preferentially blocked the dexamethasone-stimulated increase in Glc-6-P dehydrogenase specific activity. Insulin failed to stimulate the induction of Glc-6-P dehydrogenase in hepatocytes obtained from normal fed rats or from fasted and fed adrenalectomized rats. However, insulin caused a significant increase in the Glc-6-P dehydrogenase specific activity of these cells when dexamethasone was concurrently added to the culture medium.
Topics: Animals; Aphidicolin; Cells, Cultured; Dexamethasone; Diterpenes; Enzyme Induction; Glucosephosphate Dehydrogenase; Insulin; Kinetics; Liver; Male; Rats
PubMed: 6793590
DOI: No ID Found -
The Journal of Cell Biology May 1964Droplets which stain like colloid occur in the cytoplasm of the thyroid follicular epithelium of the rat following stimulation of the gland by thyroid-stimulating...
Droplets which stain like colloid occur in the cytoplasm of the thyroid follicular epithelium of the rat following stimulation of the gland by thyroid-stimulating hormone (TSH). The occurrence of droplets was remarkably reduced when the lumen became depleted of colloid. Acid phosphatase and esterase were localized in the thyroid droplets and, in addition, in granules largely around the nucleus. Stimulation by TSH resulted in an increase in the number of droplets containing enzyme. Twenty-four hours after hypophysectomy, enzyme-associated granules were localized at the basal end of the cell and droplets were absent. Intravenous injection of TSH resulted in formation of droplets at the apical end of the cell and migration of enzyme-associated granules toward the apical end of the cell. The droplets were first observed approximately 10 minutes after TSH administration and at this time did not appear to contain enzyme. Within 15 minutes many droplets contained enzyme. The granules were largely localized near the nucleus on its apical side 30 minutes after a dose of 25 milliunits of TSH, but were less well localized following one-tenth this dose. These results indicate that the epithelial cell of the thyroid gland contains preformed hydrolytic enzymes associated with granules (lysosomes). When the gland is stimulated by TSH, droplets are formed from colloid derived from the lumen (phagosomes), and hydrolytic enzymes are transferred from granules to the droplets. The droplets may be intracellular organelles for hydrolysis of colloid and liberation of thyroxine prior to the release of thyroxine into the blood.
Topics: Acid Phosphatase; Colloids; Cytoplasm; Cytoplasmic Granules; Epithelium; Esterases; Histological Techniques; Hydrolases; Hypophysectomy; Lysosomes; Pharmacology; Rats; Research; Thyroid Gland; Thyrotropin; Thyroxine
PubMed: 14153481
DOI: 10.1083/jcb.21.2.191 -
Journal of Biochemistry Sep 1992Clofibrate increased oligomycin-resistant ATPase activity in peroxisomes more than 17-fold (5.15 +/- 0.71 milliunits/mg protein) in rat liver. The activity was dependent...
Clofibrate increased oligomycin-resistant ATPase activity in peroxisomes more than 17-fold (5.15 +/- 0.71 milliunits/mg protein) in rat liver. The activity was dependent on divalent cations (Mg2+ > Ca2+) with an optimum pH of 7.5. This activity was partially inhibited by N-ethylmaleimide (NEM), 4,4'-dithiocyanatostilbene-2,2'-disulfonic acid (DIDS), silicotungstic acid (STA), and high concentrations of N,N'-dicyclohexylcarbodiimide (DCCD). Proteinase K digestion of intact peroxisomes severely reduced the NEM-sensitive activity, but little affected the NEM-resistant activity. NEM-sensitive and -resistant ATPases showed Km values for ATP of 780 and 73 microM, respectively. The NEM-sensitive activity was inhibited completely by DIDS, 7-chloro-4- nitrobenzo-2-oxa-1,3-diazole (NBD-Cl), tributyltin chloride (TBT), and quercetin, and partially by DCCD and STA, whereas the NEM-resistant activity was totally insensitive to these chemicals except for STA. These activities had unique requirements for divalent cations, anions, and substrates, respectively. They were partially separated by gel filtration chromatography and had molecular masses of 520 kDa (NEM-sensitive enzyme) and 450 kDa (NEM-resistant enzyme), respectively.
Topics: Adenosine Triphosphatases; Animals; Cations; Clofibrate; Drug Resistance; Endopeptidase K; Enzyme Induction; Ethylmaleimide; In Vitro Techniques; Kinetics; Liver; Male; Microbodies; Rats; Rats, Wistar; Serine Endopeptidases; Substrate Specificity
PubMed: 1429526
DOI: 10.1093/oxfordjournals.jbchem.a123908 -
Journal of Food Protection Jul 2011Alkaline phosphatase is a ubiquitous milk enzyme that historically has been used to verify adequate pasteurization of milk for public health purposes. Current approved...
Alkaline phosphatase is a ubiquitous milk enzyme that historically has been used to verify adequate pasteurization of milk for public health purposes. Current approved methods for detection of alkaline phosphatase in milk include the use of enzyme photoactivated substrates to give readings in milliunits per liter. The U.S. and European public health limit for alkaline phosphatase in pasteurized drinks is 350 mU/liter. A modified chemiluminescent method, fast alkaline phosphatase, was compared with the approved fluorometric and chemiluminescent alkaline phosphatase methods to determine whether the modified method was equivalent to the approved methods and suitable for detecting alkaline phosphatase in milk. Alkaline phosphatase concentrations in cow's, goat's, and sheep's milk and in flavored drinks and cream were determined by three methods. Evaluations in each matrix were conducted with pasteurized samples spiked with raw milk to produce alkaline phosphatase concentrations of 2 to 5,000 mU/liter. The tests were performed by the method developer and then reproduced at a laboratory at the National Center for Food Safety and Technology following the criteria for a single laboratory validation. The results indicated that the fast alkaline phosphatase method was not significantly different from the approved chemiluminescent method, with a limit of detection of 20 to 50 mU/liter in all the studied matrices. This modified chemiluminescent method detects alkaline phosphatase in the 350 mU/liter range with absolute differences from triplicate data that are lower and within the range of the allowed intralaboratory repeatability values published for the approved chemiluminescent method.
Topics: Alkaline Phosphatase; Animals; Cattle; Consumer Product Safety; Dairy Products; Food Contamination; Food Handling; Goats; Humans; Luminescent Measurements; Milk; Public Health; Reproducibility of Results; Sensitivity and Specificity; Sheep; Species Specificity
PubMed: 21740717
DOI: 10.4315/0362-028X.JFP-10-422 -
Proceedings of the National Academy of... Nov 1994In contrast to most vertebrate species that possess one oxytocin-like hormone and one vasopressin-like hormone, a few groups, such as marsupials or cartilaginous fishes,... (Comparative Study)
Comparative Study
Special evolution of neurohypophysial hormones in cartilaginous fishes: asvatocin and phasvatocin, two oxytocin-like peptides isolated from the spotted dogfish (Scyliorhinus caniculus).
In contrast to most vertebrate species that possess one oxytocin-like hormone and one vasopressin-like hormone, a few groups, such as marsupials or cartilaginous fishes, are endowed with two peptides of either or both types, suggesting possible gene duplications. We have now isolated two oxytocin-like hormones from the pituitary of the spotted dogfish Scyliorhinus caniculus (suborder Galeoidei). Microsequencing as well as chromatographic and pharmacological comparisons with synthetic peptides show that these peptides are [Asn4,Val8]oxytocin (asvatocin) and [Phe3,Asn4,Val8]-oxytocin (phasvatocin). Asvatocin and phasvatocin display oxytocic activity on rat uterus, about 80 and 5 milliunits per nmol, respectively, and virtually no pressor activity on anesthetized rats. They occur in roughly equal molar amounts in the gland; vasotocin is also present in a proportional amount that is lower by about a factor of 20. In addition to the duality, conservative amino acid substitutions are observed in the two oxytocic peptides in positions 4 (Gln-4-->Asn) and 8 (Leu-8-->Val), when compared with oxytocin. Furthermore, replacement of the isoleucine residue found in position 3 of all other oxytocin-like hormones by phenylalanine in phasvatocin is exceptional; it determines a dramatic decrease of the oxytocic activity. Preservation of the C-terminal-amidated nonapeptide pattern in the 12 vertebrate neurohypophysial hormones known to date suggests that both precursors and processing enzymes have coevolved tightly. On the other hand, whereas the great evolutionary stability of the mature hormones (generally observed in vertebrates) suggests a strict messenger-receptor coevolution, the exceptional diversity found in cartilaginous fishes (six oxytocin-like peptides identified out of eight known) might be due to a looseness of selective constraints, perhaps in relationship with their specific urea osmoregulation.
Topics: Amino Acid Sequence; Animals; Biological Evolution; Dogfish; Molecular Sequence Data; Oxytocin; Pituitary Gland, Posterior
PubMed: 7972045
DOI: 10.1073/pnas.91.23.11266 -
The Journal of Biological Chemistry Jun 1976A reversible interconversion of two kinetically distinct forms of hepatic pyruvate kinase regulated by glucagon and insulin is demonstrated in the perfused rat liver....
A reversible interconversion of two kinetically distinct forms of hepatic pyruvate kinase regulated by glucagon and insulin is demonstrated in the perfused rat liver. The regulation does not involve the total enzyme content of the liver, but rather results in a modulation of the substrate dependence. The forms of pyruvate kinase in liver homogenates are distinguished by measurements of the ratio of the enzyme activity at a subsaturating concentration of P-enolpyruvate (1.3 mM) to the activity at a saturating concentration of this substrate (6.6 mM). A low ratio form of pyruvate kinase (ratio between 0.1 and 0.2) is obtained from livers perfused with 10(-7) M glucagon or 0.1 mM adenosine 3':5'-monophosphate (cyclic AMP). A high ratio form of the enzyme is obtained from livers perfused with no hormone (ratio = 0.35 to 0.45). The regulation of pyruvate kinase by glucagon and cyclic AMP occurs within 2 min following the hormone addition to the liver. Insulin (22 milliunits/ml) counteracts the inhibition of pyruvate kinase caused by 5 X 10(-11) M glucagon, but has only a slight influence on the enzyme properties in the absence of the hyperglycemic hormone. The low ratio form of pyruvate kinase obtained from livers perfused with glucagon or cyclic AMP is unstable in liver extracts and will revert to a high ratio form within 10 min at 37 degrees or within a few hours at 0 degrees. Pyruvate kinase is quantitatively precipitated from liver supernatants with 2.5 M ammonium sulfate. This precipitation stabilizes the enzyme and preserves the kinetically distinguishable forms. The kinetic properties of the two forms of rat hepatic pyruvate kinase are examined using ammonium sulfate precipitates from the perfused rat liver. At pH 7.5 the high ratio form of the enzyme has [S]0.5 = 1.6 +/- 0.2 mM P-enolpyruvate (n = 8). The low ratio form of enzyme from livers perfused with glucagon or cyclic AMP has [S]0.5 = 2.5 +/- 0.4 mM P-enolpyruvate (n = 8). The modification of pyruvate kinase induced by glucagon does not alter the dependence of the enzyme activity on ADP (Km is approximately 0.5 mM ADP for both forms of the enzyme). Both forms are allosterically modulated by fructose 1,6-bisphosphate, L-alanine, and ATP. The changes in the kinetic properties of hepatic pyruvate kinase which follow treating the perfused rat liver with glucagon or cyclic AMP are consistent with the changes observed in the enzyme properties upon phosphorylation in vitro by a clyclic AMP-stimulated protein kinase (Ljungström, O., Hjelmquist, G. and Engström, L. (1974) Biochim. Biophys. Acta 358, 289--298). However, other factors also influence the enzyme activity in a similar manner and it remains to be demonstrated that the regulation of hepatic pyruvate kinase by glucagon and cyclic AMP in vivo involes a phosphorylation.
Topics: Adenosine Triphosphate; Alanine; Animals; Cyclic AMP; Fasting; Glucagon; Insulin; Kinetics; Liver; Male; Perfusion; Phosphoenolpyruvate; Pyruvate Kinase; Rats; Time Factors
PubMed: 180008
DOI: No ID Found -
The Journal of Biological Chemistry Jan 1990The purpose of this study was to simultaneously isolate skeletal muscle plasma and microsomal membranes from the hind limbs of male Sprague-Dawley rats perfused either...
The purpose of this study was to simultaneously isolate skeletal muscle plasma and microsomal membranes from the hind limbs of male Sprague-Dawley rats perfused either in the absence or presence of 20 milliunits/ml insulin and to determine the effect of insulin on the number and distribution of glucose transporters in these membrane fractions. Insulin increased hind limb glucose uptake greater than 3-fold (2.4 +/- 0.7 versus 9.2 +/- 1.0 mumol/g x h, p less than 0.001). Plasma membrane glucose transporter number, measured by cytochalasin B binding, increased 2-fold (9.1 +/- 1.0 to 20.4 +/- 3.1 pmol/mg protein, p less than 0.005) in insulin-stimulated muscle while microsomal membrane transporters decreased significantly (14.8 +/- 1.6 to 9.8 +/- 1.4 pmol/mg protein, p less than 0.05). No change in the dissociation constant (Kd approximately 120 nm) was observed. K+-stimulated-p-nitrophenol phosphatase, 5'-nucleotidase, and galactosyltransferase specific activity, enrichment, and recovery in the plasma and microsomal membrane fractions were not altered by insulin treatment. Western blot analysis using the monoclonal antibody mAb 1F8 (specific for the insulin-regulatable glucose transporter) demonstrated increased glucose transporter densities in plasma membranes from insulin-treated hind limb skeletal muscle compared with untreated tissues, while microsomal membranes from the insulin-treated hind limb skeletal muscle had a concomitant decrease in transporter density. We conclude that the increase in plasma membrane glucose transporters explains, at least in part, the increase in glucose uptake associated with insulin stimulation of hind limb skeletal muscle. Our data further suggest that these recruited transporters originate from an intracellular microsomal pool, consistent with the translocation hypothesis.
Topics: 5'-Nucleotidase; Adipose Tissue; Animals; Blotting, Western; Body Weight; Cell Membrane; Cytochalasin B; Electrophoresis, Polyacrylamide Gel; Galactosyltransferases; Glucose; Insulin; Male; Microsomes; Monosaccharide Transport Proteins; Muscles; Organ Size; Rats; Rats, Inbred Strains
PubMed: 2104834
DOI: No ID Found -
The Journal of Biological Chemistry Nov 1988Phosphorylation of pure fructose-6-phosphate,2-kinase:fructose-2,6-bisphosphatase from bovine heart by cAMP-dependent protein kinase and protein kinase C was...
Phosphorylation of myocardial fructose-6-phosphate,2-kinase: fructose-2,6-bisphosphatase by cAMP-dependent protein kinase and protein kinase C. Activation by phosphorylation and amino acid sequences of the phosphorylation sites.
Phosphorylation of pure fructose-6-phosphate,2-kinase:fructose-2,6-bisphosphatase from bovine heart by cAMP-dependent protein kinase and protein kinase C was investigated. The major enzyme form (subunit Mr of 58,000) was rapidly phosphorylated by both cAMP-dependent protein kinase and protein kinase C, incorporating 0.8 and 1.0 mol/mol of subunit, respectively. The rate of phosphorylation of the heart enzyme by cAMP-dependent protein kinase was 10 times faster than that of the rat liver enzyme. The minor enzyme (subunit Mr of 54,000), however, was phosphorylated only by protein kinase C and was phosphorylated much more slowly with a phosphate incorporation of less than 0.1 mol/mol of subunit. Phosphorylation by either cAMP-dependent protein kinase or protein kinase C activated the enzyme, but each phosphorylation affected different kinetic parameters. Phosphorylation by cAMP-dependent protein kinase lowered the Km value for fructose 6-phosphate from 87 to 42 microM without affecting the Vmax, whereas the phosphorylation by protein kinase C increased the Vmax value from 55 to 85 milliunits/mg without altering the Km value. The phosphorylated peptides were isolated, and their amino acid sequences were determined. The phosphorylation sites for both cAMP-dependent protein kinase and protein kinase C were located in a single peptide whose sequence was Arg-Arg-Asn-Ser-(P)-Phe-Thr-Pro-Leu-Ser-Ser-Ser-Asn-Thr(P)-Ile-Arg-Arg-Pro. The seryl residue nearest the N terminus was the residue specifically phosphorylated by cAMP-dependent protein kinase, whereas the threonine residue nearest the C terminus was phosphorylated by protein kinase C.
Topics: Amino Acid Sequence; Animals; Binding Sites; Cattle; Enzyme Activation; Liver; Molecular Sequence Data; Molecular Weight; Myocardium; Phosphofructokinase-2; Phosphoric Monoester Hydrolases; Phosphorylation; Phosphotransferases (Alcohol Group Acceptor); Protein Kinase C; Protein Kinases
PubMed: 2846551
DOI: No ID Found -
The Journal of Biological Chemistry Feb 1986Removal of cell surface sialic acid from adipocytes with neuraminidase inhibits insulin action. Here, we have examined the effects of mild neuraminidase treatment (5...
Removal of cell surface sialic acid from adipocytes with neuraminidase inhibits insulin action. Here, we have examined the effects of mild neuraminidase treatment (5 milliunits/ml, 12 degrees C, 15 min) on insulin receptor structure and function. Neuraminidase treatment sufficient to cause greater than 90% loss of insulin stimulatable lipogenesis had no effect on 125I-insulin binding, tyrosine kinase activity of partially purified insulin receptors, insulin receptor phosphorylation in intact cells, or insulin-induced receptor internalization. However, recycling of the insulin receptor to the plasma membrane was inhibited by 50%. Recycled receptors in neuraminidase-treated cells were unable to mediate insulin action in contrast to recycled receptors from non-neuraminidase-treated cells. Furthermore, when insulin receptors were protected from exposure to neuraminidase, by inducing receptor internalization prior to neuraminidase treatment, the cells were still unable to respond to insulin. Analysis of the alpha and beta subunits of the receptor from neuraminidase-treated cells, affinity-labeled with 125I-insulin, or labeled by autophosphorylation, and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis failed to indicate any changes in the holoreceptor or the individual subunits. This suggests there was no detectable release of sialic acid from the receptor. From this data we conclude that loss of sialic acid from nonreceptor glycoconjugates leads to loss of insulin action and inhibition of receptor recycling. The insulin receptor does not appear to be involved in this inhibitory effect. These findings suggest that an uncharacterized plasma membrane glycoprotein is essential in transmitting the "signal" of insulin binding to the cellular effector system.
Topics: Adipose Tissue; Affinity Labels; Animals; Electrophoresis, Polyacrylamide Gel; Insulin; Male; N-Acetylneuraminic Acid; Neuraminidase; Phosphorylation; Protein-Tyrosine Kinases; Rats; Rats, Inbred Strains; Receptor, Insulin; Sialic Acids; Surface Properties
PubMed: 3512542
DOI: No ID Found