scholarly journals Counter-regulation by insulin and isoprenaline of a prominent fat-associated phosphoprotein doublet in rat adipocytes

1991 ◽  
Vol 274 (2) ◽  
pp. 433-438 ◽  
Author(s):  
R A Mooney ◽  
K L Bordwell
Keyword(s):  
Cyclic Amp ◽  
Metabolic Regulation ◽  
Sequence Similarity ◽  
Subcellular Fractionation ◽  
Hormonal Control ◽  
Common Mechanism ◽  
Absolute Increase ◽  
Rat Adipocytes ◽  
32P Incorporation ◽  
Close Sequence

1. In the adipocyte, phosphorylation/dephosphorylation of regulatory proteins is a common mechanism of metabolic regulation. We have observed a very prominent phosphoprotein doublet of 61 kDa and 63 kDa in rat adipocytes that is markedly responsive to hormones. The 63 kDa band was the predominant phosphoprotein in the cell in response to 0.1 microM-isoprenaline, whereas the 61 kDa band was nearly absent. Insulin alone did not alter 32P incorporation into the doublet, but partially counteracted the effects of isoprenaline, decreasing label in the 63 kDa band by as much as 50% and resulting in the reappearance of the 61 kDa band. 2. Subcellular fractionation demonstrated that both phosphoprotein bands were fat-associated. Neither insulin nor isoprenaline altered this localization. Peptide maps (one-dimensional) of the 61/63 kDa bands demonstrated close sequence similarity. Amino acid analysis revealed the presence of phosphoserine and phosphothreonine. The latter was more prominent in the 61 kDa band. Isoprenaline caused an absolute increase in both phosphoamino acids. 3. Permeabilization of 32P-labelled isoprenaline-treated cells with digitonin initiated rapid dephosphorylation of the 63 kDa band, with reappearance of the 61 kDa band. Insulin increased the rate of dephosphorylation by 2-3-fold when present with isoprenaline before permeabilization. 4. In permeabilized adipocytes, cyclic AMP (1 microM-1 mM) increased phosphorylation of the 61/63 kDa doublet by 4-10-fold in the presence of [gamma-32P]ATP, but insulin had no effect. 5. We conclude that this prominent phosphoprotein, migrating as a 61/63 kDa doublet, is coupled to the cyclic AMP-dependent protein kinase and is associated with an insulin-stimulated phosphoprotein phosphatase activity. This fat-associated phosphoprotein, which is under counter-regulatory hormonal control, may play a role in hormone-dependent lipid metabolism.

Possible Roles for Protein Phosphorylation in Platelet Responses

1979 ◽  
Author(s):  
R.J. Haslam ◽  
J.E.B. Fox ◽  
S.E. Salama ◽  
J.A. Lynham
Keyword(s):  
Cyclic Amp ◽  
Protein Phosphorylation ◽  
Protein Kinases ◽  
Platelet Function ◽  
Subcellular Fractionation ◽  
Human Platelets ◽  
Type I ◽  
Sds Page ◽  
Dependent Protein ◽  
32P Incorporation

The relationships between the phosphorylation of specific platelet polypeptides and platelet function were studied using washed human platelets labelled by preincubation with [32p] Pi. Platelet polypeptides were separated by SDS-PAGE and 32P incorporation into them determined by autoradiography. Whereas induction of platelet aggregation alone did not affect protein phosphorylation, induction of the release reaction increased 3P incorporation into several polypeptides (P75,P47,P40,P27,P20,P19), including the P-light chain of platelet myosin (P20). These changes were inhibited by drugs that blocked Ca2 movements and may be due to activation of Ca2+-dependent protein kinases. Compounds that inhibited platelet function by increasing cyclic AMP (e.g. PCE1) also suppressed these reactions but, in addition, increased phosphorylation of other polypeptides (P50,P49,P36,P24,P22). Type I and Type II cyclic AMP-dependent protein kinases were present in platelets and may mediate Che latter effects of cyclic AMP. Subcellular fractionation of 32p-labelled platelets that had been exposed to PCE1 showed that P24 was present in membranes that could take up Ca2+ by an ATP-dependent mechanism. Membranes from PCE1-treated platelets took up Ca2+ more rapidly than control membranes. Thus, the cyclic AMP-dependent phosphorylation of P24 may stimulate the removal of Ca2+ from platelet cytosol and suppress Ca2+-dependent phosphorylation reactions necessary for release of granule constituents.

scholarly journals Reversible inactivation by noradrenaline of long-chain fatty acyl-CoA synthetase in rat adipocytes

1985 ◽  
Vol 226 (1) ◽  
pp. 275-282 ◽  
Author(s):  
M Hall ◽  
E D Saggerson
Keyword(s):  
Cyclic Amp ◽  
Microsomal Protein ◽  
Subcellular Fractionation ◽  
Fatty Acyl ◽  
Reversible Inactivation ◽  
Rat Adipocytes ◽  
Wide Range ◽  
Dependent Protein ◽  
Triton X ◽  
Subsequent Addition

Incubation of rat adipocytes with the same range of noradrenaline concentrations that stimulate lipolysis caused a rapid and stable decrease in the activity of fatty acyl-CoA synthetase. Corticotropin, glucagon and dibutyryl cyclic AMP also decreased the activity of the enzyme. The effect of noradrenaline was apparent over a wide range of concentrations for the three substrates of the enzyme. A novel fluorescence assay of fatty acyl-CoA synthetase using (1,N6-etheno)-CoA is described. The effect of noradrenaline was not abolished by inclusion of albumin in homogenization buffers, persisted through subcellular fractionation and isolation of microsomes (microsomal fractions) and even survived treatment of microsomes with Triton X-100. The effect of noradrenaline was rapidly reversed within cells by the subsequent addition of insulin or propranolol. The inclusion of fluoride in homogenization buffers did not alter the observed effect of noradrenaline. Additions of cyclic AMP-dependent protein kinase to adipocyte microsomes caused considerable phosphorylation of microsomal protein by [gamma-32P]ATP, but did not affect the activity of fatty acyl-CoA synthetase.

scholarly journals Characterization and metabolic regulation of a liver-specific 5.4-kilobase mRNA whose synthesis is transcriptionally induced by carbohydrates and repressed by glucagon and cyclic AMP

1985 ◽  
Vol 226 (3) ◽  
pp. 637-644 ◽  
Author(s):  
A L Pichard ◽  
A Munnich ◽  
M C Meienhofer ◽  
S Vaulont ◽  
M P Simon ◽  
...  
Keyword(s):  
Cyclic Amp ◽  
Metabolic Regulation ◽  
Hormonal Control ◽  
Control Of Gene Expression ◽  
Transcription System ◽  
Rna Transcripts ◽  
Eukaryotic Genes ◽  
Liver Cdna Library ◽  
Protein Rich

Four clones derived from a carbohydrate-induced rat liver cDNA library were found to hybridize with a 5.4-kilobase mRNA species encoding a 36 kDa protein. This mRNA was abundant in the liver, barely detectable in adipocytes and kidney, and absent from the other tissues tested. In the liver, the mRNA was fully induced by a carbohydrate-rich diet, but was undetectable during both starvation and feeding with a protein-rich or lipid-rich diet. Adrenalectomized, thyroidectomized and diabetic animals did not express the mRNA in their liver when re-fed with the carbohydrate-rich diet. When these animals were given the missing hormone, the amount of hybridizable RNA returned to normal values, but administration of the hormone alone failed to induce mRNA synthesis in starved animals. Both glucagon and its second messenger, cyclic AMP, abolished the induction of the mRNA in re-fed animals. Exogenous insulin, whatever the dose, did not reverse the inhibitory action of glucagon. In an isolated nuclei transcription system, no detectable RNA transcripts were found in starved animals, whereas feeding the animals with the carbohydrate-rich diet led to a maximum rate of gene transcription. Although unidentified, this mRNA proves to be a remarkable marker of dietary and hormonal control of gene expression in vivo. It will provide a useful model for further analysis of the role of cyclic AMP in regulating the transcription of eukaryotic genes.

scholarly journals Ca2+-induced insulin secretion from electrically permeabilized islets. Loss of the Ca2+-induced secretory response is accompanied by loss of Ca2+-induced protein phosphorylation

1992 ◽  
Vol 285 (3) ◽  
pp. 973-978 ◽  
Author(s):  
P M Jones ◽  
S J Persaud ◽  
S L Howell
Keyword(s):  
Protein Kinase C ◽  
Insulin Secretion ◽  
Protein Kinase ◽  
Cyclic Amp ◽  
Protein Phosphorylation ◽  
Prolonged Exposure ◽  
Kinase C ◽  
Dependent Protein ◽  
32P Incorporation ◽  
Rat Islets Of Langerhans

Increasing the cytosolic Ca2+ concentration of electrically permeabilized rat islets of Langerhans caused rapid increases in insulin secretion and in 32P incorporation into islet proteins. However, the secretory responsiveness of permeabilized islets was relatively transient, with insulin secretion approaching basal levels within 20-30 min despite the continued presence of stimulatory concentrations of Ca2+. The loss of Ca2(+)-induced insulin secretion was accompanied by a marked reduction in Ca2(+)-dependent protein phosphorylation, but not in cyclic AMP-dependent protein phosphorylation. Similarly, permeabilized islets which were no longer responsive to Ca2+ were able to mount appropriate secretory responses to cyclic AMP and to a protein kinase C-activating phorbol ester. These results suggest that prolonged exposure to elevated cytosolic Ca2+ concentrations results in a specific desensitization of the secretory mechanism to Ca2+, perhaps as a result of a decrease in Ca2(+)-dependent kinase activity. Furthermore, these studies suggest that secretory responses of B-cells to cyclic AMP and activators of protein kinase C are not dependent upon the responsiveness of the cells to changes in cytosolic Ca2+.

THE EFFECTS OF MANDUCA SEXTA DIURETIC HORMONE ON FLUID TRANSPORT BY THE MALPIGHIAN TUBULES AND CRYPTONEPHRIC COMPLEX OF MANDUCA SEXTA

1993 ◽  
Vol 178 (1) ◽  
pp. 231-243 ◽  
Author(s):  
N. Audsley ◽  
G. M. Coast ◽  
D. A. Schooley
Keyword(s):  
Cyclic Amp ◽  
Manduca Sexta ◽  
Fluid Transport ◽  
Basal Membrane ◽  
Fluid Secretion ◽  
Hormonal Control ◽  
Malpighian Tubules ◽  
Proximal Tubules ◽  
Fluid Absorption ◽  
Diuretic Hormone

1. Manduca sexta diuretic hormone (Mas-DH) stimulates fluid secretion by adult Malpighian tubules of M. sexta, demonstrating its site of diuretic action in M. sexta for the first time. It was not possible to develop a suitable bioassay to measure fluid secretion in larval proximal tubules. 2. Mas-DH has an antidiuretic action on the cryptonephric complex of larval M. sexta because it increases fluid absorption from the rectum. It appears that in this complex Mas-DH is acting on a Na+/K+/2Cl- co-transporter, presumably on the basal membrane of the cryptonephric Malpighian tubules, because Mas-DH-stimulated fluid absorption by the cryptonephric complex is inhibited by bumetanide or the removal of Cl-, Na+ or K+ from the haemolymph side of the tissue. This is the first demonstration of hormonal control of fluid absorption by the cryptonephric complex. 3. Concomitant with the stimulation of fluid transport, Mas-DH increases the amount of cyclic AMP secreted by adult Malpighian tubules and the cryptonephric complex. In addition, Mas-DH promotes cyclic AMP production by the larval proximal tubules.

Adaptations to a terrestrial existence in the robber crab Birgus latro L. IX. Hormonal control of post-renal urine reprocessing and salt balance in the branchial chamber

2000 ◽  
Vol 203 (2) ◽  
pp. 389-396 ◽  
Author(s):  
S. Morris ◽  
P. Greenaway ◽  
A.M. Adamczewska ◽  
M.D. Ahern
Keyword(s):  
Cyclic Amp ◽  
Ion Transport ◽  
Fresh Water ◽  
Sea Water ◽  
Inhibitory Effect ◽  
Hormonal Control ◽  
Uptake System ◽  
Dietary Salt ◽  
Artificial Urine ◽  
Brachyuran Crabs

The terrestrial robber crab Birgus latro L. regulates the composition of its final excretory product (termed P) depending on the availability of dietary salt by reabsorbing ions from urine passed over the gills. Laboratory and field-based studies investigated the nature of the mechanisms of control of this branchial ion uptake. B. latro were prepared such that their branchial chambers could be perfused with artificial urine, and the rate of ion transport from the artificial urine was determined. For B. latro acclimated to drinking fresh water, the rates of Na(+) and Cl(−) uptake were more than four times those of crabs drinking 70 % sea water. Crabs were injected with either saline carrier or the same solution containing either dopamine or dibutyryl cyclic AMP (db-cAMP) (final concentration 8.7×10(−)(7)mol l(−)(1)haemolymph). Dopamine and db-cAMP inhibited Na(+) and Cl(−) uptake in animals acclimated to fresh water and markedly reduced their gill Na(+)/K(+)-ATPase activity. Dopamine stimulated the production of cyclic AMP within the branchial epithelial cells. Dopamine, released from the pericardial organs, acts as a primary messenger, and cyclic AMP acts as a second messenger most likely promoting phosphorylation of membrane proteins. In contrast to aquatic brachyuran crabs, ion transport in B. latro, an anomuran, is controlled via an inhibitory effect. Terrestrial crabs normally have access to fresh water and must salvage salt from their urine, and a mechanism to down-regulate a normally active uptake system seems more appropriate to their ecology. Whether the control is stimulatory or inhibitory in the various air-breathing crabs may depend on the osmoregulatory abilities of their aquatic ancestors, but in either case has significant implications for the evolution of crustaceans to life on land. Further work must establish whether terrestrial brachyuran crabs are similar to B. latro and whether this crab is unique amongst the anomuran crabs.

scholarly journals Microbiological Oxidation of Antimony(III) with Oxygen or Nitrate by Bacteria Isolated from Contaminated Mine Sediments

2015 ◽  
Vol 81 (24) ◽  
pp. 8478-8488 ◽  
Author(s):  
Lee R. Terry ◽  
Thomas R. Kulp ◽  
Heather Wiatrowski ◽  
Laurence G. Miller ◽  
Ronald S. Oremland
Keyword(s):  
Sequence Similarity ◽  
Primary Electron ◽  
Nitrate Respiration ◽  
Bacterial Strains ◽  
Bacterial Oxidation ◽  
Terminal Electron Acceptor ◽  
Content Type ◽  
Variovorax Paradoxus ◽  
Microbiological Oxidation ◽  
Close Sequence

ABSTRACTBacterial oxidation of arsenite [As(III)] is a well-studied and important biogeochemical pathway that directly influences the mobility and toxicity of arsenic in the environment. In contrast, little is known about microbiological oxidation of the chemically similar anion antimonite [Sb(III)]. In this study, two bacterial strains, designated IDSBO-1 and IDSBO-4, which grow on tartrate compounds and oxidize Sb(III) using either oxygen or nitrate, respectively, as a terminal electron acceptor, were isolated from contaminated mine sediments. Both isolates belonged to theComamonadaceaefamily and were 99% similar to previously described species. We identify these novel strains asHydrogenophagataeniospiralisstrain IDSBO-1 andVariovorax paradoxusstrain IDSBO-4. Both strains possess a gene with homology to theaioAgene, which encodes an As(III)-oxidase, and both oxidize As(III) aerobically, but only IDSBO-4 oxidized Sb(III) in the presence of air, while strain IDSBO-1 could achieve this via nitrate respiration. Our results suggest that expression ofaioAis not induced by Sb(III) but may be involved in Sb(III) oxidation along with an Sb(III)-specific pathway. Phylogenetic analysis of proteins encoded by theaioAgenes revealed a close sequence similarity (90%) among the two isolates and other known As(III)-oxidizing bacteria, particularlyAcidovoraxsp. strain NO1. Both isolates were capable of chemolithoautotrophic growth using As(III) as a primary electron donor, and strain IDSBO-4 exhibited incorporation of radiolabeled [14C]bicarbonate while oxidizing Sb(III) from Sb(III)-tartrate, suggesting possible Sb(III)-dependent autotrophy. Enrichment cultures produced the Sb(V) oxide mineral mopungite and lesser amounts of Sb(III)-bearing senarmontite as precipitates.

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