scholarly journals The mode of action of adenosine 3′:5′-cyclic monophosphate in mammalian islets of Langerhans. Preparation and properties of islet-cell protein phosphokinase

1972 ◽  
Vol 129 (3) ◽  
pp. 551-560 ◽  
Author(s):  
W. Montague ◽  
S. L. Howell
Keyword(s):  
Cyclic Amp ◽  
Islets Of Langerhans ◽  
Islet Cell ◽  
Association Constant ◽  
Cell Protein ◽  
Cyclic Monophosphate ◽  
Endogenous Protein ◽  
Dependent Protein ◽  
Protein Components ◽  
Independent Protein

1. A protein was demonstrated in mammalian islets of Langerhans that after purification appeared as a single component possessing both cyclic-AMP (adenosine 3′:5′-cyclic monophosphate)-binding and cyclic-AMP-dependent protein phosphokinase activities. 2. The protein had an intrinsic association constant for cyclic AMP of 1.15×10−8m, which was similar to the Km for cyclic AMP (1.11×10−8m) of the protein phosphokinase activity. 3. Incubation of the protein in the presence of cyclic AMP resulted in its dissociation into cyclic-AMP-independent protein phosphokinase (catalytic) and cyclic-AMP-binding (receptor) subunits, which could be separated on Sephadex G-200. 4. The cyclic-AMP-dependent protein phosphokinase was capable of phosphorylating a variety of proteins, the most readily phosphorylated being histone, casein and protein components of sub-cellular fractions prepared from islets of Langerhans. 5. The cyclic-AMP-dependent phosphorylation of histone had a Km for ATP of 1.1×10−5m. 6. The endogenous protein phosphokinase activity in rat islets incubated with agents that are known to alter the intracellular concentration of cyclic AMP was investigated. Theophylline and 3-isobutyl-1-methylxanthine, agents that raise cyclic AMP concentrations in islets, increased the activity of the protein phosphokinase, whereas adrenaline, which lowers islet cyclic AMP concentrations, decreased its activity. 7. It is suggested that cyclic AMP may exert its effects on insulin release by increasing the activity of a protein phosphokinase and may thereby promote the phosphorylation and activity of a rate-determining component of the secretory mechanism.

scholarly journals Glucose-stimulated protein phosphorylation in the pancreatic islet

1984 ◽  
Vol 220 (2) ◽  
pp. 529-537 ◽  
Author(s):  
J R Colca ◽  
N Kotagal ◽  
P E Lacy ◽  
C L Brooks ◽  
L Norling ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Protein Kinase ◽  
Cyclic Amp ◽  
Islet Cell ◽  
Cell Protein ◽  
Dependent Protein Kinase ◽  
Cell Homogenate ◽  
Dependent Protein ◽  
Cell Cytosol ◽  
Hexose Phosphates

A glucose-dependent phosphorylation of a 68kDa islet-cell protein was observed in islet-cell homogenates. In the presence of [gamma-32P]ATP the protein was phosphorylated only in the presence of alpha-D-glucose; other sugars were ineffective. Activation of the phosphorylation was half-maximal at 0.34 mM-glucose, 7 microM-ATP and 0.3 mM-Mg2+. Although the addition of glucose 6-phosphate in this design did not stimulate phosphorylation of the islet-cell protein, addition of glucose 6-phosphate to the radioactively labelled 68kDa protein rapidly removed (chased) the 32P label. The addition of presynthesized glucose 6-[32P]phosphate phosphorylated the 68kDa band in the islet-cell homogenate and also phosphorylated purified skeletal-muscle phosphoglucomutase. Phosphoglucomutase labelled thus by 32P was indistinguishable from the islet-cell phosphoprotein on electrophoretic gels. The 32P incorporated into both the islet-cell protein and the purified skeletal-muscle phosphoglucomutase was chased similarly by hexose phosphates. The purified phosphoglucomutase could also be phosphorylated by cyclic AMP-dependent protein kinase or by a mannoheptulose-insensitive process by the islet-cell cytosol. The phosphoenzyme formed thus was also dephosphorylated by D-glucose 6-phosphate and alpha-D-glucose 1-phosphate, suggesting that this may be a mechanism for generation of glucose 1,6-bisphosphate.

scholarly journals Protein kinase activities in rat pancreatic islets of Langerhans

1979 ◽  
Vol 180 (1) ◽  
pp. 219-229 ◽  
Author(s):  
M C Sugden ◽  
S J Ashcroft ◽  
P H Sugden
Keyword(s):  
Protein Kinase ◽  
Cyclic Amp ◽  
Islets Of Langerhans ◽  
Kinase Inhibitor ◽  
Protein Kinase Inhibitor ◽  
Dependent Protein Kinase ◽  
Rat Islets ◽  
Dependent Protein ◽  
Rat Islets Of Langerhans ◽  
Independent Protein

1. Protein kinase activities in homogenates of rat islets of Langerhans were studied. 2. On incubation of homogenates with [gamma-32P]ATP, incorporation of 32P into protein occurred: this phosphorylation was neither increased by cyclic AMP nor decreased by the cyclic AMP-dependent protein kinase inhibitor described by Ashby & Walsh [(1972) J. Biol. Chem. 247, 6637–6642]. 3. On incubation of homogenates with [gamma-32P]ATP and histone as exogenous substrate for phosphorylation, incorporation of 32P into protein was stimulated by cyclic AMP (approx. 2.5-fold) and was inhibited by the cyclic AMP-dependent protein kinase inhibitor. In contrast, when casein was used as exogenous substrate, incorporation of 32P into protein was not stimulated by cyclic AMP, nor was it inhibited by the cyclic AMP-dependent protein kinase inhibitor. 4. DEAE-cellulose ion-exchange chromatography resolved four peaks of protein kinase activity. One species was the free catalytic subunit of cyclic AMP-dependent protein kinase, two species corresponded to ‘Type I’ and ‘Type II’ cyclic AMP-dependent protein kinase holoenzymes [see Corbin, Keely & Park (1975) J. Biol. Chem. 250, 218–225], and the fourth species was a cyclic AMP-independent protein kinase. 5. Determination of physical and kinetic properties of the protein kinases showed that the properties of the cyclic AMP-dependent activities were similar to those described in other tissues and were clearly distinct from those of the cyclic AMP-independent protein kinase. 6. The cyclic AMP-independent protein kinase had an s20.w of 5.2S, phosphorylated a serine residue(s) in casein and was not inhibited by the cyclic AMP-dependent protein kinase inhibitor. 7. These studies demonstrate the existence in rat islets of Langerhans of multiple forms of cyclic AMP-dependent protein kinase and also the presence of a cyclic AMP-independent protein kinase distinct from the free catalytic subunit of cyclic AMP-dependent protein kinase. The presence of the cyclic AMP-independent protein kinase may account for the observed characteristics of 32P incorporation into endogenous protein in homogenates of rat islets.

scholarly journals Effects of dehydrouramil on protein phosphorylation and insulin secretion in rat islets of Langerhans

1986 ◽  
Vol 237 (1) ◽  
pp. 191-196 ◽  
Author(s):  
D E Harrison ◽  
M Poje ◽  
B Rocic ◽  
S J H Ashcroft
Keyword(s):  
Insulin Secretion ◽  
Protein Kinase ◽  
Cyclic Amp ◽  
Protein Phosphorylation ◽  
Islets Of Langerhans ◽  
Dependent Protein Kinase ◽  
Tetradecanoylphorbol Acetate ◽  
Endogenous Protein ◽  
Dependent Protein ◽  
Rat Islets Of Langerhans

Dehydrouramil hydrate hydrochloride (DHU), a stable analogue of alloxan, inhibited the phosphorylation of an endogenous protein of Mr 53,000 catalysed by a Ca2+-calmodulin-dependent protein kinase in extracts of islets of Langerhans. The concentration of DHU required for 50% inhibition was 0.09 mM. DHU did not inhibit islet cyclic AMP-dependent protein kinase and caused only slight inhibition of Ca2+-phospholipid-dependent protein kinase. Inhibition of Ca2+-calmodulin-dependent protein kinase was neither prevented nor reversed by dithiothreitol. DHU did not affect the ability of calmodulin to activate cyclic AMP phosphodiesterase. In intact islets, pre-exposure to DHU impaired the insulin-secretory response to glucose and blocked the potentiatory effect on insulin secretion of forskolin, an activator of adenylate cyclase, and of tetradecanoylphorbol acetate (TPA), an activator of Ca2+-phospholipid-dependent protein kinase. The increase in islet cyclic AMP elicited by forskolin was not affected by DHU. The data are consistent with the hypothesis that protein phosphorylation catalysed by a Ca2+-calmodulin-dependent protein kinase may play a central role in the regulation of insulin secretion.

scholarly journals Properties of neurofilament protein kinase

1986 ◽  
Vol 235 (1) ◽  
pp. 283-289 ◽  
Author(s):  
D Toru-Delbauffe ◽  
M Pierre ◽  
J Osty ◽  
F Chantoux ◽  
J Francon
Keyword(s):  
Protein Kinase ◽  
Cyclic Amp ◽  
Kinase Activity ◽  
Specific Activity ◽  
Maximal Activity ◽  
Brain Cell ◽  
Neurofilament Protein ◽  
Dependent Protein ◽  
Protein Components ◽  
Cytoskeletal Elements

Neurofilament (NF) protein kinase, partially purified from NF preparations [Toru-Delbauffe & Pierre (1983) FEBS Lett. 162, 230-234], was found to be distinct from both the casein kinase present in NFs and the cyclic AMP-dependent protein kinase which is able to phosphorylate NFs. NF-kinase phosphorylated the three NF protein components. The amount of phosphate incorporated per molecule was higher for NF 200 than for NF 145 and NF 68. Other proteins present in the NF preparations were also used as NF-kinase substrates. Two of them might correspond to the myelin basic proteins with Mr values of 18,000 and 21,000. Four other substrates in the NF preparation were not identified (respective Mr values 53,000, 55,000, 65,000 and greater than 300,000). NF kinase also phosphorylated two additional brain-cell cytoskeletal elements: GFAp and vimentin. Casein, histones and phosvitin, currently used as substrates for protein kinase assays, were very poor phosphate acceptors. Half-maximal NF-kinase activity was obtained at an NF protein concentration of about 0.25 mg/ml in heated, salt-washed, NF preparations. The specific activity was about 5 pmol of 32P incorporated/min per microgram of NF kinase preparation protein. ATP was a phospho-group donor (Km 8 × 10(-5) M), but GTP was not. NF-kinase activity remained stable at 65 degrees C for more than 1 h. The enzyme was not degraded by storage at −20 degrees C for several months in a buffer containing 50% (w/v) sucrose. Maximal activity was obtained with 5 mM-Mg2+ (Mg2+ could be replaced by Co2+); Zn2+ and Cu2+ inhibited the reaction. NF-kinase was not dependent on cyclic AMP, cyclic GMP, Ca2+ or Ca2+ plus dioleoylglycerol and phosphatidylserine.

scholarly journals Substrates for cyclic AMP-dependent protein kinase in islets of Langerhans. Studies with forskolin and catalytic subunit

1985 ◽  
Vol 227 (3) ◽  
pp. 727-736 ◽  
Author(s):  
M R Christie ◽  
S J Ashcroft
Keyword(s):  
Protein Kinase ◽  
Cyclic Amp ◽  
Insulin Release ◽  
Islets Of Langerhans ◽  
Plasma Membranes ◽  
Sodium Dodecyl ◽  
Catalytic Subunit ◽  
Subcellular Fractions ◽  
Dependent Protein Kinase ◽  
Dependent Protein

To investigate substrates for cyclic AMP-dependent protein kinase in intact islets of Langerhans, batches of islets were incubated with [32P]Pi for 1 h in the presence of 10 mM-glucose; the adenylate cyclase activator forskolin, which in parallel experiments was shown to increase islet cyclic AMP content and insulin release, was then added. Islets were homogenized and subcellular fractions prepared by differential centrifugation. Phosphopeptides were electrophoresed on sodium dodecyl sulphate/polyacrylamide gels and quantified by autoradiography and densitometry. Within 5 min forskolin caused increased labelling of Mr-25 000 and −30 000 cytosolic and Mr-23 000 and −32 000 particulate peptides; a rapid decrease in phosphorylation of Mr-18 000 and −34 000 cytosolic peptides was also observed. In addition, rather slower phosphorylation occurred of the Mr-15 000 peptide previously identified as histone H3 [Christie & Ashcroft (1984) Biochem. J. 218, 87-99]. When similar subcellular fractions were incubated with [gamma-32P]ATP and purified catalytic subunit of cyclic AMP-dependent protein kinase, peptides phosphorylated included cytosolic species of Mr 25 000 and 30 000 and particulate species of Mr 23 000 and 32 000. The distribution of RNA in the subcellular fractions suggested that the Mr-32 000 species could be a ribosomal protein. The 24 000 g pellet was heterogeneous, as judged by marker assays, and was therefore fractionated further by Percoll-density-gradient centrifugation. The peak containing the Mr-23 000 peptide was resolved from marker enzymes for plasma membranes, mitochondria and endoplasmic reticulum and coincided with a peak for insulin: hence the Mr-23 000 peptide is likely to be a secretory-granule component. The study demonstrates that the potentiation of insulin release that occurs when islet cyclic AMP is increased is accompanied by rapid phosphorylation of specific islet substrates for cyclic AMP-dependent protein kinase. The data are consistent with the hypothesis that protein phosphorylation is involved in the regulation of insulin secretion.

REGULATION OF REPRESSIBLE ACID PHOSPHATASE BY CYCLIC AMP IN SACCHAROMYCES CEREVISIAE

Genetics ◽  
1984 ◽  
Vol 108 (1) ◽  
pp. 53-66 ◽  
Author(s):  
Kunihiro Matsumoto ◽  
Isao Uno ◽  
Tatsuo Ishikawa
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Acid Phosphatase ◽  
Protein Kinase ◽  
Cyclic Amp ◽  
Adenylate Cyclase ◽  
Dependent Protein Kinase ◽  
Dependent Protein ◽  
Low Levels ◽  
Constitutive Synthesis ◽  
Independent Protein

ABSTRACT One of the cyr1 mutants (cyr1-2) in yeast produced low levels of adenylate cyclase and cyclic AMP at 25° and was unable to derepress acid phosphatase. Addition of cyclic AMP to the cyr1-2 cultures elevated the level of repressible acid phosphatase activity. The bcy1 mutation, which suppresses the cyr1-2 mutation by allowing activity of a cyclic AMP-independent protein kinase, also allows acid phosphatase synthesis without restoring adenylate cyclase activity. The CYR3 mutant had structurally altered cyclic AMP-dependent protein kinase and was unable to derepress acid phosphatase. The cyr1 locus was different from pho2, pho4 and pho81, which were known to regulate acid phosphatase synthesis. Mutants carrying cyr1-2 and pho80, PHO81c, PHO82 or pho85 mutations, which confer constitutive synthesis of repressible acid phosphatase, produced acid phosphatase. The cyr1-2 mutant produced significantly low levels of invertase and α- d-glucosidase. These results indicated that cyclic AMP-dependent protein kinase exerts its function in the synthesis of repressible acid phosphatase and other enzymes.

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