scholarly journals Identification and characterization of Ca2+-phospholipid-dependent protein kinase in rat islets and hamster β-cells

1984 ◽  
Vol 219 (2) ◽  
pp. 547-551 ◽  
Author(s):  
J M Lord ◽  
S J H Ashcroft
Keyword(s):  
Protein Kinase ◽  
Beta Cells ◽  
Kinetic Properties ◽  
Dependent Protein Kinase ◽  
Protein Substrates ◽  
Rat Islets ◽  
Endogenous Protein ◽  
Dependent Protein ◽  
Rat Islets Of Langerhans

We show that extracts of rat islets of Langerhans and of cloned hamster beta-cells (HIT-T15 cells) contain Ca2+-phospholipid-dependent protein kinase and endogenous protein substrates for the kinase. We purified Ca2+-phospholipid-dependent protein kinase from HIT-T15 beta-cells and report here its physical and kinetic properties.

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.

Parathyroid hormone modulates protein kinase in giant cell tumors of human bone

1980 ◽  
Vol 239 (2) ◽  
pp. E144-E149 ◽  
Author(s):  
D. A. Ausiello ◽  
M. Rosenblatt ◽  
J. M. Dayer
Keyword(s):  
Parathyroid Hormone ◽  
Protein Kinase ◽  
Cyclic Amp ◽  
Giant Cell ◽  
Giant Cell Tumors ◽  
Dependent Protein Kinase ◽  
Protein Substrates ◽  
Endogenous Protein ◽  
Dependent Protein ◽  
Tumors Of Bone

The physiological effects of parathyroid hormone (PTH) in bone are mediated at least in part by cyclic AMP. The biochemical events subsequent to this step have not been well characterized in this tissue. Giant cell tumors of bone (GT) increase cyclic AMP in response to PTH. This response can be inhibited by an analogue of bovine PTH, [Nle8, Nle18, Tyr34] bPTH-(3-34) amide (PTH-Inh). Cyclic AMP content and cyclic AMP-dependent protein kinase (cAMP-PK) were assayed in fresh tumors and cells in culture incubated with 1 microgram/ml of bPTH and/or PTH-Inh. PTH fully activated cAMP-PK in GT, and PTH-Inh completely inhibited PTH-stimulated increases in cyclic AMP content and cAMP-PK activity. When endogenous protein substrates were sought for cAMP-PK, three phosphoproteins of 55,000, 43,000, and 38,000 mol wt maximally increased their phosphorylation by 30% after 12-min incubation with bPTH. Dephosphorylation of proteins of 200,000 and 120,000 mol wt was also observed. These data are consistent with the hypothesis that PTH action in bone is mediated by the phosphorylation and dephosphorylation of specific substrates.

scholarly journals Characterization of 6-phosphofructo-2-kinase from foetal-rat liver

1992 ◽  
Vol 281 (2) ◽  
pp. 457-463 ◽  
Author(s):  
P Martín-Sanz ◽  
M Cascales ◽  
L Boscá
Keyword(s):  
Protein Kinase ◽  
Cyclic Amp ◽  
Gel Filtration ◽  
Kinetic Properties ◽  
Dependent Protein Kinase ◽  
Kinase C ◽  
Sds Page ◽  
Dependent Protein ◽  
Molecular Masses

Foetal and adult liver 6-phosphofructo-2-kinase (PFK-2) were purified by identical protocols. The native molecular masses of both enzymes were determined by gel filtration and were 89.1 and 100.0 kDa respectively. No differences were found in SDS/PAGE in 10%-acrylamide gel (55 kDa per subunit). The kinetic properties displayed by both enzymes were similar, except for the sensitivity to inhibition by sn-glycerol 3-phosphate. Foetal PFK-2 was a good substrate for phosphorylation by cyclic AMP-dependent protein kinase and protein kinase C, whereas the adult enzyme was phosphorylated only by cyclic AMP-dependent protein kinase. However, the phosphorylation affected only the kinetic properties of the adult enzyme, suggesting the presence in both enzymes of different sites of phosphorylation by cyclic AMP-dependent protein kinase. These differences in primary structure were consistent with the distinct chromatographic profiles of the phosphopeptides after digestion of the protein with CNBr. Western-blot analysis with antibodies specific for the N-terminal region of the liver-type PFK-2 poorly recognized the foetal enzyme, suggesting that both enzymes differ at least in the N-terminal sequence.

Sign in / Sign up

Export Citation Format

Share Document