Radiolabelled Phosphate Research Articles

Vascular calcification maladaptively participates in acute phosphate homeostasis.

Non-renal extravasation of phosphate from the circulation and transient accumulation into tissues and extracellular fluid is a regulated process of acute phosphate homeostasis that is not well understood. This process is especially relevant in the setting of chronic kidney disease (CKD), where exposure to increased phosphate is prolonged due to inefficient kidney excretion. Furthermore, CKD-associated mineral dysregulation induces pathological accumulation of phosphate causing vascular calcification (VC). Our objective was to determine whether the systemic response to acute phosphate challenges is altered by VC. After bolus phosphate administration, circulating and tissue deposition of this challenge was assessed in two rat models of VC using a radiolabelled phosphate tracer. In an adenine-induced model of CKD (N = 70), animals with VC had a blunted elevation of circulating 33PO4 following oral phosphate administration (P < 0.01), and the discordant deposition could be traced to the calcified arteries (11.4 [7.5-13.1] vs.43.0 [35.5-53.7] pmol/ng tissue, P < 0.001). In a non-CKD model of VC, calcification was induced with 0.5 ug/kg calcitriol and then withdrawn (N = 24). New phosphate uptake by the calcified vasculature correlated to the pre-existing burden of calcification (r = 38, P < 0.001) and was substantially attenuated in the absence of calcification stimulus (P < 0.01). Phosphate accrual was stimulated by the phosphate challenge and not present to the same degree during passive disposition of circulating phosphate. Further, the form of phosphate that deposited to the vasculature was predominately amorphous inorganic phosphate and not that which was bound in matured calciprotein particles. In the process of calcification, arteries acutely deposit substantial amorphous phosphate while blunting the elevation in the circulation, thereby altering the systemic disposition of phosphate and identifying VC as a participatory mineral homeostatic organ. This study demonstrates the negative vascular consequence of acute fluctuations in circulating phosphate, and supports the importance of phosphate bioavailability and diet management in CKD patients as a mediator of cardiovascular risk.

MK2 Phosphorylates Caspase‐3, Facilitates Nuclear Translocation of Caspase 3, and Regulates Apoptosis

BackgroundApoptosis is a necessary and key pathologic feature in the development of acute lung injury (ALI). Previous research has identified MAPK Activated Protein Kinase 2 (MK2) as a potential regulator in the development of apoptosis, by facilitating the nuclear translocation of active caspase‐3, the executioner of apoptosis. Specifically, loss of MK2 results in cytoplasmic sequestration of active caspase‐3. Since MK2 is a kinase, we hypothesize that MK2 phosphorylates caspase‐3 leading to its nuclear translocation and ultimately executing apoptosis.MethodsCaspase‐3 phosphorylation was determined by 2‐D gel electrophoresis with and without phosphatases, in addition to direct protein kinase assays using radiolabelled phosphate. Caspase‐3 localization was determined by performing nuclear and cytoplasmic extractions, and subsequent measurement of caspase‐3 activity. Apoptosis was determined by flow cytometry. NCI‐H23 (non‐small cell cancer, NSLC) cells were used as a model due to their ability to resist apoptosis, as well as their ability to sequester active‐caspase‐3 in the cytoplasm.Results2‐D gel electrophoresis showed that the p‐19 fragment of cleaved caspase‐3, the fragment that translocates to the nucleus, has a shift in isoelectric focus that is absent with the loss of MK2. In addition, 2‐D gel analyses of lung homogenates from WT mice exposed to LPS and treated with phosphatase have similar isoelectric foci for caspase 3 as lung homogenates from MK2‐/‐ mice exposed to LPS. Furthermore, phosphor‐imaging shows that MK2 directly phosphorylates caspase‐3. Interestingly, immunoblots of NCI‐H23 cells (which sequester active‐caspase‐3 in the cytoplasm) have lower MK2 expression than the small‐cell lung cancer cell line, H446. Flow cytometry with DAPI staining demonstrated increased apoptosis following etoposide treatment in cells over‐expressing MK2 compared to control transfections. Over‐expression of MK2 led to an increase in nuclear caspase‐3 activity. Additionally, overexpression of nuclear localization sequence (NLS) tagged caspase 3 led to increased etoposide induced apoptosis compared to WT caspase 3 or control transfections.ConclusionCombined these results strongly suggest that MK2 phosphorylates caspase‐3, which may facilitate the nuclear translocation of caspase‐3, thereby promoting the execution of apoptosis. Further work is needed to understand the mechanisms by which MK2 regulates the trafficking of active caspase‐3.

Forecasting future diabetes incidence in Canada using FINDRISC

| 227 assessed by immunoblot analysis. Cdk2 activity was determined in cdk2 immunoprecipates, by measuring the incorporation of radiolabelled phosphate into histone H1 by autoradiography. results: The inhibition of adipogenesis by J774-MacCM was confirmed, shown by the 87% reduction in triacylglycerol accumulation (p<0.05; n=3). There were no apparent changes in cdk2 expression over the first 48 hrs following addition of adipogenic inducers, in the absence or presence of J774-MacCM. Activity of cdk2 was measured at 20 hrs following induction of differentiation. Under standard differentiating conditions, cdk2 activity increased 5-fold (p<0.01; n=3) from control (non-diffferentiating). In the presence of J774-MacCM, the increase in cdk2 activity was strongly inhibited by 83% (p< 0.01; n=3). conclusion: J774-MacCM inhibits the increase in cdk2 activity and Rb phosphorylation that normally occurs in response to adipogenic inducers. reference: 1) Constant et al, Diabetologia 2006 49:1402-11 2) Yarmo et al, Exp Cell Res 2009 315:411-8 Insulin action glucose transport, receptors, cellular mechanisms No conflict of interest

Membrane phospholipids as a phosphate reserve: the dynamic nature of phospholipid‐to‐digalactosyl diacylglycerol exchange in higher plants

It is well established that phosphate deficiency induces the replacement of membrane phospholipid with non-phosphorous lipids in extra-plastidial membranes (e.g. plasma membrane, tonoplast, mitochondria). The predominant replacement lipid is digalactosyl diacylglycerol (DGDG). This paper reports that the phospholipid-to-DGDG replacement is reversible, and that when oat seedlings are re-supplied with radio-labelled phosphate, it is initially recovered primarily in phosphatidylcholine (PC). Within 2 d, the shoot contains more than half of the lipid-associated radiolabel, reflecting phosphate translocation. Oat was also cultivated in different concentrations of phosphate and the DGDG/PC ratio in roots and phospholipase activities in isolated plasma membranes was assayed after different times of cultivation. The DGDG/PC ratio in root tissue correlated more closely with plasma membrane-localized phospholipase D, yielding phosphatidic acid (PA), than with plasma membrane-localized PA phosphatase, the activity that results in a decreased proportion of phospolipids. The lipid degradation data did not reflect a significant involvement of phospholipase C, although a putative phospholipase C analogue, non-specific phospholipase C4 (NPC4), was present in oat roots. The correlation between increased phospholipase D activity and DGDG/PC ratio is consistent with a model where phospholipid-to-DGDG replacement involves formation of PA that readily is removed from the plasma membrane for further degradation elsewhere.

Pharmacological activators of AMP‐activated protein kinase have different effects on Na+ transport processes across human lung epithelial cells

AMP-activated protein kinase (AMPK) is activated by metformin, phenformin, and the AMP mimetic, 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside (AICAR). We have completed an extensive study of the pharmacological effects of these drugs on AMPK activation, adenine nucleotide concentration, transepithelial amiloride-sensitive (I(amiloride)) and ouabain-sensitive basolateral (I(ouabain)) short circuit current in H441 lung epithelial cells. H441 cells were grown on permeable filters at air interface. I(amiloride), I(ouabain) and transepithelial resistance were measured in Ussing chambers. AMPK activity was measured as the amount of radiolabelled phosphate transferred to the SAMS peptide. Adenine nucleotide concentration was analysed by reverse phase HPLC and NAD(P)H autofluorescence was measured using confocal microscopy. Phenformin, AICAR and metformin increased AMPK (alpha1) activity and decreased I(amiloride). The AMPK inhibitor Compound C prevented the action of metformin and AICAR but not phenformin. Phenformin and AICAR decreased I(ouabain) across H441 monolayers and decreased monolayer resistance. The decrease in I(amiloride) was closely related to I(ouabain) with phenformin, but not in AICAR treated monolayers. Metformin and phenformin increased the cellular AMP:ATP ratio but only phenformin and AICAR decreased cellular ATP. Activation of alpha1-AMPK is associated with inhibition of apical amiloride-sensitive Na(+) channels (ENaC), which has important implications for the clinical use of metformin. Additional pharmacological effects evoked by AICAR and phenformin on I(ouabain), with potential secondary effects on apical Na+ conductance, ENaC activity and monolayer resistance, have important consequences for their use as pharmacological activators of AMPK in cell systems where Na+K+ATPase is an important component.

Nicotinamide prevents the development of hyperphosphataemia by suppressing intestinal sodium-dependent phosphate transporter in rats with adenine-induced renal failure

Nicotinamide has been shown to inhibit intestinal sodium-dependent phosphate transport activity in normal rats. It was reported recently that type IIb sodium-dependent phosphate co-transporter (NaPi-2b) is a carrier of intestinal phosphate absorption, and that its expression level is regulated by serum 1,25-dihydroxyvitamin D [1,25(OH)(2)D] and Pi levels in normal rats. However, in chronic renal failure (CRF), serum 1,25(OH)(2)D and Pi levels are often abnormal. In a rat model of CRF, we investigated whether short-term nicotinamide administration was effective in reducing intestinal phosphate absorption and, if so, whether the effect was mediated by intestinal NaPi-2b. Adenine-induced CRF rats were given a single daily intrapenitoneal administration of nicotinamide or vehicle solution for 6 days, and time course changes in serum Pi, Ca, blood urea nitrogen (BUN) and creatinine levels were monitored. Intestinal phosphate absorption was examined by oral administration of radiolabelled phosphate on the final day. In addition, NaPi-2b protein content in jejunum brush border membranes was determined. Nicotinamide prevented the progressive increase in serum Pi associated with renal failure and significantly inhibited intestinal Pi absorption as assessed by the influx of orally administered radiolabelled phosphate into the circulation. This effect was accompanied by a decrease in NaPi-2b expression in jejunum brush border membranes. In addition, nicotinamide treatment was also associated with less marked elevations in BUN and serum creatinine and a higher creatinine clearance. Nicotinamide inhibited intestinal Pi absorption in a rat model of CRF, at least in part by inhibiting the expression of NaPi-2b, and appeared to protect against the deterioration of renal function.

Groundwater flow and phosphate dynamics surrounding a high discharge wastewater disposal well in the Florida Keys

Two artificial tracer experiments (June 1998 and September 1999) were conducted in a groundwater system surrounding a high-volume (500–1200 m 3/d) wastewater disposal well in the Florida Keys to determine the fate of wastewater borne phosphate in the subsurface. SF 6 served as the conservative groundwater tracer in both experiments. Groundwater transport rates are bimodal in nature, both horizontally and vertically. Diffusive/dispersive-type flow attributed to the limestone's primary porosity was estimated to have rates below 0.3 m/d. The most rapid conduit flow due to the limestone's secondary porosity was as high as 20 m/d in the well field and even higher, up to 123.3 m/d, immediately adjacent to the point of injection. Typical transport rates were approximately five times higher in the 1999 experiment than those observed in 1998 commensurate with a wastewater discharge increase from 0.63×10 6 to 2.32×10 6 l/d. The second experiment used radiolabelled phosphate as the reactive tracer and showed that 32PO 4 was rapidly adsorbed onto Key Largo limestone. Recirculation experiments using core material from the site and phosphate-rich water showed a rapid initial uptake of phosphate followed by a slower adsorption until an equilibrium concentration of approximately 26 μM was reached. Addition of phosphate-free water to the same core material showed a release of PO 4 into solution until the same equilibrium concentration was reached. The limestone matrix underlying the study site appears to act as a phosphate buffer once exposed to phosphate-rich water. The mechanisms controlling this buffering capacity are poorly understood at this time.

Does phosphate adsorption onto Saharan dust explain the unusual N/P ratio in the Mediterranean Sea?

A Saharan soil, considered as a proxy for Saharan aerosols, was used to perform radio-labelled phosphate adsorption experiments using 33PO 4 3–: leached particles were exposed to poisoned western Mediterranean seawater for varying lengths of time. The measured adsorption capacity of Saharan dust for phosphate was 0.13 μmol g –1. Considering this value and an annual Saharan dust deposition of 12.5 t km –2 year –1, we show that Saharan particles do not represent a significant sink for seawater phosphate in the western Mediterranean Sea. This result is in agreement with that determined from a similar approach conducted in the eastern basin. As a consequence, the unusual N/P ratio measured in the whole Mediterranean Sea (up to 29) cannot be explained by the adsorption process of seawater phosphate onto Saharan dust.

The activation of nuclear phosphoinositide 3-kinase C2β in all- trans-retinoic acid-differentiated HL-60 cells

The activity of nuclear phosphoinositide 3-kinase C2β (PI3K-C2β) was investigated in HL-60 cells induced to differentiate along granulocytic or monocytic lineages. A significant increase in the activity of immunoprecipitated PI3K-C2β was observed in the nuclei and nuclear envelopes isolated from all- trans-retinoic acid (ATRA)-differentiated cells which was inhibited by the presence of PI3K inhibitor LY 294002. High-performance liquid chromatography analysis of inositol lipids showed an increased incorporation of radiolabelled phosphate in both PtdIns(3)P and PtdIns(3,4,5)P 3 with no changes in the levels of PtdIns(4)P, PtdIns(3,4)P 2 and PtdIns(4,5)P 2. Western blot analysis of the PI3K-C2β immunoprecipitates with anti-P-Tyr antibody revealed a significant increase in the level of the immunoreactive band corresponding to PI3K-C2β in the nuclei and nuclear envelopes isolated from ATRA-differentiated cells.

Phosphate exchange and ATP synthesis by DMSO-pretreated purified bovine mitochondrial ATP synthase

Purified soluble bovine mitochondrial F1Fo-ATP synthase contained 2mol of ATP, 2mol of ADP and 6mol of Pi/mol. Incubation of this enzyme with 1mM [32P]Pi caused the exchange of 2mol of Pi/mol of F1Fo-ATP synthase. The labelled phosphates were not displaced by ATP. Transfer of F1Fo-ATP synthase to a buffer containing 30% (v/v) DMSO and 1mM [32P]Pi resulted in the loss of bound nucleotides with the retention of 1mol of ATP/mol of F1Fo-ATP synthase. Six molecules of [32P]Pi were incorporated by exchange with the existing bound phosphate. Removal of the DMSO by passage of the enzyme through a centrifuged column of Sephadex G-50 resulted in the exchange of one molecule of bound [32P]Pi into the bound ATP. Azide did not prevent this [32P]Pi ↔ ATP exchange reaction. The bound labelled ATP could be displaced from the enzyme by exogenous ATP. Addition of ADP to the DMSO-pretreated F1Fo-ATP synthase in the original DMSO-free buffer resulted in the formation of an additional molecule of bound ATP. It was concluded that following pretreatment with and subsequent removal of DMSO the F1Fo-ATP synthase contained one molecule of ATP at a catalytic site which was competent to carry out a phosphateŐATP exchange reaction using enzyme-bound inorganic radiolabelled phosphate. In the presence of ADP an additional molecule of labelled ATP was formed from enzyme-bound Pi at a second catalytic site. The bound phosphateŐATP exchange reaction is not readily accommodated by current mechanisms for the ATP synthase.

Phosphate exchange and ATP synthesis by DMSO-pretreated purified bovine mitochondrial ATP synthase.

Purified soluble bovine mitochondrial F(1)F(o)-ATP synthase contained 2 mol of ATP, 2 mol of ADP and 6 mol of P(i)/mol. Incubation of this enzyme with 1 mM [(32)P]P(i) caused the exchange of 2 mol of P(i)/mol of F(1)F(o)-ATP synthase. The labelled phosphates were not displaced by ATP. Transfer of F(1)F(o)-ATP synthase to a buffer containing 30% (v/v) DMSO and 1 mM [(32)P]P(i) resulted in the loss of bound nucleotides with the retention of 1 mol of ATP/mol of F(1)F(o)-ATP synthase. Six molecules of [(32)P]P(i) were incorporated by exchange with the existing bound phosphate. Removal of the DMSO by passage of the enzyme through a centrifuged column of Sephadex G-50 resulted in the exchange of one molecule of bound [(32)P]P(i) into the bound ATP. Azide did not prevent this [(32)P]P(i)<-->ATP exchange reaction. The bound labelled ATP could be displaced from the enzyme by exogenous ATP. Addition of ADP to the DMSO-pretreated F(1)F(o)-ATP synthase in the original DMSO-free buffer resulted in the formation of an additional molecule of bound ATP. It was concluded that following pretreatment with and subsequent removal of DMSO the F(1)F(o)-ATP synthase contained one molecule of ATP at a catalytic site which was competent to carry out a phosphate-ATP exchange reaction using enzyme-bound inorganic radiolabelled phosphate. In the presence of ADP an additional molecule of labelled ATP was formed from enzyme-bound P(i) at a second catalytic site. The bound phosphate-ATP exchange reaction is not readily accommodated by current mechanisms for the ATP synthase.

Effect of ethanol, propylene glycol and glycerol on the interaction of methyl and propyl p-hydroxybenzoate with Staphylococcus aureus and Pseudomonas aeruginosa

Co-solvents ethanol, propylene glycol and glycerol were used to increase the aqueous concentrations of methyl and propyl p-hydroxybenzoate above their saturation solubility. The increased aqueous concentration of parabens in co-solvent solutions was associated with increased antibacterial activity against Staphylococcus aureus and Pseudomonas aeruginosa, but the extent of this effect increased with increasing hydrophobicity of the co-solvent. Addition of co-solvents to aqueous solutions of parabens caused a reduction in their octanol/water partitioning and a reduction in the uptake of the parabens by the cells, the extent of the reduction increasing as the hydrophobicity of the co-solvent increased. The addition of co-solvents also potentiated cell membrane damage as evidenced by leakage of radio-labelled phosphate from parabens-treated cells, but in this situation the extent of the damage was correlated with increasing hydrophobicity of the co-solvent. The results indicate that the increase in antibacterial activity of parabens preservatives in co-solvent solutions can largely be accounted for by their combined effects on the integrity of the cell membrane.

Natural-product inhibitors of human DNA ligase I

Enzymatic activity mediated by recombinant human DNA ligase I (hLI), in conjunction with tannin removal procedures, has been applied to a natural-product screen involving approximately 1000 plant extracts and various pure compounds. The primary hLI activity assay involved the measurement of the amount of radiolabelled phosphate in a synthetic nucleic acid hybrid that becomes resistant to alkaline phosphatase as a result of ligation. A bioactivity-guided fractionation scheme resulted in the isolation of ursolic [IC50=100 micrograms/ml (216 microM)] and oleanolic [IC50=100 micrograms/ml (216 microM)] acids from Tricalysia niamniamensis Hiern (Rubiaceae), which demonstrated similar DNA ligase inhibition profiles to other triterpenes such as aleuritolic acid. Protolichesterinic acid [IC50=6 micrograms/ml (20 microM)], swertifrancheside [IC50 = 8 micrograms/ml(11)microM)] and fulvoplumierin [IC50=87 micrograms/ml (357 microM)] represent three additional natural-product structural classes that inhibit hLI. Fagaronine chloride [IC50=10 micrograms/ml (27 micronM] and certain flavonoids are also among the pure natural products that were found to disrupt the activity of the enzyme, consistent with their nucleic acid intercalative properties. Further analyses revealed that some of the hLI-inhibitory compounds interfered with the initial adenylation step of the ligation reaction, indicating a direct interaction with the enzyme protein. However, in all cases, this enzyme-inhibitor interaction did not disrupt the DNA relaxation activity mediated by hLI. These results indicate that, although the same enzyme active site may be involved in both enzyme adenylation and DNA relaxation, inhibitors may exert allosteric effects by inducing conformational changes that disrupt only one of these activities. Studies with inhibitors are important for the assignment of specific cellular functions to these enzymes, as well as for their development into clinically useful antitumour agents.

Polyunsaturated fatty acids do not activate protein kinase C in the testis of the goldfish (Carassius auratus)

Earlier studies have established that polyunsaturated fatty acids (PUFA) such as eicosapentaenoic acid and docosahexaenoic acid inhibit steroid production in the goldfish testis. As PUFA inhibit testicular steroidogenesis in the rat through activation of protein kinase C (PKC), the present studies were undertaken to characterize the properties of PKC in the goldfish testis and to test the effects of selected PUFA on PKC activity. PKC activity was quantified in goldfish testis homogenate following partial purification by DEAE-cellulose chromatography by determining the transfer of radiolabelled phosphate from [γ - (32)P]ATP to histone III-S. Testicular PKC activity was defined by the amount of protein phosphorylation in the presence of phosphatidylserine, phasphatidylcholine, Ca(2+) ions and diolein (a 1,2-diacylglycerol analog) above that obtained in response to Ca(2+) ions alone. Western blot analysis of a crude testis homogenate using an antibody specific to the α and β isoforms of mammalian PKC led to the identification a single band of protein (80 kD) that co-migrated with PKC from rabbit brain cytosol. Addition of arachidonic, eicosapentaenoic or docosahexaenoic acids failed to activate PKC. However, PKC activity stimulated by phospholipid, Ca(2+) ions and diolein was inhibited in a dose related fashion by all of these fatty acids. These studies suggest that the inhibitory effects of EPA and DHA on testicular steroidogenesis are not mediated by activation of PKC. The lack of effect of PUFA on PKC activity in the goldfish testis suggests that either the distribution of PKC isoforms differs between the testis of mammals and fish or that PKC is not activated by PUFA in the goldfish.

Phosphate influx into ectomycorrhizal and saprotrophic fungal hyphae in relation to phosphate supply; a potential method for selection of efficient mycorrhizal species

The application of phosphorus bioassays developed to identify the nutritional status of plant roots have been applied to ectomycorrhizal and saprotrophic fungal hyphae. With increasing phosphate supply, a decrease in influx of radiolabelled phosphate solution in the test was seen in all fungal species, similar to the response found in plant roots. The ectomycorrhizal species Hebeloma crustuliniforme had more than twice the P influx rates of the other fungi, suggesting a potentially greater scavenging ability than the other species tested. There appears to be no significant difference in influx rates between saprotrophic and mycorrhizal fungal species. The range of influx values between isolates of the same ectomycorrhizal fungal species was generally less than that between species. This technique could be used as a means of selection of efficient ectomycorrhizal species and isolates for inoculation programmes.

Quantitation by fast-atom bombardment mass spectrometry: assay of cytidine 3',5'-cyclic monophosphate-responsive protein kinase.

A protein kinase, stimulated by cytidine 3',5'-cyclic monophosphate, is conventionally assayed by monitoring the incorporation of radiolabelled phosphate from adenosine triphosphate into a histone substrate. Here the assay of the protein kinase is carried out by positive-ion fast-atom bombardment mass spectrometric analysis of the enzyme incubation mixture after the reaction has been terminated. The data so obtained show good agreement with data obtained by the conventional radiometric assay: the intrinsic advantage of the mass spectrometric assay is the capacity for multiple component monitoring; the ability of the kinase to bind competing cyclic nucleotides together with integral adenosine triphosphatase (ATPase) and phosphodiesterase activity can also be assessed.

Relationships between heterogeneity of the PSII core complex from grana particles and phosphorylation

Isoelectrofocusing of photosystem II enriched membranes from spinach reveals the presence of at least four different populations of PSII core complex. The four bands are neither equally populated nor equally active in electron transport from diphenylcarbazide to 2,6-dichlorophenolindophenol. Under conditions of a low and high phosphorylation level a change in the relative populations of the PSII core isoforms is observed and the amount of radiolabelled phosphate incorporated into the four types of complexes is correlated to the value of their isoelectric point suggesting that the origin of the heterogeneity evidenced in vitro is at least partially due to different levels of light-induced phosphorylation. A 9 KD phosphoprotein, previously described in PSII, is found in our core complex preparation at a concentration which decreases as the total phosphorylation level on D 1 D 2 polypeptides increases.

Collagen-platelet interaction: Type XI collagen-induced platelet aggregation

Type XI collagen in its native fibrillar but not in soluble monomeric form mediates human platelet aggregation and release of adenosine triphosphate in a dose-dependent manner. Its action is inhibited by aspirin. Type XI collagen also increased radiolabelled phosphate incorporation into protein bands with molecular weights of 42 KDa and 22 KDa, respectively. In contract, these events were not observed in platelets incubated with type IX collagen. These results suggest that the fibrillar type XI collagen has the same ability as other types of collagen to induce human platelet aggregation.

Protein kinase activity and endogenous protein phosphorylation in rat liver plasma membranes.

When rat liver plasma membranes were incubated with [gamma-32P]ATP radio-labelled phosphate was incorporated into endogenous protein and exogenous substrate by a membrane-bound protein kinase activity. A high ATP/membrane protein ratio was required for optimum incorporation conditions. Cyclic AMP did not affect the incorporation of phosphate. The protein kinase activity was extracted from the membranes by 1% Triton X-100 and a high concentration of KCl. The solubilised enzyme resolved into two fractions on DEAE-cellulose chromatography. One enzyme fraction had the same properties as the catalytic subunit of cytosolic cyclic AMP-dependent protein kinases. Endogenously phosphorylated proteins were resolved by SDS-polyacrylamide gel electrophoresis into five major and additional minor phosphorylated components. Three of the major phosphorylated components were tightly bound to the membrane material and were not extracted by 1% Triton X-100 and 1M KCl.