Metabolism of S-adenosylmethionine in rat hepatocytes: transfer of methyl group from S-adenosylmethionine by methyltransferase reactions

Treatment of rats with a methionine diet leads not only to a marked increase of S-adenosylmethionine synthetase in liver, but also to the increase of glycine, guanidoacetate and betaine-homocysteine methyltransferases. The activity of tRNA methyltransferase decreased with the increased amounts of methionine in the diets. However, the activities of phospholipids and S-adenosylmethionine-homocysteine methyltransferases did not show any significant change. When hepatocarcinogenesis induced by 2-fluorenylacetamide progresses, the activities of glycine and guanidoacetate methyltransferases in rat liver decreased, and could not be detected in tumorous area 8 months after treatment. The levels of S-adenosylmethionine in the liver also decreased to levels of one-fifth of control animals at 8 months. The uptake and metabolism of [methyl-3H]-methionine and -S-adenosylmethionine have been investigated by in vivo and isolated hepatocytes. The uptake of methionine and transfer of methyl group to phospholipid in the cells by methionine were remarkably higher than those by S-adenosylmethionine. These results indicate that phospholipids in hepatocytes accept methyl group from S-adenosylmethionine immediately, when it is synthesized from methionine, before mixing its pool in the cells.     

A radioactive assay for guanidoacetate methyltransferase.

A radioactive assay for guanidoacetate methyltransferase is described. It is based on a separation by an AG 50 (NH₄⁺) column, on which [methyl-¹⁴C]Adenosylmethionine is adsorbed, in contrast to [methyl-¹⁴C]creatine which can be collected in the effluent fraction. Guanidoacetate methyltransferase is sensitive to inhibition by adenosylhomocysteine and 3-deazaadenosylhomocysteine.     

Guanidoacetate methyltransferase. Purification and molecular properties.

Guanidoacetate methyltransferase has been purified about 140-fold from pig liver. Polyacrylamide gel electrophoresis of the purified enzyme showed four protein bands, each of which is associated with guanidoacetate methyltransferase activity. During gel electrophoresis at pH 3 in 8 M urea, guanidoacetate methyltransferase migrated as a single component. The molecular weight of the purified guanidoacetate methyltransferase was estimated to be 31,000 by sodium dodecyl sulfate-gel electrophoresis, which also showed only one protein component with guanidoacetate methyltransferase activity. This molecular weight is in agreement with that estimated by Sephadex G-75 chromatography. Guanidoacetate methyltransferase is inhibited by adenosylhomocysteine, 3-deazaadenosylhomocysteine, and sinefungin with Ki values of 16 microM, 39 microM, and 18 microM, respectively.     

Detection and characterization of the Ah receptor for 2,3,7,8-tetrachlorodibenzo-p-dioxin in the human colon adenocarcinoma cell line LS180.

The Ah (aromatic hydrocarbon) receptor mediates induction of aryl hydrocarbon hydroxylase (AHH; an enzyme activity associated with cytochrome P450IA1) by polycyclic aromatic hydrocarbon carcinogens such as 3-methylcholanthrene (MC) and benzo[a]pyrene (BP) and the halogenated toxin 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Until recently the AhR seemed to be present only at very low levels in human cells and tissue. With a modified assay (the presence of sodium molybdate and a reduction in the amount of charcoal used to adsorb "excess" ligand) we found that cytosol from LS180 cells contains a high concentration of AhR (400-500 fmol/mg cytosolic protein) when detected by [3H]TCDD or [3H]MC. Cytosolic receptor also was detected with [3H]BP but at a level that was 35% of that detected with [3H]TCDD or [3H]MC. These levels are similar to those found in mouse Hepa-1 hepatoma cells in which AhR has been extensively characterized. The apparent binding affinity (Kd) of the cytosolic receptor for [3H]TCDD and for [3H]MC was about 5 nM. As with Hepa-1, the human LS180 cytosolic AhR sedimented at about 9 S on sucrose gradients when detected with [3H]TCDD, [3H]BP or [3H]MC. The nuclear-associated ligand.receptor complex recovered from cells incubated in culture with [3H]TCDD sedimented at about 6.2 S. The 9.8 S cytosolic form corresponds to a multimeric protein of a relative molecular mass (Mr) of about 285,000 whereas the 6.2 S nuclear receptor corresponds to a multimeric protein of Mr 175,000. The smallest specific ligand-binding subunit (detected by sodium dodecyl sulfate-polyacrylamide electrophoresis under denaturing conditions of receptor photoaffinity labeled with [3H]TCDD) was about Mr 110,000. AHH activity was induced in cells exposed in culture to TCDD or benz[a]anthracene (BA). The EC50 was 4 x 10(-10) M for TCDD and 1.5 x 10(-5) M for BA. For both inducers the EC50 in LS180 cells was shifted about one log unit to the right as compared to the EC50 for AHH induction in mouse Hepa-1 cells. The lower sensitivity of the LS180 cells to induction of AHH activity by TCDD or BA is consistent with the lower affinity of TCDD and MC for binding to human AhR. The ligand-binding properties, physicochemical properties, and mode of action of the AhR in this human cell line are therefore very similar to those of the extensively characterized AhR in rodent cells and tissues.     

Intracellular compartmentation of deoxycytidine nucleotide pools in S phase mouse 3T3 fibroblasts

We labeled mouse 3T3 fibroblasts, synchronized in G0 or S phase, from [3H]cytidine or [3H]deoxycytidine and measured the flow of isotope into and through deoxycytidine nucleotide pools, including the two deoxyliponucleotides dCDP choline and dCDP ethanolamine. Compared to G0 cells, S phase cells had much larger pools with a 20-40-fold faster turnover. The dCTP pool of S phase cells during steady state conditions attained a 6-fold higher specific activity than the pool of G0 cells when labeled from cytidine but a 10-fold lower specific activity when labeled from deoxycytidine. The dCTP pool of G0 cells showed a slow but measurable turnover indicating a limited amount of de novo synthesis also in resting cells. The labeling pattern of dCTP and deoxyliponucleotides of G0 cells was compatible with a simple precursor-product relationship. In S phase cells, however, dCDP choline had a 4-6 times higher specific activity during steady state conditions than dCTP and dCMP when the cells were labeled with [3H]deoxycytidine. We suggest that 3T3 cells contain two distinct intracellular dCTP pools, one labeled preferentially from cytidine and used for DNA replication, the other labeled from deoxycytidine and used for deoxyliponucleotide synthesis. We speculate that the latter pool during S phase may be temporarily sequestered in the cell's membrane fraction before equilibration with the much larger dCTP pool originating in S phase cells from the reduction of CDP.     

Phospholipase A2 inhibitory activity in thymocytes of dexamethasone-treated mice--possible implication of lipocortins

The cellular phospholipase A2 activity of mouse thymocytes was estimated in vitro by the release of [3H]-Arachidonic acid from labeled and calcium ionophore A23187-stimulated cells. This activity was decreased in thymocytes from dexamethasone-treated mice. Thus, the presence of phospholipase A2 inhibitory proteins in mouse thymus was investigated. Three main proteins (36 kDa I, 36 kDa II, 73 kDa) were purified. These proteins were able to inhibit both phospholipase A2 in vitro, and the release of [3H]-Arachidonic acid from labeled and stimulated mouse thymocytes. Biochemical analysis revealed that the three proteins were lipocortin-like proteins. Our results show that in vivo dexamethasone treatment induces a phospholipase A2 inhibitory activity in mouse thymus, such an inhibition can be reproduced on isolated thymocytes by purified thymic lipocortins, known as glucocorticosteroid-inducible proteins.     

Increased ATP and creatine phosphate turnover in phagocytosing mouse peritoneal macrophages.

Resident and thioglycollate-elicited macrophages maintained in culture for 24 h contain approximately 5 x 10(-16) and 12 x 10(-16) mol of ATP per cell, respectively. During particle ingestion, the levels of ATP in these cells did not change. However, the specific activity of ATP extracted from macrophages labeled with [32P]Pi during phagocytosis was 40% lower than ATP extracted from control cells. These results suggested that macrophages contain a high energy phosphate reservoir, in addition to the ATP pool(s). A search for such a reservoir led to the identification of creatine phosphate in both resident and thioglycollate-elicited macrophages at concentrations that are in 3- to 5-fold-molar excess over ATP. Creatine phosphate levels in phagocytosing resident macrophages decreased by 45%, while creatine phosphate levels in phagocytosing thioglycollate-elicited macrophages did not change. Creatine phosphate turnover was measured in macrophages prelabeled with [14C]creatine. Over 90% of the intracellular label was in the form of creatine phosphate. During phagocytosis, there was a 40% decrease in intracellular [14C]creatine phosphate in both resident and thioglycollate-elicited macrophages. These results indicate that creatine phosphate turns over more rapidly during phagocytosis and replenishes the ATP consumed.     

Steroid metabolism and binding activity in a murine renal tumor cell line

The purpose of this study was to partially characterize the steroid binding activity of murine renal tumor cells in continuous culture. The steroid receptor content of a cloned renal tumor cell line (RAG) and a subline RAG-2 was examined by sucrose gradient analysis, hydroxylapatite and dextran-coated charcoal methods. The RAG cells lacked estrogen- and progestin-binding activity, whereas specific 5 alpha-dihydrotestosterone (DHT) and dexamethasone (Dx) binding activities were detected as 8S peaks on low salt gradients. The specificity of DHT binding was examined by sucrose gradient analysis: DHT, R1881 and ORG2058 all completely inhibited [3H]DHT binding whereas diethylstilbestrol and Dx were ineffective. The androgen receptor content of the RAG cells was approx. 15 fmol/mg cytosol protein by the hydroxylapatite-filter assay, with an estimated Kd for methyltrienolone (R1881) of 5 nM at 0 degrees C. Scatchard analysis of [3H]Dx binding by RAG cytosol showed a Kd of 6 nM for Dx and 44 nM for corticosterone at 0 degrees C. Glucocorticoid receptor levels were estimated to be 182 fmol/mg cytosol protein by dextran-coated charcoal assay. Metabolism of [3H]testosterone and [3H]DHT by RAG cells was examined 1, 4 and 6 h after exposure to labeled hormone. Radioactive DHT was the primary intracellular metabolite recovered after exposure to [3H]testosterone. There was little conversion of DHT to androstanediol.     

Assignment of A and B Types of Monoamine Oxidase in NCB20 Hybrid Cells to Those of the Parental Cells by Peptide Mapping

The structures of [3H]pargyline-labeled, flavin-containing polypeptides of monoamine oxidase (MAO) from hybrid NCB20 cells, and their parental cells, A/J mouse brain cells and Chinese hamster brain cells, were analyzed and compared by using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and limited proteolysis and one-dimensional peptide mapping in SDS gels. After preincubation of mitochondrial preparations with deprenyl or clorgyline, the flavin-containing polypeptide of type A or type B MAO was selectively labeled with [3H]pargyline. SDS-PAGE of [3H]pargyline-labeled mitochondrial samples revealed that the polypeptide with apparent Mr of 62,000 was associated with type A activity in the three types of cells, and that the polypeptide with apparent Mr of 61,000 or 58,000 was associated with type B activity in Chinese hamster brain cells and NCB20 cells or A/J mouse brain cells, respectively. Chymotrypsin digestion of the [3H]pargyline-labeled polypeptides and the peptide mapping in SDS gels from A/J mouse and Chinese hamster brain cells produced identical map patterns between the two type A MAOs, almost the same map patterns (with the exception of one additional peptide fragment) between the two type B MAOs, and different map patterns between type A and type B MAOs. The results of identical treatments of the [3H]pargyline-labeled polypeptides of MAOs in NCB20 cells showed that type A and type B MAO in NCB20 cells were similar to type A MAO of A/J mouse and Chinese hamster brain cells and to type B MAO of Chinese hamster brain cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Evidence for a signal sequence at the N terminus of the common precursor to adrenocorticothrophin and beta-lipotropin in mouse pituitary cells

The precursor to corticotropin and beta-endorphin was synthesized in a reticulocyte cell-free system under the direction of mRNA from mouse AtT-20 pituitary tumor cells in the presence of [3H]proline, [3H]phenylalanine, [3H]leucine, [3H]valine, [3H]isoleucine or [35S]methionine. Automatic Edman degradation of the radioactive cell-free product showed the following N-terminal sequence: Pro-1, Met-2, Leu-11, Leu-12, Leu-13, Leu-15, Leu-16, Leu-17, Ile-21 and Val-23. The corticotropin-endorphin precursor was also labeled in AtT-20 cells with [3H]valine, [3H]leucine, [3H]tryptophan, [3H]serine, [35S]methionine or [35S]cysteine. Automatic Edman degradation of the radioactive intact cell form gave the following N-terminal sequence: Trp-1, Cys-2, Leu-3, Ser-5, Ser-6, Val-7, Cys-8, Leu-11, Leu-17, Leu-18 and tentatively Met-27. The sequence of the intact cell form from AtT-20 cells matches the sequence of the cell-free form of bovine pituitary precursor beginning at Trp-27, as determined by recombinant DNA technology [Nakanishi, S., Inoue, A., Kita, T., Nakamura, M., Chang, A. C. Y., Cohen, S. N., and Numa, S. (1979) Nature (Lond.) 278, 423-427]. The sequence of the mouse pituitary mRNA-directed cell-free translation product also matches the bovine precursor beginning at Pro-2. The results suggest that both the mouse and bovine precursors possess a signal sequence of 26 amino acids which is cleaved in intact cells. CNBr cleavage of [35S]cysteine-labelled intact cell precursor gave rise to an N-terminal fragment of a size compatible with the presence of a methionyl residue at or near position 27.     

Differential processing and regulation of thyroid-stimulating hormone subunit carbohydrate chains in thyrotropic tumors and in normal and hypothyroid pituitaries

Thyroid-stimulating hormone (TSH) alpha- and beta-subunit glycosylation was investigated in mouse thyrotropic tumor and in normal and hypothyroid pituitary cells for various periods of time in the presence of [3H]mannose or [3H]galactose. After sequential precipitation with anti-alpha and anti-beta sera, subunits were treated with Pronase followed by endo-beta-N-acetylglucosaminidase H (Endo H) and analyzed by paper chromatography. In primary cultures of thyrotropic tumor cells incubated for 60 min with [3H]mannose, primarily Man9GlcNAc and Man8GlcNAc were found on TSH + alpha subunits, whereas Glc1Man9GlcNAc and Man9GlcNAc were prominent on free beta subunits. After preincubation of cells for 16 h in the presence or absence of glucose followed by a 60-min pulse of [3H]mannose, there was an 8-fold increase in labeled TSH + alpha but only a minimal change in free beta or total proteins. In the absence of glucose, there was a selective accumulation of Man8GlcNAc on TSH + alpha but not on free beta or total proteins; however, there was no detectable accumulation of Endo H resistant forms during glucose starvation on TSH subunits or total proteins. Normal mouse and rat pituitary minces incubated for 60 min with either [3H]mannose or [3H]galactose showed no glucose-containing species on TSH subunits, but equal amounts of Man9GlcNAc and Man8GlcNAc on TSH + alpha, and mostly Man9GlcNAc on free beta subunits. In contrast, hypothyroid mouse and rat pituitaries exhibited an increase in Glc1Man9NAc and Glc1Man8GlcNAc on free beta but not on TSH + alpha or total proteins.(ABSTRACT TRUNCATED AT 250 WORDS)     

Transfer of purine metabolites between cells through the medium and via cell contacts in cocultures of HGPRT+ and HGPRT- cells

Cells with and without hypoxanthine-guanine phosphoribosyltransferase (HGPRT) activity were used to examine the transfer of purine metabolites through the medium and via cell contacts. HGPRT- Chinese hamster and human fibroblasts were able to incorporate 3H-labeled purine metabolite(s) from medium in which mouse HGPRT+ B82 cells had been grown for 24 h with [3H]hypoxanthine, but mouse A9 fibroblasts that were deficient in HGPRT, adenine phosphoribosyltransferase (APRT), and methylthioadenosine phosphorylase (MTAP) were unable to incorporate these metabolites. This suggests that in recipient cells incorporation is due to [3H]MTA, which has been shown previously to be the major 3H-labeled purine metabolite to accumulate in B82 medium, being cleaved by MTAP to [3H]adenine, which is phosphoribosylated by APRT to [3H]AMP. Incorporation by recipient cells of metabolites from the medium is referred to as contact-independent metabolite transfer (CIMT). In autoradiograms of B82/A9 cocultures that were labeled with [3H]hypoxanthine, grains were found over A9 that were not in contact with B82, although A9 did not act as recipients of CIMT. This is termed proximity-dependent metabolite transfer (PDMT). Both CIMT and PDMT interfered with the assessment of nucleotide exchange between HGPRT+ and HGPRT- cells through cell contacts, which is referred to as contact-dependent metabolite transfer (CDMT). These problems were unique to HGPRT+ mouse L cells. However, HGPRT- mouse L cells, A9, could be used as potential recipients. A9 were positive recipients of CDMT with only one of five cell lines tested, which suggested that these cells were selective communicators. CDMT could not be studied with [3H]guanine because the nuclei of HGPRT- cells became labeled.     

Biosynthesis and acquisition of biological activity of the fibronectin receptor

The biosynthesis of the 140-kilodalton fibronectin receptor complex by cultured 3T3 mouse cells was characterized and compared with that of chick embryo fibroblasts. Three murine glycoprotein components of 140-150 kilodaltons (band 1), 125 kilodaltons (band 2), and 105 kilodaltons (band 3) could be immunoprecipitated from metabolically labeled 3T3 cells using polyclonal antibodies. In pulse-chase experiments, bands 1 and 3 of the mouse receptor were labeled to maximal levels immediately after completion of the labeling pulse. However, band 2 was not detected at short chase times, and it reached maximal labeling only after approximately 12 h of chase. The appearance of band 2 occurred at the same rate as the disappearance of band 3. Only bands 1 and 2 could be affinity purified by binding to immobilized fibronectin cell-binding fragment, indicating that they represent mature functional receptor components. When 3T3 cells were incubated with radioactive sugars, band 1 of the receptor labeled well with both [3H]mannose and [3H]glucosamine. However, band 2 labeled well with [3H]glucosamine but contained low amounts of mannose, and band 3 labeled well with [3H]mannose but contained low amounts of glucosamine. Digestion of both bands 2 and 3 with endoglycosidase F yielded similar-sized products of approximately 88,000 daltons, suggesting that post-translational asparagine-linked oligosaccharide processing can account for most of the size difference between these bands. These data suggest that in the mouse fibronectin receptor, band 3 is a biologically inactive precursor of band 2 that does not appear on the cell surface. In contrast, pulse-chase experiments using chicken embryo fibroblasts indicated that the three components of the chicken 140k complex were distinct moieties. Our results demonstrate distinct types of processing for fibronectin receptor complexes from different species. In mammalian cells, this receptor undergoes a surprisingly long (20 h) maturation process involving asparagine-linked oligosaccharides before reaching its final, biologically active form.     

Localization of the estrogen receptor in uterine cells by affinity labeling with [3H]tamoxifen aziridine

The possibility that estrogen receptors may exist in uterine plasma membranes was investigated by covalent labeling of estrogen receptors in mouse uterine cells with [3H]tamoxifen aziridine (TA). Isolated epithelial and stromal cells of immature mice were incubated with [3H]TA in the presence or absence of unlabeled tamoxifen, homogenized and separated into nuclear, cytosolic and microsomal fractions by differential centrifugation. These fractions were subjected to SDS-polyacrylamide gel electrophoresis and the proteins labeled covalently with TA were visualized by autoradiography. Proteins labeled specifically with [3H]TA were observed almost exclusively in the nuclear fraction of both epithelial and stromal cells. In contrast, very little labeled protein was detected in the cytosolic or microsomal fraction. Although these data do not preclude the possibility that estrogen binding sites are present in plasma membranes of uterine cells, this cellular fraction is definitely not labeled to a significant extent by [3H]TA. Thus, if membrane estrogen binding sites exist, their structural conformations may be different from that of nuclear estrogen receptors.     

Stimulus-specific induction of phospholipid and arachidonic acid metabolism in human neutrophils

Phospholipid remodeling resulting in arachidonic acid (AA) release and metabolism in human neutrophils stimulated by calcium ionophore A23187 has been extensively studied, while data obtained using physiologically relevant stimuli is limited. Opsonized zymosan and immune complexes induced stimulus-specific alterations in lipid metabolism that were different from those induced by A23187. [3H]AA release correlated with activation of phospholipase A2 (PLA2) but not with cellular activation as indicated by superoxide generation. The latter correlated more with calcium-dependent phospholipase C (PLC) activation and elevation of cellular diacylglycerol (DAG) levels. When cells that had been allowed to incorporate [3H]AA were stimulated with A23187, large amounts of labeled AA was released, most of which was metabolized to 5-HETE and leukotriene B4. Stimulation with immune complexes also resulted in the release of [3H]AA but this released radiolabeled AA was not metabolized. In contrast, stimulation with opsonized zymosan induced no detectable release of [3H]AA. Analysis of [3H]AA-labeled lipids in resting cells indicated that the greatest amount of label was incorporated into the phosphatidylinositol (PI) pool, followed closely by phosphatidylcholine and phosphatidylserine, while little [3H]AA was detected in the phosphatidylethanolamine pool. During stimulation with A23187, a significant decrease in labeled PI occurred and labeled free fatty acid in the pellet increased. With immune complexes, only a small decrease was seen in labeled PI while the free fatty acid in the pellets was unchanged. In contrast, opsonized zymosan decreased labeled PI, and increased labeled DAG. Phospholipase activity in homogenates from human neutrophils was also assayed. A23187 and immune complexes, but not zymosan, significantly enhanced PLA2 activity in the cell homogenates. On the other hand, PLC activity was enhanced by zymosan and immune complexes. Stimulated increases in PLC activity correlated with enhanced superoxide generation induced by the stimulus.     

Analyses of the interactions between retinoid-binding proteins and embryonal carcinoma cells

[3H]Retinoic acid (RA) and [3H]retinol bind in an unsaturable manner to isolated nuclei from Nulli-SCC1 and PCC4.aza1R embryonal carcinoma (EC) cells. When nuclei are challenged with the same labeled retinoids on their respective binding proteins (CRABP and CRBP), much less binding is observed and the binding is saturable. RA-CRABP does not compete with [3H]retinol-CRBP for binding to specific Nulli-SCC1 nuclear sites, whereas retinol-CRBP (but not apo-CRBP) actually potentiates the binding of [3H]RA-CRABP to these nuclei. The binding of [3H]RA-CRABP and [3H]retinol-CRBP is not dramatically affected by prior removal of the outer nuclear membrane with Triton X-100. However, treatment with the detergent after the binding reaction is complete removes about half of the bound [3H]RA-CRABP and almost all of the bound [3H]retinol-CRBP. We measured specific retinoid-binding activities in nucleoplasmic extracts of Nulli-SCC1 and PCC4.aza1R cells. The only readily detectable specific binding activity in nucleoplasmic extracts from untreated cells was for [3H]retinol in PCC4.aza1R preparations. Nucleoplasmic extracts from Nulli-SCC1 and PCC4.aza1R cells pretreated with RA had considerable levels of specific [3H]RA-binding activity with little or no increase in [3H]retinol binding. By contrast, similar extracts from Nulli-SCC1 cells treated with retinol bound large amounts of both [3H]retinol and [3H]RA. Under the same conditions, PCC4.aza1R extracts also contained [3H]RA-binding activity with no increase in [3H]retinol binding above the high endogenous levels. Although these results might reflect translocation of binding proteins from cytoplasm to nucleus, other interpretations must be considered since we often observed an increase, rather than the expected reduction, in cytoplasmic retinoid-binding protein levels.     

Induction of rat liver glycine methyltransferase by high methionine diet

Dietary experiments were carried out to evaluate the physiological role of glycine methyltransferase. When rats received a 18% casein diet containing excess methionine, the activity of the enzyme in liver extracts increased with increasing methionine content in the diet. Adenosylmethionine synthetase and adenosylhomocysteinase activities were also elevated, while guanidoacetate methyltransferase activity showed no significant change. The glycine methyltransferase activity reached a maximal level after 4–6 days on the 3% methionine diet. Immunological titration showed that the increase in activity was associated with the increase in amount of the enzyme.     

Immunological and molecular characterization of the cAMP-dependent protein kinases in AtT20 cells

The properties of the cAMP-dependent protein kinases in AtT20 mouse pituitary tumor cells were characterized by a combination of immunological and biochemical techniques. Ninety per cent of the total cAMP-dependent protein kinase was in the 40,000 X g supernatant fraction. Protein kinases I and II were immunoprecipitated with specific antisera directed against their regulatory subunits. The immunoprecipitated kinases bound [3H]cAMP and were catalytically active when incubated with [gamma-32P]ATP-Mg and protamine or histone H2B. Immunoprecipitated protein kinases I and II bound [3H]cAMP with apparent Kb values of 1.5 and 15 nM, respectively. Regulatory subunit concentrations in AtT20 cells were measured by immunoprecipitation of [3H]cAMP-R complexes. R-I and R-II levels were 2.7 and 3.0 pmol of [3H]cAMP binding activity per mg of cytosolic protein, respectively, however, the ratio of protein kinase II to protein kinase I was 2.5 indicating the presence of a significant amount of free R-I. This was confirmed by DEAE-cellulose chromatography and the isolation of immunoreactive R-I devoid of protein kinase activity. A significant amount of R-I also coeluted with protein kinase II when AtT20 cell extracts were subjected to DEAE-cellulose chromatography. In quantitative immunoprecipitation experiments, 0.1 microliter of anti-brain R-II serum complexed up to 0.5 pmol of the [3H]cAMP-binding activity of protein kinase II prepared from bovine and rat brain, and AtT20 cells while 2 microliter of anti-brain R-II serum was required to precipitate an equal amount of protein kinase II from bovine skeletal muscle showing that the protein kinase II in AtT20 cells contained the neural-specific R-II subunit.     

Lithium-induced accumulation of inositol 1-phosphate during cholecystokinin octapeptide- and acetylcholine-stimulated phosphatidylinositol breakdown in dispersed mouse pancreas acinar cells

Dispersed mouse pancreas acinar cells were prepared in which phosphatidylinositol had been labeled with myo[2-3H]inositol. During incubation with 0.3 microM cholecystokinin octapeptide (CCK-8) for 15 min, there was a loss of [3H]phosphatidylinositol radioactivity (23%) and a 3-fold gain in trichloroacetic acid-soluble radioactivity. Replacement of NaCl by up to 58 mM LiCl did not significantly affect the amount of CCK-8-stimulated [3H]phosphatidylinositol breakdown or the gain in acid-soluble radioactivity. However, in normal medium, the product of phosphatidylinositol breakdown was almost all inositol, whereas in Li+-containing medium, the product was almost all inositol 1-phosphate. Similar results were obtained with acetylcholine which, in the presence of Li+, gave a dose-responsive increase in inositol 1-phosphate over the concentration range of 0.1 to 10 microM. No increased accumulation of [3H]inositol diphosphate or [3H]inositol triphosphate was detected in stimulated cells. Time courses in the presence of Li+ indicated that the formation of inositol 1-phosphate preceded the formation of inositol. Addition of up to 50 mM myoinositol to the incubation medium showed no diluting effect on the amount of [3H]inositol 1-phosphate found. The accumulation of inositol 1-phosphate is presumably due to the known ability of Li+ to inhibit myoinositol 1-phosphatase. The results provide clear evidence that stimulated phosphatidylinositol breakdown involves a phospholipase C type of phosphodiesterase activity. 1.25 mM Li+ gave half-maximal inositol 1-phosphate accumulation. This is close to the range of plasma Li+ levels which is used therapeutically in psychiatric disorders. In unstimulated cells, [3H]inositol 1-phosphate accumulation in the presence of Li+ corresponded to a breakdown rate for [3H]phosphatidylinositol of 2 to 3%/h.