Crude Cytosolic Fractions Research Articles

Pentylenetetrazole-induced chemoshock affects protein kinase C and substrate proteins in mouse brain.

Protein kinase C (PKC) activity, western blot analysis of PKC alpha, beta, gamma, epsilon, and zeta by isozyme-specific antibodies, and in vitro phosphorylation of endogenous substrate proteins were studied in the mice brain after pentylenetetrazole-induced chemoshock. The PKC isozymes and endogenous substrates in the crude cytosolic and membrane fractions were partially purified by DE-52 columns eluted with buffer A containing 100 or 200 mM KCl. This method consistently separates cytosolic and membrane proteins and various PKC isoforms. The 100 mM KCl eluates from DE-52 columns contain more PKC alpha and beta in both cytosol and membrane than the 200 mM KCl eluates, whereas PKC gamma, epsilon, and zeta appear in equal amounts in these two eluates. The kinase activity assayed by phosphorylation of exogenous histone was increased in the chemoshocked mice in both the cytosol and membrane of 200 mM KCl eluates. In further analysis by immunoblotting, this increased activity was found to be due to the increase in content of PKC gamma isozyme. As for novel-type epsilon and zeta isozymes, they were not altered in the chemoshocked mice. From autoradiography, the endogenous substrate 17-kDa neurogranin, which was shown below 21 kDa, was mostly eluted by 100 mM KCl from the DE-52 column, whereas 43-kDa neuromodulin, which was also demonstrated in sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis, only appeared in the 200 mM KCl eluates. The in vitro phosphorylation of neuromodulin was found to be increased in the chemoshocked mice. Therefore, the increased phosphorylation of neuromodulin and increased content of the PKC gamma isoform were involved in the pentylenetetrazole-induced chemoshock.

Alterations of protein kinase C isozyme and substrate proteins in mouse brain after electroconvulsive seizures

Protein kinase C (PKC) activity, Western blot analysis of PKCα, β, γ, ε and ζ with isozyme-specific antibodies, endogenous substrate protein phosphorylation, and Western blot analysis of neuromodulin, were studied in mouse brain after repeated electroconvulsive shock. The PKC isozymes and endogenous substrates in the crude cytosolic and membrane fractions were partially purified on DE-52 columns eluted with buffer containing 100 or 200 mM KCl. The kinase activity assayed by phosphorylation of exogenous histone was increased in the 200 mM KCl eluates of both the cytosol and membrane fractions from electroshocked mice. Further analysis by immunoblotting demonstrated that this increased activity was due to an increase in the PKCγ isozyme. The level of the novel type isozymes, ε and ζ, was not altered in electroshocked mice. An in vitro phosphorylation study showed that the endogenous substrate, 17 kDa neurogranin, was mostly eluted by 100 mM KCl. In contrast, the 43 kDa neuromodulin only appeared in the 200 mM KCl eluate, according to autoradiography, SDS-PAGE and Western blot analysis; its level was found to be increased in the membrane fraction of electroshocked mice, as demonstrated by in vitro phosphorylation studies. Therefore, an increase in both PKCγ and neuromodulin contributed to the increased phosphorylation of neuromodulin during electroshock seizure.

Arachidonyl trifluoromethyl ketone, a potent inhibitor of 85-kDa phospholipase A2, blocks production of arachidonate and 12-hydroxyeicosatetraenoic acid by calcium ionophore-challenged platelets.

Arachidonyl trifluoromethyl ketone (AACOCF3) is a potent and selective slow binding inhibitor of the 85-kDa cytosolic phospholipase A2 (cPLA2) (Street, I. P., Lin, H.-K., Laliberté, F., Ghomashchi, F., Wang, Z., Perrier, H., Tremblay, N. M., Huang, Z., Weech, P. K., and Gelb, M. H. (1993) Biochemistry 32, 5935-5940). AACOCF3 and a number of its structural analogues have been used to investigate the role of cPLA2 in the cellular generation of free arachidonic acid (AA) and in eicosanoid biosynthesis. AACOCF3 inhibited the release of AA from calcium ionophore-challenged U937 cells (IC50 = 8 microM, 2 x 10(6) cells ml-1) and from platelets (IC50 = 2 microM, 4 x 10(7) cells ml-1). Arachidonyl methyl ketone (AACOCH3) and AACH(OH)CF3, both of which are noninhibitory to the purified cPLA2, did not inhibit the production of AA in the ionophore-challenged cells. In addition to the release of AA, AACOCF3 also inhibited the production of 12-hydroxyeicosatetraenoic acid (12-HETE) and thromboxane B2, two of the major metabolites of AA produced by platelets. The inhibition of 12-HETE biosynthesis showed a dose dependence similar to that of AA release in ionophore-challenged platelets; however, when platelet 12-HETE production was stimulated with 10 microM AA to circumvent the PLA2-dependent step, AACOCF3 no longer inhibited the production of 12-HETE. In contrast, AACOCF3 blocked thromboxane B2 formation by both calcium ionophore- and AA-challenged platelets, indicating that the compound affects the cyclooxygenase pathway in addition to AA release. The crude cytosol and membrane fractions from platelets were assayed for calcium-dependent and calcium-independent PLA2 activities and for the susceptibility of each to inhibition by AACOCF3. At AACOCF3 concentrations as high as 10 mol %, only one of the observed PLA2 activities was inhibited by more than 25%. The AACOCF3-susceptible PLA2 (77% inhibition at 1.6 mol %) was found in the cytosolic platelet fraction and showed the functional characteristics of the cPLA2. These results suggest that the cPLA2 plays an important role in the generation of free AA for 12-HETE biosynthesis in platelets.

Affinity of hepatic glucocorticoid receptors is influenced by energy/feeding status

Genetically obese animals have been shown to have a reduced number and affinity of glucocorticoid receptors. The relationship between the alterations in receptor binding and the regulation of energy balance is not known. We sought to determine the role of body energy/feeding status on the binding characteristics of glucocorticoid receptors. To accomplish this, we examined the effect of long-term food restriction on the number and affinity of hepatic glucocorticoid receptors from Sprague-Dawley rats. After 3 weeks of food restriction (40% of ad lib), animals were bilaterally adrenalectomized. Livers were removed, a crude cytosolic glucocorticoid receptor fraction was isolated, and radioreceptor assays were performed. Glucocorticoid receptors from food-restricted rats showed a significant reduction in the dissociation constant ( K d) as compared to receptors derived from free-feeding controls. No difference in receptor number was observed. These results suggest that energy or feeding status of the animal may influence the affinity of hepatic glucocorticoid receptors, while receptor number may be independent of this status.

Self-peptides from four HLA-DR alleles share hydrophobic anchor residues near the NH2-terminal including proline as a stop signal for trimming.

Naturally processed MHC class II-associated peptides proved to be heterogeneous in size, varying from 13 to 25 amino acids. Truncation variants suggested sequence motifs that afford the amino termini to be shifted for obtaining an alignment: a 9- to 11-residue core region that is bordered by primary anchor residues is surrounded by extra sequences of variable lengths and hitherto unknown functions. Herein we present bulk sequencing analyses of self-peptides from four HLA-DR alleles and HLA-DQw7 clearly showing that the length of most of the NH2-terminal preanchor sequence is limited to 1 to 3 residues. Most strikingly, proline is the dominant residue reappearing at positions 2 and 3 in any allele. Proline revealed to function as a stop signal for NH2-terminal trimming as well as a secondary anchor: crude cytosolic and endosomal peptide fractions could be processed by aminopeptidases in vitro, whereupon DR1 binding peptides with increased affinity were generated. In addition, aminopeptidase treatment of DR1: self-peptide complexes implied that proline together with sterical constraints of the MHC molecule do protect the peptides' NH2-termini from further processing, whereas their COOH-termini were accessible to cathepsin B processing. Finally, bulk sequencing profiles contained signals from further putative anchor residues clustering in the NH2-terminal region:tyrosine, phenylalanine, leucine, isoleucine, and valine are enriched at positions 2 to 4 in DR1, DR5, and DR6, however, at positions 4 to 6 in DR3. Isotype-specificity is demonstrated by DQw7 displaying glutamine and asparagine at position 2. Obviously, the degenerate occurrence of aromatic or aliphatic side chains close to the NH2-terminal guarantees for essential interactions with a hydrophobic pocket of the investigated DR molecules. Most probably, this pocket is located in the nonpolymorphic DR alpha-chain rationalizing previous findings of promiscuous peptide binding to different DR alleles.

A possible pathway of phosphoinositide metabolism through EDTA-insensitive phospholipase A1 followed by lysophosphoinositide-specific phospholipase C in rat brain.

Incubation of [2-3H]glycerol-labeled phosphatidylinositol with a crude cytosol fraction of rat brain in the presence of EDTA yielded [3H]lysophosphatidylinositol predominantly without accumulation of labeled monoacylglycerol and diacylglycerol. The pH optimum of this phospholipase A activity was 8.0. The activity for phosphatidylinositol was twofold higher than for phosphatidylethanolamine, whereas phosphatidylcholine, phosphatidylserine, and phosphatidic acid were not hydrolyzed significantly under the conditions used. The phospholipase A activity for phosphatidylethanolamine was resolved in part from that for phosphatidylinositol by ammonium sulfate fractionation of the cytosol, indicating the existence of at least two forms of EDTA-insensitive phospholipase A. The positional specificity of the phosphatidylinositol-hydrolyzing activity was found to be that of a phospholipase A1, as radioactive lysophosphatidylinositol was produced from 1-stearoyl-2-[1-14C]arachidonyl-sn-glycero-3-phosphoinositol without release of free arachidonate. A phospholipase C activity specific for lysophosphoinositides was found in a membrane fraction from rat brain, which was similar to that characterized in porcine platelets. The phospholipase C was demonstrated to hydrolyze the 2-acyl isomer as well as the 1-acyl isomer of lysophosphatidylinositol. Taken together, our results suggest a possible pathway through which phosphatidylinositol is selectively degraded to the 2-acyl isomer of lysophosphatidylinositol in a Ca(2+)-independent manner, and subsequently converted to a 2-monoacylglycerol in rat brain.

The kinetics of mebendazole binding to Haemonchus contortus tubulin

The kinetics of the binding of mebendazole (MBZ) to tubulin from the third-stage (L3) larvae of the parasitic nematode. Haemonchus contortus, have been characterized. In partially purified preparations, the association of [ 3H]MBZ to nematode tubulin was rapid, k 1 = (2.6 ± 0.3) × 10 5 m 1min 1, but dissociation was slow, k 1, = (1.58 ± 0.02) × 10 3min 1. The affinity constant ( K a) for the interaction, determined by the ratio k 1/ k 1, was (1.6 ± 0.2) × 10 8 m 1. Similar results were obtained with crude cytosolic fractions. In equilibrium studies, performed with partially purified nematode tubulin under similar conditions, a K a of (5.3 ± 1.6) × 10 6 m 1 was obtained. The best estimate for the K a of the MBZ-nematode tubulin interaction is considered to be the ‘kinetic’ value determined from the ratio of rate constants. The slow dissociation of MBZ from nematode tubulin. which contrasts with the rapid dissociation of MBZ from mammalian tubulin, supports the hypothesis that the selective toxicity of the benzimidazole anthelmintics results from a difference between the affinities of mammalian and nematode tubulins for these drugs.

The major endogenous bovine brain protein kinase C inhibitor is a heat-labile protein

A crude cytosolic fraction prepared from bovine brain contained protein kinase C, as shown by immunoblotting, but its activity was undetectable, suggesting the presence of interfering factors. Phosphatase, ATPase and protease activities did not account for the absence of detectable protein kinase C activity. The major contributing factor was found to be a heat-labile protein which was separated from the kinase by ion-exchange chromatography. The contribution to the total inhibitory activity of heat-stable proteins was relatively minor, suggesting that they may not function physiologically as protein kinase C inhibitors.

A protein kinase C inhibitory activity is present in the human T lymphoblast cell line Jurkat

The human lymphoblast cell line Jurkat is widely used as a model system for studying signal transduction pathways during lymphocyte activation. We report the presence of a potent endogenous inhibitor of protein kinase C (PKC) in the cytosolic fraction of Jurkat cells. This inhibitor is not diffusible and is thermolabile; it is assumed to be a protein. It was separated from PKC by ion-exchange chromatography on DEAE-cellulose. The inhibitory activity was partially reversed by increasing the concentration of the PKC substrate; increasing that of PKC activators (calcium and phospholipids) was without effect. PKC activity was inhibited by more than 90% in the crude cytosolic fraction but the inhibition could be completely reversed by diluting the cell extract. This inhibitory activity could not be detected in the cytosol from normal lymphocytes or from lymphoblasts from leukemic patients.

Characterization of nitric oxide synthases in non‐adrenergic non‐cholinergic nerve containing tissue from the rat anococcygeus muscle

Tissue homogenates prepared from rat anococcygeus muscle converted L-arginine to L-citrulline indicating the presence of nitric oxide (NO) synthase. NO synthase activity was also found in crude and partially-purified soluble and particulate fractions prepared from the homogenates. Both soluble and particulate NO synthase were dependent on NADPH, 5,6,7,8-tetrahydrobiopterin and calcium, and inhibited by NG-nitro-L-arginine. Tissue homogenates or crude cytosolic and membrane fractions from rat vas deferens, which does not contain NO releasing non-adrenergic non-cholinergic neurones, had no NO synthase activity.

Kinetic properties of a high molecular mass arachidonoyl-hydrolyzing phospholipase A2 that exhibits lysophospholipase activity

The first step in the production of eicosanoids and platelet-activating factor is the hydrolysis of arachidonic acid from membrane phospholipid by phospholipase A2. We previously purified from the macrophage cell line RAW 264.7 an intracellular phospholipase A2 that preferentially hydrolyzes sn-2-arachidonic acid. The enzyme exhibits a molecular mass of 100 kDa and an isoelectric point of 5.6. When assayed for other activities, the phospholipase A2 was found to exhibit lysophospholipase activity against palmitoyllysoglycerophosphocholine, and both activities copurified to a single band on silver-stained sodium dodecyl sulfate-polyacrylamide gels. An antibody against the macrophage enzyme was found to quantitatively immunoprecipitate both phospholipase A2 and lysophospholipase activities from a crude cytosolic fraction. When the immunoprecipitated material was analyzed on immunoblots, a single band at 100 kDa was evident, further suggesting that a single protein possessed both enzyme activities. When assayed as a function of palmitoyllysoglycerophosphocholine concentration and plotted as a double-reciprocal plot, two different slopes were apparent, corresponding to concentrations above and below the critical micellar concentration (7 microM) of the substrate. Above the critical micellar concentration, lysophospholipase exhibited an apparent Km of 25 microM and a Vmax of 1.5 mumol/min/mg. Calcium was not required for lysophospholipase activity, in contrast to phospholipase A2 activity. The enzyme, when assayed as either a phospholipase A2 or lysophospholipase, exhibited nonlinear kinetics beyond 1-2 min despite low substrate conversion. Readdition to more substrate after the activity plateaued did not result in further enzyme activity, ruling out substrate depletion. Readdition of enzyme, however, resulted in another burst of enzyme activity. The results are not consistent with product inhibition, but suggest that the enzyme may be subject to inactivation during catalysis.

Evidence for one or more Raf-1 kinase kinase(s) activated by insulin and polypeptide growth factors

The protein product of the Raf-1 proto-oncogene is a protein serine/threonine kinase that is activated after stimulation of cells with insulin and other mitogens. To investigate the mechanism of this activation, we used purified Raf-1 expressed in E. coli as a substrate for a putative Raf-1 protein kinase kinase. In three different insulin-sensitive cell types, insulin activated Raf-1 kinase kinase activity in crude cytosolic cellular fractions. The insulin stimulation of this activity was evident as early as 2 min after exposure to insulin, maximal at 5-8 min, and inapparent at 15 min. Phosphoamino acid analysis of phosphorylated Raf-1 revealed that serine was the primary phosphate acceptor for the insulin-activated kinase or kinases; small amounts of phosphothreonine were also detected. The insulin effect occurred in cells depleted of protein kinase C, and in extracts depleted of endogenous Raf-1 kinase by immunodepletion; these data argue against protein kinase C or Raf-1 kinase itself being the insulin-stimulated activity. The insulin-activated kinase or kinases phosphorylated the Raf-1 protein on multiple sites in vitro, as evidenced by tryptic mapping; at least some of these appeared to overlap with sites phosphorylated in response to serum in intact cells. Several other mitogens and growth factors stimulated Raf-1 kinase kinase activity, including epidermal growth factor, platelet-derived growth factor, fibroblast growth factor, serum, and phorbol 12-myristate 13-acetate. This insulin- and mitogen-stimulated Raf-1 kinase kinase activity may play a role in mediating the phosphorylation and possibly the activation of the Raf-1 kinase by insulin and other growth factors.

Cytoplasmic 8 S glucocorticoid receptor binds to actin filaments through the 90-kDa heat shock protein moiety

The glucocorticoid receptor exists in the cytoplasm of hormone-untreated cells as a complex with the 90-kDa heat shock protein (HSP90). Glucocorticoids induce dissociation of the glucocorticoid binding protein from HSP90 and translocation of the receptor to the nucleus. HSP90 binds to actin filaments, and calmodulin or tropomyosin inhibits the binding. We present here evidence that the HSP90-containing glucocorticoid receptor complexes (8 S receptor) bind to filamentous actin in vitro while the HSP90-free form of the receptor does not. The binding was detectable for both the crude cytosolic fractions and the partially purified 8 S glucocorticoid receptor. Purified HSP90 or tropomyosin completely abolished the binding. Calmodulin also inhibited the binding in a Ca(2+)-dependent manner. From these results, we conclude that the glucocorticoid receptor complex is able to bind actin filaments via the HSP90 moiety. The binding may provide an anchoring mechanism for the glucocorticoid receptor in the cytoplasm.

A myo-inositol d-3 hydroxykinase activity in Dictyostelium

A soluble ATP-dependent enzyme which phosphorylates myo-inositol has been characterized in Dictyostelium. The myo-inositol kinase activity was partially purified from amoebae by chromatography on DEAE-Sepharose and phenyl-Sepharose columns. The product of both the partially purified activity and of a crude cytosolic fraction was myo-inositol 3-phosphate. The partially purified preparations of myo-inositol kinase (a) possessed a Km for myo-inositol of 120 microM (in the presence of 5 mM-ATP) and for ATP of 125 microM (in the presence of 1 microM-myo-inositol), (b) did not recognize allo-, epi-, muco-, neo-, scyllo-, 1 D-chiro or 1 L-chiro-inositol as substrates, (c) were competitively inhibited by three naturally occurring analogues of myo-inositol: 1 L-chiro-inositol (Ki 49.5 +/- 0.7 microM: the structural equivalent of myo-inositol, except that the D-3 hydroxy moiety is axial), D-3-deoxy-myo-inositol [Ki 103 +/- 1 microM: (-)-viburnitol], and sequoyitol (Ki 271 +/- 7 microM; unlike 1 L-chiro-inositol and D-3-deoxy-myo-inositol, this was a substrate for the kinase), and finally (d) were apparently non-competitively inhibited by myo-inositol 3-phosphate. The product of myo-inositol kinase could be detected in intact amoebae and was a substrate for the first in a series of inositol polyphosphate kinases present in Dictyostelium which ultimately yield myo-inositol hexakisphosphate. The activity of myo-inositol D-3-hydroxykinase in Dictyostelium lysates showed evidence of developmental regulation.

Evidence for In Vivo Synthesis of Thiamin Triphosphate by Cytosolic Adenylate Kinase in Chicken Skeletal Muscle

We showed previously that cytosolic adenylate kinase (AK1) purified from pig skeletal muscle catalyzes in vitro formation of thiamin triphosphate (TTP) from thiamin diphosphate (TDP) and ADP in addition to ATP formation from ADP [Shikata, H. et al. (1989) Biochem. Int. 18, 933-942]. To obtain evidence for in vivo synthesis of TTP by AK1, changes in TTP content and AK1 activity were determined in chicken skeletal muscle during development after hatching. Thiamin phosphate metabolism in chicken skeletal muscle was also studied. i) An extremely high TTP content, 81% of total thiamin (thiamin plus thiamin phosphates), was detected in the white (fast-twitch) muscle of adult normal chicken (5th to 9th month) compared with a relatively high TTP content of 31% in the red (slow-tonic) muscle. Since approximately equivalent amounts of total thiamin were present in the two types of muscle, the ratio of TTP to TDP was high (5.0) in the white muscle and low (0.41) in the red muscle. ii) Rabbit anti-chicken AK1 antiserum against the purified chicken cytosolic AK1 preparation was obtained. Both AK1 activity and TTP-synthesizing activity in crude cytosol fraction of adult chicken white muscle were inhibited in parallel by the antiserum. iii) In the white muscle of normal chicken, the TTP content and AK1 activity responsible for forming either ATP or TTP were increased in a parallel manner up to day 16 after hatching, after which both remained constant. In the red muscle, on the other hand, both the TTP content and the AK1 activity were low in comparison with those in the white muscle, and were almost constant after hatching.(ABSTRACT TRUNCATED AT 250 WORDS)

Affinity chromatography of estrogen- and progesterone-binding proteins of human uterus

The purification of estrogen- and progesterone-binding proteins of human uterus by employing affinity resins coupled with steroid-bovine serum albumin conjugates, led to the isolation of preparations with estrogen- and progesterone-binding sites havingKd values in the range of 0.96 to 1.20 × 10-9 M. These were different from theKd values of 10-10 M and 10-8 M obtained for two types of binding sites present in the crude cytosolic and nuclear fractions. The purified proteins sedimented on sucrose gradient withS values in the range of 3.6–4.4.

Identification of the clathrin assembly protein AP180 in crude calf brain extracts by two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis

We present a two-dimensional gel electrophoretic method which affords a diagnostic means for the identification of the neuron-specific clathrin assembly protein AP180 in crude cytosolic and microsomal fractions of bovine brain. The method is based on the finding that in the presence of sodium dodecyl sulfate (SDS) in a newly developed continuous high salt Tris-acetate-EDTA buffer system protein AP180 migrates at a rate corresponding to its molecular weight of ‖120,000, while in other more commonly used SDS-polyacrylamide gel electrophoresis methods it behaves anomalously as a 170- to 180-kDa polypeptide. By combining electrophoresis in the Tris-acetate-EDTA system in the first dimension with either the electrophoretic system of Laemmli [Laemmli, U. K. (1970) Nature (London) 227, 680–685] or that of Neville [Neville, D. M. (1971) J. Biol. Chem. 246, 6328–6334] in the second dimension, it is possible to identify AP180 in complex protein mixtures, because it is the only major protein that fell significantly off a diagonal defined by other proteins. A comparison of the microsomal and soluble fractions examined in this manner reveals that most of the AP180 is present in the soluble fraction.

Kinetic characteristics of a thioredoxin-activated rat hepatic and renal low-Km iodothyronine 5'-deiodinase.

The properties and kinetic characteristics of a non-GSH NADPH-dependent cofactor system activating rat hepatic and renal 5'-deiodinase (5'-DI), which we have previously demonstrated with partially purified cytosol Fractions A and B [Sawada, Hummel & Walfish (1986) Biochem. J. 234, 391-398], were examined further. Although microsomal fractions prepared from either rat liver or kidneys could be activated by crude cytosol Fractions A and B from those tissues as well as from rat brain and heart, a homologous hepatic or renal system was the most potent in producing 5'-deiodination of reverse tri-iodothyronine (rT3). At nanomolar concentrations both rT3 and thyroxine (T4) were deiodinated but with a much greater substrate preference for rT3 than for T4. However, at micromolar concentrations of these substrates no activation of 5'-DI could be detected. In this deiodinative system, T4 and tri-iodothyronine (T3) competitively inhibited 5'-deiodination of rT3. Dicoumarol, iopanoate, arsenite and diamide were also inhibitory to the activation of hepatic or renal 5'-deiodination by this cofactor system. Purification of cofactor components in hepatic crude cytosolic Fractions A and B to near homogeneity, as assessed by their enzymic and physical properties, indicated that these co-purified with and were therefore identical with thioredoxin reductase and thioredoxin respectively, and accounted almost entirely for the observed activation of rT3 5'-DI. When highly purified liver cytosolic thioredoxin reductase and thioredoxin were utilized to determine the kinetic characteristics of the reaction, evidence for a sequential mechanism operative at nanomolar but not micromolar concentrations of rT3 and T4 was obtained. The Km for rT3 was 1.4 nM. Inhibition by 6-n-propyl-2-thiouracil (Ki 6.7 microM) was competitive with respect to thioredoxin and non-competitive with respect to rT3, whereas inhibition by T4 (Ki 1.3 microM) was competitive. Since rT3 is a potent inhibitor of T4 5'-deiodination, this thioredoxin system activating deiodination of rT3 may play an important role in regulating the rate of intracellular production of T3 from T4.

Subcellular distribution of protein kinase C in rat adrenal glomerulosa cells

With the aid of a synthetic nonapeptide which is a selective substrate for protein kinase C the activity of this enzyme was determined in the crude cytosolic and particulate fractions of rat adrenal glomerulosa cells. When the cells were sonicated in the presence of Ca2+ chelators 65 per cent of their total protein kinase C activity was found in the cytosolic extract. The treatment of cells with angiotensin II under conditions where the maximal stimulation of inositol-lipid hydrolysis was observed did not cause a statistically significant change in the apparent subcellular distribution of protein kinase C. However, when the cytosolic extract was prepared in the presence of Ca2+ the protein kinase C activity was recovered nearly exclusively from the particulate fraction.

Regulation of protein kinase C by cyclic adenosine 3':5'-monophosphate and a tumor promoter in skeletal myoblasts.

The specific activity of protein kinase C in rat skeletal myoblasts decreased when they were exposed for very short periods to isoproterenol, forskolin, dibutyryl cyclic AMP (Bt2cAMP), or the phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA). In the presence of Bt2cAMP or forskolin only the cytosolic but not the membrane-bound kinase activity was found to decrease. Treatment with TPA, however, led to a decrease in the activity of the enzyme both in the cytosolic as well as the membrane fractions. The effects observed in vivo could be duplicated in crude extracts of myoblasts incubated with cAMP analogues or TPA. In the presence of ATP, protein kinase C activity decreased considerably in crude cytosolic fractions treated with the cAMP analogues, but a requirement for ATP was not evident for the decrease in activity brought about by TPA. For the cAMP analogues the decrease in protein kinase C was also prevented by incubation of the extracts with an inhibitor of cAMP-dependent protein kinase. The regulation of protein kinase C by Bt2cAMP (but not by TPA) was altered in Rous sarcoma virus-transformed myoblasts. It is considered likely that a component affected by cAMP (probably a substrate for cAMP-dependent protein kinase) participates in the regulation of protein kinase C activity, and it is altered in unknown ways in transformed myoblasts.