Crude Cytosolic Fractions Research Articles

Interleukin 2 and diacylglycerol stimulate phosphorylation of 40 S ribosomal S6 protein. Correlation with increased protein synthesis and S6 kinase activation.

Interleukin 2 (IL-2) and the synthetic diacylglycerol, 1-oleoyl-2-acetylglycerol (OAG), a direct activator of protein kinase C, induce phosphorylation of the ribosomal S6 protein in a murine IL-2-dependent lymphocyte clone. The phosphorylation of S6 protein was correlated with increased protein synthesis in this cell line. Using cell-free assay systems, two unique kinases capable of phosphorylating the S6 protein were identified, namely, a calcium/phospholipid-dependent phosphotransferase, protein kinase C, and a second phospholipid-independent kinase detected in crude cytosolic fractions. Peptide mapping of the S6 protein demonstrated that the degree of S6 phosphorylation stimulated by IL-2 and OAG was similar to that achieved using the second (calcium/phospholipid-independent) kinase but not to the level of phosphorylation achieved with protein kinase C. The kinase responsible for phosphorylating S6 was soluble in stimulated cells and was induced in a time-dependent manner by either IL-2 or diacylglycerol treatment of intact cells. These data support the notion that, although protein kinase C is activated by IL-2 or OAG, subsequent events such as S6 phosphorylation may be the result of the activation of secondary phosphotransferase systems regulated by protein kinase C.

Differential effects of manoalide on secreted and intracellular phospholipases

Manoalide, a novel nonsteroidal sesterterpenoid, is a potent inhibitor of phospholipase A 2 isolated from bee and cobra venoms. This report compares the inhibition by manoalide of phospholipase A 2 in crude cytosol fractions from four mammalian tissues with that of four purified extracellular phospholipase A 2's. Phospholipase A 2 isolated from bee venom ( Apis mellifera) was the most sensitive to inactivation by manoalide ( ic 50 ⋍ 0.12 μ M ). Extracellular phospholipase A 2 from rattlesnake and cobra venom was intermediate in sensitivity to manoalide ( ic 50 values of 0.7 and 1.9 μM respectively). Porcine pancreatic phospholipase A 2 was relatively resistant to inactivation by manoalide ( ic 50 ⋍ 30 μ M ). The phospholipase A 2 assayed in crude cytosol fractions from four mammalian tissues exhibited ic 50 values of 30 μM or greater. Cytosolic proteins as well as bovine serum albumin and poly-L-lysine ( Mr = 57,000) protected purified bee venom phospholipase A 2 from inactivation by manoalide. In contrast, amino acids such as lysine and alanine failed to protect the purified enzyme from inactivation. Proteins and certain amino acids, such as lysine, formed a chromogenic product when incubated with manoalide. These data suggest that lysine is capable of reacting with manoalide, but only when it is present in macromolecules is it capable of protecting phospholipase A 2 from inactivation by manoalide. Because cellular proteins protect PLA 2 from inactivation by manoalide, high concentrations of manoalide must be applied topically to produce statistically significant inactivation of intracellular phospholipase A 2. Finally, a chemical model is presented which explains the formation of a chromogenic product when manoalide is incubated with proteins and amino acids.

Phosphorylation of a high molecular weight DNA polymerase alpha.

Anti-human DNA polymerase alpha murine IgG SJK-287-38 [Tanaka, S., Hu, S.-Z., Wang, T. S.-F. & Korn, D. (1982) J. Biol. Chem. 257, 8386-8390] neutralized DNA polymerase alpha activity from rat embryonic fibroblasts infected with a temperature-sensitive transformation mutant of Rous sarcoma virus (tsLA24). After centrifugation of a crude cytosol fraction from log-phase cells in a 5-20% linear sucrose gradient, polypeptides of Mr approximately equal to 185,000 and 220,000 were immunoprecipitated only from gradient fractions containing DNA polymerase alpha activity. When similar cultures were incubated in medium containing [32P]orthophosphate, it was found that the Mr 220,000 protein was phosphorylated but that the other peptides specific for polymerase alpha activity did not contain detectable amounts of phosphate. Phospho amino acid analysis of the high molecular weight immunoprecipitable proteins indicated that the labeled amino acid was phosphoserine. Incubation of 2.5 units of crude DNA polymerase alpha with 4 units of agarose-immobilized alkaline phosphatase resulted in a nearly complete inhibition of DNA polymerase alpha activity. Subsequent incubation of this preparation with 5 or 50 microM ATP, but not the nonhydrolyzable analog adenosine 5'-[gamma-thio]triphosphate, restored the in vitro DNA polymerizing activity. These results demonstrate that a high molecular weight DNA polymerase alpha (Mr approximately equal to 220,000) is phosphorylated in cultured cells and that this protein is a substrate for a serine kinase rather than the tyrosine-specific protein kinase of Rous sarcoma virus. The results suggest that phosphorylation/dephosphorylation reactions modulate the activity of this polymerase.

Newly synthesized Na,K-ATPase alpha-subunit has no cytosolic intermediate in MDCK cells.

Recently published data indicate that the alpha-subunit of Na,K-ATPase, a transmembrane protein of animal cell plasma membranes, is synthesized as a soluble precursor. In the present experiments we demonstrate that an apparent "soluble" form can indeed be detected in crude cytosolic fractions prepared by centrifugation from MDCK cells disrupted by sonication. We find, however, that this form has no precursor-product relationship with membrane-associated alpha-subunit. The quantity of unsedimentable alpha-subunit can be greatly diminished by increasing the centrifugal field employed to remove membranous vesicles from the cytosol fraction. Sonication of membrane pellets generates alpha-subunit which, like the "soluble" form, resists pelleting. Finally, cytosol fractions prepared from cells disrupted by sonication contain membrane-bound vesicles which can be immunoadsorbed on Staphylococcus aureus cells coated with a monoclonal antibody directed against alpha-subunit. We find, therefore, that the previously observed soluble precursor of alpha-subunit is actually a component of small unpelleted membrane vesicles generated by harsh disruption conditions. When cells are disrupted by less violent techniques no newly synthesized alpha-subunit can be detected in the cytosol fraction. We calculate that to escape detection under our experimental conditions a bona fide soluble precursor of alpha-subunit must have a cytosolic t1/2 less than 20 s. We conclude that the alpha-subunit is most probably inserted into the bilayer cotranslationally.

Adenosine 3',5'-monophosphate (cAMP)-binding protein and cAMP-dependent protein kinase in human placenta.

cAMP modulates estrogen, hCG, and lactate syntheses by human placenta, cAMP presumably exerts its major intracellular effect by binding to cAMP-dependent protein kinase (cAMP-PK), which, in turn, phosphorylates regulatory proteins within the target cell. cAMP binding and cAMP-PK have not been previously identified in placenta. [3H]cAMP binding to crude cytosol fractions of term placenta was rapid, saturable, and reversible. Scatchard analyses of saturation experiments of [3H]cAMP binding to placental cytosol were linear (Kd = 1.13 +/- 0.11 x 10(-8) M; n = 5). The binding capacity was 1.27 +/- 0.18 pmol/mg protein. Competition for the [3H]cAMP-binding site followed the potency order cAMP much greater than cGMP much greater than (Bu)2cAMP, analogous to cAMP binding to cAMP-PK in other tissues. ADP, ATP, and adenosine did not compete for the [3H]cAMP-binding site. cAMP significantly enhanced phosphorylation of histone protein by placental cytosol (activity ratio, 0.57 +/- 0.04; P less than 0.01). Two peaks of [3H]cAMP binding and coincident cAMP-PK activity were identified by DEAE-cellulose column chromatography of placental cytosol corresponding to classical type I and type II cAMP-PK. While the majority of the cAMP-PK was found in placental cytosol, cAMP-PK was also demonstrated in crude microsomal and microvillous brush border membranes of human placenta after solubilization with Triton X-100 (P less than 0.05). Regulation of placental function by catecholamines and other hormones known to mediate cAMP levels may be accomplished through the phosphorylation of cellular proteins by cAMP-dependent protein kinases.

50] Lipid transfer proteins: Overview and applications

This chapter provides an overview of lipid transfer proteins and its application. The chapter discusses various purified lipid transfer proteins, their molecular weights, and their isoelectric points. Both beef and rat liver cytosol contain a transfer protein that is quite specific for the transfer of phosphatidylcholine. Examples of animal and plant tissues that appear to contain lipid transfer activities are discussed. The lipid transfer activities are widely distributed among cell types. Thus far, there is no indication as to whether a specific type of transfer activity is associated with a particular cell type or with a specialized cell function. The transfer activity of a purified transfer protein, or of a crude cytosol fraction, is measured by determining the transfer of an isotopically labeled lipid from a donor to an acceptor particle. The donor or acceptor may be an intact cell, an organelle, a plasma membrane, a synthetic vesicle, a lipoprotein, or a monolayer. Transfer proteins are used as modifiers of membrane composition to study the dependence of enzyme activity on membrane lipid composition. The phosphatidylcholine-specific transfer protein is used to supply labeled phosphatidylcholine to various membrane fractions to study the conversion of phosphatidyl[ 3 H]choline to sphingomyelin.

Somatostatin and vitamin D3 metabolites in rat calvarium: in vitro evidence for physiological interaction.

The hypotheses that bone is a target organ for somatostatin and that vitamin D3 metabolites interact with this peptide on bone were tested in vitro, using calvaria from newborn rats with the following results. 1) [125I]iV-Tyr1 -somatostatin bound specifically to intact calvaria tissue and cells (0.1–7.5% at the 30th–50th min of incubation) and to the crude cytosol fraction (23.6 ± 4.8% of the total radioactivity incubated with cytosol). Three binding sites with different affinities could be detected; the Kd of one of them was 1.17 ± 0.17 × 10-10 M, with a binding site capacity of 0.34 ± 0.05 pmol/mg protein. 2) Low doses of somatostatin (6 × 10-9 to 3 × 10-7 M) affected the cAMP content of calvaria incubated in a protein-free medium for 5–60 min. The somatostatin-induced change was calcium dependent; there was an increase in cAMP with 0 or 1 mM calcium and a decrease with 3 mM calcium. 3) Somatostatin and vitamin D3 metabolites influenced the effects of one another on cAMP content and phosphatase activities. In the presence of 2.4 × 10-9 M 1,25-dihydroxyvitamin D3, the somatostatin affected cAMP levels at a concentration (6 × 10-11 M) that was 100 times lower than that necessary in the absence of 1,25-dihydroxyvitamin D3. The cAMP response to somatostatin was also influenced by 24,25-dihydroxyvitamin D3 (2.4 × 10-9 M), but in a different manner, with a decrease rather than an increase using 1 mM calcium. The stimulation of phosphatase activities by both vitamin D3 metabolites was decreased by somatostatin. These results support the hypotheses of a physiological role for somatostatin on bone and an interaction of this peptide with vitamin D3 metabolites.

Carbohydrate energy metabolism in Fasciola gigantica (trematoda)

Umezurike G. M. and Anya A. O. 1980. Carbohydrate energy metabolism in Fasciola gigantica (Trematoda). International Journal for Parasitology 10: 175–180. Adult Fasciola gigantica contained 4.49 ± 0.06 % (mean ± S.D.) wet weight glycogen. Tissue homogenates contained high levels of malate dehydrogenase (MDH), NAD-linked malic enzyme (ME), Phosphoenolpyruvate carboxykinase (PEPCK) and lactate dehydrogenase (LDH). MDH, PEPCK and ME activities appeared to be localized in both cytosolic and mitoehondrial fractions, fumarase activity appeared to be predominantly mitochondrial whereas LDH and pyruvate kinase activities were cytosolic in distribution. Polyacrylamide gel electrophoresis revealed the predominance of LDH-1, LDH-2 and LDH-3 but only traces of LDH-4 and LDH-5 isoenzymes in the crude cytosolic fraction. LDH activity in the crude sample was inhibited by excess substrate (pyruvate). The mitoehondrial system showed NADH -cytochrome c oxidoreductase, succinate-cytochrome c oxidoreductase, NADH oxidase and some cytochrome c-oxygen oxidoreductase activities. Under anaerobic conditions, NADH-fumarate oxidoreductase and succinate-NAD + oxidoreductase activities of mitoehondrial preparations were stimulated in the presence of ADP and ATP respectively. Isolated mitochondria contained rhodoquinone and no ubiquinone, and isolated rhodoquinone was readily reduced by succinate in the presence of submitochondrial particles. Hydrogen peroxide was produced by submitochondrial particles in the presence or absence of KCN or in the presence of fumarate.

Regulation of microsomal stearoyl-coenzyme A desaturase. Purification of a non-substrate-binding protein that stimulates activity.

Crude cytosolic fraction from rat liver was examined for proteins that may be involved in regulation of microsomal stearoyl-CoA desaturase activity. Gel filtration revealed the presence of several components that either stimulate or inhibit this enzyme. In addition, other components bind the acyl-CoA substrate, thus affecting observed activities in vitro. A protein that stimulates stearoyl-CoA desaturase but does not bind substrate was purified approx. 1100-fold. The purified protein had no visible absorption spectrum and an approximate mol.wt. of 26500. Maximal stimulation of desaturase activity occurred with less than 2 micrometer purified protein. The protein was heat-labile and exhibited neither catalase nor glutathione peroxidase activity. Addition of the cytosolic protein produced no effect on the desaturase reaction stoicheiometry; the proportions O2 consumed/NADH oxidized/stearoyl-CoA desaturated remained 1:1:1. Because the Km' for stearoyl-CoA was also unchanged by addition of the cytosolic protein, no change in substrate affinity was suggested. Furthermore addition of the cytosolic protein also produced no effect on desaturase inhibition by oleoyl-CoA, which suggested that the protein does not simply relieve apparent product inhibition. These results indicate that, in analogy to other cytosolic proteins that stimulate microsomal oxidase activities, the protein may have a regulatory function, perhaps related to activity modulation via organization of the multienzymic desaturase in the membrane.