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J J Sando

Publications and source records attributed to J J Sando.

52 records · Page 3Linked to original sources
PubMed1985

Stimulation of porcine jejunal ion secretion in vivo by protein kinase-C activators.

Microbial toxins act through cyclic nucleotide dependent (cAMP or cGMP) or cyclic nucleotide independent pathways to cause intestinal ion secretion. To explore the calcium dependent, cyclic nucleotide independent pathway that is postulated to involve protein kinase C activation, we measured protein kinase C activity and phorbol ester binding in isolated intestinal epithelial cells and examined the effects of the C-kinase activators, phorbol myristate acetate, phorbol dibutyrate, and 4-beta-phorbol-12,13-didecanoate, in weaned pig jejunum in vivo. We demonstrated both protein kinase C activity and specific phorbol ester binding in porcine jejunal epithelial cells. Phorbol myristate acetate, phorbol dibutyrate, and 4-beta-phorbol-12,13-didecanoate (10(-5) M) each caused striking secretory responses at 5 h with accumulation of Na+, K+, Cl-, and HCO3- intraluminally. In contrast, 4-alpha-phorbol and 4-alpha-phorbol-12,13-didecanoate, which do not affect protein kinase C, allowed normal net absorption of all electrolytes from the intestinal lumen equivalent to controls with only Ringer's lactate. Time course studies revealed significant secretion within 30 min after exposure to the C-kinase activators. These data suggest an important role for protein kinase C activation in intestinal ion secretion.

Animals↗
PubMed1983

Characterization of the chromobindins. Soluble proteins that bind to the chromaffin granule membrane in the presence of Ca2+.

A group of proteins that bind to the chromaffin granule membrane in the presence of Ca2+ has been isolated by affinity chromatography of bovine adrenal medullary cytosol on granule membranes coupled to Sepharose 4B. Twenty-two of these proteins were resolved into classes depending upon the Ca2+ concentration at which they were eluted from the affinity column (40 or 0.1 microM), upon their affinities for native granule membranes or for liposomes prepared from extracted granule lipids, and upon the requirement of seven of the proteins for ATP in the cytosol fraction and column buffers to promote binding. The molecular weights and isoelectric points of these proteins were determined by two-dimensional electrophoresis. Two of the granule-binding proteins were identified: synexin and calmodulin. Calmodulin was found to bind to seven specific granule membrane proteins after diffusion of 125I-labeled calmodulin into an acrylamide gel of membrane proteins separated by electrophoresis in the presence of sodium dodecyl sulfate. A phospholipid-activated protein kinase activity, possibly due to protein kinase C, was present in the granule-binding fraction. Two major granule-binding proteins were found to present a pattern in two-dimensional electrophoresis that was very similar to but shifted slightly toward the basic end of the gel from the pattern generated by light chains associated with clathrin in adrenal medullary coated vesicles. In the chromaffin cell, these proteins, by associating with the granule membrane in the presence of an increased cytosolic Ca2+ concentration, might play a variety of roles in the process of exocytosis.

Adenosine Triphosphate↗
PubMed1983

Identification of high-affinity phorbol ester receptor in cytosol of EL4 thymoma cells: requirement for calcium, magnesium, and phospholipids.

A specific high-affinity phorbol ester binding component has been identified in the cytosol of an EL4 mouse thymoma line by using conditions similar to those for demonstrating activity of a calcium/phospholipid-dependent protein kinase. Specific binding is absolutely dependent on acidic phospholipids (maximal binding at 96 micrograms of phosphatidylserine per ml or 200 micrograms of phosphatidylinositol per ml) and is greatly enhanced by addition of calcium (0.5 mM) and magnesium (75 mM). Scatchard analysis of the cytosolic binding component indicated a Kd of 4.2 +/- 2.5 nM with 1.8 +/- 0.6 x 10(4) sites per cell compared to a Kd of 11.9 +/- 4.4 nM and 4.1 +/- 1.0 x 10(4) sites per cell for the membrane receptor. Consistent with the existence of at least two phorbol ester binding sites in EL4 cells was the observation of curvilinear Scatchard plots for binding to whole homogenates. The cytosolic binding showed the same order of potency for binding phorbol ester analogs as has been observed for intact cells. These results further support the similarity between phorbol ester receptors and the calcium/phospholipid-dependent protein kinase and suggest that the cytosolic receptor may be involved in subsequent phorbol ester effects in EL4 cells including loss of the kinase activity from the cytosol and production of T-cell growth factor.

Animals↗
PubMed1982

Identification of phorbol ester receptors in T-cell growth factor-producing and -nonproducing EL4 mouse thymoma cells.

Two lines of EL4 mouse thymoma cells, one which responds to phorbol ester tumor promoters with production of T-cell growth factor and inhibition of proliferation and one which does not respond, have been examined for the presence of specific phorbol ester-binding components. Both lines displayed saturable specific binding as determined by using [20-3H]phorbol 12,13-dibutyrate in a whole-cell-binding assay. Specific binding in each line was maximal within 10 min at 37 degrees but rapidly decreased to about 30% within 30 min. Maximal binding at 4 degrees was reached after 50 min and was stable for at least 8 hr; however, this level was only about 30% of the maximum obtained at 37 degrees. Saturation of the specific binding after 3 hr at 4 degrees obtained at a concentration (30 to 50 nM) which is consistent with that yielding maximal T-cell growth factor production in the responding line but greater than that at which inhibition of proliferation was detected in those cells. Scatchard analysis of these data was consistent with the existence of a single class of binding sites with a Kd of 11 +/- 0.6 (S.D.) nM for the T-cell growth factor-producing cells and 18 +/- 1.9 nM for the nonproducing line. The numbers of sites per cell measured in the responding and nonresponding lines were 5.6 +/- 1.3 x 10(4) and 7.1 +/- 0.2 x 10(4), respectively. Competition by a series of phorbol esters for [20-3H]phorbol 12,13-dibutyrate binding in the responding line showed the same order of potency as production of T-cell growth factor and inhibition of proliferation by the analogs. These data support identification of the phorbol ester-binding component in the responding cells as the receptor mediating T-cell growth factor production. The presence of binding components in unresponsive cells in numbers and with characteristics indistinguishable from those of responsive cells suggests that the failure to respond is due to modification of a step which succeeds the initial binding event.

Animals↗
PubMed1981

Specific receptors for phorbol esters in lymphoid cell populations: role in enhanced production of T-cell growth factor.

Phorbol ester tumor promoters act synergistically with concanavalin A to cause production of T-cell growth factor by normal human peripheral blood lymphocytes. A specific, saturable, binding component which may mediate the phorbol ester effect has been identified by using [20-3H]phorbol 12,13-dibutyrate in a whole-cell binding assay. Specific binding is maximal with 5 min at 37 or 23 degrees C but the level of bound ligand rapidly decreases to about 50% within 1 hr. At 4 degrees C, 2 hr are required to reach maximal binding, and the binding is stable for at least 20 hr. Binding is reversible at 37 and 4 degrees C with time courses similar to those for initial binding at the respective temperatures. Saturation of the specific binding occurs at a concentration (approximately 30 nM) consistent with that producing maximal T-cell growth factor activity. Scatchard analysis of the binding after 30 min at 37 degrees C demonstrates a lower Kd (9 nM) than that determined after 2 hr at 4 degrees C (22 nM). The median number of sites per cell for six donors was 2 X 10(5) (range, 1.3-4 X 10(5). Other tumor-promoting phorbol esters compete for [20-3H]phorbol 12,13-dibutyrate binding in approximate proportion to their activity in stimulating T-cell growth factor production. Phorbol, 4-alpha-phorbol didecanoate, dexamethasone, retinoic acid, butyric acid, and dimethyl sulfoxide do not compete for specific binding.

Caenorhabditis elegans Proteins↗
PubMed1979

Molybdate inhibition of glucocorticoid receptor inactivation and transformation.

The inactivation of glucocorticoid receptors that occurs when cytosol is heated at 25 degrees C is blocked reversibly by molybdate and slowed by some other phosphatase inhibitors such as fluoride and glucose 1-phosphate. Molybdate is also capable of preventing nonenzymatic inactivation of unbound receptors caused by exposure to salt or precipitation with ammonium sulfate at 0 degrees C. Inactivation of unbound receptors caused by Sephadex G-50 gel filtration is prevented by all three inhibitors. Both molybdate and tungstate block temperature-dependent transformation of glucocorticoid.receptor complexes to the DNA-binding state, where fluoride and glucose 1-phosphate have no effect. Transformation brought about at 0 degrees C by salt, ammonium sulfate precipitation, or gel filtration is also blocked by both molybdate and tungstate. Tungstate differs from molybdate in that it has little or no effect on receptor inactivation. Fluoride and glucose 1-phosphate do not inhibit transformation. These observations support the proposal that molybdate and tungstate are interacting through a reversible association with the glucocorticoid receptor itself. We propose that they may act by forming a complex with a phosphate moiety on the receptor.

Adrenalectomy↗
PubMed1979

ATP-dependent activation of L cell glucocorticoid receptors to the steroid binding form.

The specific glucocorticoid binding capacity in cytosols prepared from L929 mouse fibroblasts (L cells) is inactivated with a half-life of approximately 2 h at 25 degrees C. As previously published, this inactivation can be prevented with 10 mM molybdate and markedly slowed by addition of other phosphatase inhibitors such as glucose 1-phosphate and fluoride. We have now found that ATP (5 to 10 mM) also slows the rate of this inactivation. After extensively inactivating the receptor by preincubating cytosol at 25 degrees C for 4 and preventing further inactivation by addition of molybdate, addition of ATP results in reactivation of the steroid binding capacity. Maximal reactivation of 40 to 70% is achieved with 5 to 10 mM ATP. The activation is temperature-dependent and specific for ATP. ADP, GTP, CTP, and UTP do not cause activation and preliminary results indicate no effect of cyclic nucleotides in this system. If activation is prevented by addition of 10 mM EDTA to the cytosol, addition of 3 to 10 mM magnesium permits ATP-dependent activation of the binding capacity. The level of reactivation can be enhanced by addition of a heat-stable factor prepared from the same L cell supernatant. These results support the proposal that L cell glucocorticoid receptors can be activated to the glucocorticoid binding state by an ATP-dependent phosphorylation mechanism.

Adenosine Triphosphate↗
PubMed1979

Activation of thymocyte glucocorticoid receptors to the steroid binding form. The roles of reduction agents, ATP, and heat-stable factors.

The specific glucocorticoid binding capacity in cytosol preparations of rat thymocytes decays with a half-life of 4 h at 0 degrees C or 20 min at 25 degrees C. Phosphatase inhibitors (molybdate, fluoride, glucose 1-phosphate) added alone do not prevent this inactivation. Dithiothreitol (2 mM) has a large stabilizing effect on the binding capacity at 0 degrees C but only a small effect at 25 degrees C. Addition of 10 mM molybdate plus 2 mM dithiothreitol totally prevents inactivation for at least 8 h at 25 degrees C as well as at 0 degrees C. Fluoride (100 mM) also retards the inactivation if added with dithiothreitol. Addition of dithiothreitol at 25 degrees C to inactivated cytosol receptors results in partial activation of the binding capacity. Addition of dithiothreitol to receptors inactivated at 25 degrees C in the presence of molybdate allows total reactivation of the binding capacity to the maximum zero time value. If binding capacity is inactivated by preincubation of the cytosol at 25 degrees C, addition of ATP with dithiothreitol enhances the activation observed with only dithiothreitol. This ATP stimulated activation is optimal at 1 to 3 mM. ATP (10 mM) is required when molybdate is added to prevent simultaneous inactivation. ADP, GTP, CTP, and UTP have some activating capacity but the effects of all nucleotides are inhibited by the ATP analog, adenyl-5'-yl (beta, gamma-methylene)diphosphonate. ATP-dependent activation can also be prevented with 50 mM EDTA, and addition of magnesium partially overcomes the EDTA inhibition. Dithiothreitol activation of thymocyte glucocorticoid binding capacity can also be enhanced by addition of a heat-stable preparation from thymocytes, L cells, or liver. Sephadex G-25 chromatography, assay of ATP, and inhibition of the activation with adenyl-5'-yl (beta, gamma-methylene)diphosphonate suggest that these preparations contain varying amounts of endogenous reducing equivalents and ATP as well as a larger heat stable factor. Maximum activation is obtained by adding dithiothreitol, ATP, molybdate, and the larger heat-stable factor. These results suggest that stabilization and activation of glucocorticoid binding capacity in thymocytes requires phosphorylation as well as reduction of the receptor itself or of some other component required for the steroid binding reaction.

Adenosine Triphosphate↗
PubMed1979

Glucocorticoid receptor inactivation and activation by phosphorylation mechanisms.

The specific glucocorticoid binding capacity of cytosol preparations is rapidly lost on incubation at 25 degrees in the absence of ligand. We have examined this process in cell-free preparations from rat thymus, rat liver and mouse fibroblasts (L 929 cells), and we have found that the unoccupied receptor is inactivated by endogenous enzymes to a form that does not bind steroids. The inactivation can be prevented by inhibitors of phosphatase action such as molybdate, fluoride and glucose-1-phosphate. On the basis of this type of evidence we propose that the receptor activity of cytosol can be rendered inactive by a dephosphorylation process. We have now been able to partially reactivate the receptor in both L cell and rat thymus cytosols in an ATP dependent manner. If fibroblast (L cell) cytosol is preincubated to permit receptor inactivation by endogenous enzyme, further inactivation can be prevented by the addition of 10 mM molybdate and reactivation of the binding capacity can be obtained by adding 5 to 10 mM ATP in addition to molybdate. ATP dependent activation is prevented with EDTA and this block is overcome by added magnesium. ADP, CTP, GTP, and UTP are inactive. After inactivating the glucocorticoid binding capacity of rat thymocyte cytosol by incubation for 45 minutes at 25 degrees, considerable reactivation is obtained by addition of dithiothreitol and ATP. This system does not absolutely require the presence of a phosphatase inhibitor in order to show activation. Thymocyte cytosol can also be activated to a steroid binding state by addition of DTT and heat-treated (90 degrees for 15 min.) cytosol from a variety of cell types. The heat-treated cytosol contains ATP, reducing equivalents, and a relatively small molecular weight heat-stable activator(s) that potentiates the reactivation process. Maximum receptor activation is obtained by adding dithiothreitol, heat-stable factor, ATP, and molybdate to the inactivated thymocyte cytosol.

Adenosine Triphosphate↗
PubMed1979

Transport of C5 dicarboxylate compounds by Pseudomonas putida.

Induced glutarate and 2-oxoglutarate uptake and transport by Pseudomonas putida were investigated in whole cells and membrane vesicles, respectively. Uptake of 2-oxoglutarate, but not glutarate, was against a concentration gradient to 1.7-fold greater than the initial extracellular concentration. Membrane vesicles transported 2-oxoglutarate and glutarate against gradients to intramembrane concentrations fivefold greater than the initial extravesicle concentrations. The rates of transport of both compounds were greatest in the presence of the artificial electron donor system phenazine methosulfate-ascorbate. Malate and D-lactate were the only naturally occurring compounds that served as electron donors. Uptake and transport were inhibited by KCN, NaN3, and 2,2-dinitrophenol. Kinetic parameters of transport were: glutarate, apparent Km--1.22 mM, Vmax--400 nmol/min per mg of membrane protein; 2-oxoglutarate, apparent Km--131 microM, Vmax--255 nmol/min per mg of membrane protein. Studies of competitive inhibition indicated a common system for transport of five C5 dicarboxylate compounds. The apparent Km and Ki values with 2-oxoglutarate as a substrate placed the substrate affinity for transport in the order 2-oxoglutarate greater than glutarate greater than D-2-hydroxyglutarate and L-2-hydroxyglutarate greater than glutaconate.

Biological Transport↗
PubMed1977

Reactivation of thymocyte glucocorticoid receptors in a cell-free system.

The specific glucocorticoid binding capacity of the 100,000 X g supernatant from thymocytes becomes rapidly inactivated with a half-life of approximately 4 h at 0 degrees. Following this inactivation, the receptor can be reactivated by a factor (or factors) present in the high speed supernatant of L929 mouse fibroblasts. The L cell activating factor is excluded from Sephadex G-25. It can be separated from the glucocorticoid receptor of L cells by chromatography of Sephadex G-150 where it elutes with the small molecular weight material. The probable molecular weight range for the activating factor is 5,000 to 20,000. Approximately 50% of the activating activity is retained after heating at 90 degrees for 15 min.

Adrenalectomy↗
PubMed1977

Evidence that dephosphorylation inactivates glucocorticoid receptors.

Highly purified alkaline phosphatase [orthophosphoric-monoester phosphohydrolase (alkaline optimum), EC 3.1.3.1] from calf intestine inactivates the glucocorticoid-binding capacity of soluble preparations from mouse fibroblasts (L cells) and rat liver. The unbound receptor is sensitive to inactivation whereas the steroid-bound receptor is unaffected. The ability of the enzyme preparation to inactivate the receptor, like its ability to dephosphorylate p-nitrophenyl phosphate, is dependent on zinc and inhibited by arsenate. Both the dephosphorylating and receptor inactivating activities coelute during DEAE-cellulose purification of the enzyme. There is no detectable proteolytic activity in the purified alkaline phosphatase preparation. In a mixed system containing both glucocorticoid and estrogen receptors, the glucocorticoid receptor is selectively inactivated. Although these observations do not prove that the receptor molecule itself is the substrate, they are consistent with the proposal that the glucocorticoid receptor can be inactivated by dephosphorylation and that only the phosphorylated form of the molecule is capable of binding steroid. A phosphorylation-dephosphorylation mechanism may be responsible for determining the level of active receptor in the cell.

Alkaline Phosphatase↗
PubMed1976

Evidence for a phospholipid requirement in the specific binding of glucocorticoids to receptors of fibroblasts and thymic lymphocytes.

The specific steroid binding capacity of soluble preparations from mouse fibroblasts and rat thymic lymphocytes is inactivated by incubation with phospholipases. Receptor binding is drastically reduced by very low concentrations of boiled phospholipase A preparations from bee venom and snake venoms. The enzyme effect is calcium-dependent and is blocked by both phospholipid and a substrate analog that is a competitive inhibitor of phospholipase A. The specific binding capacity is also sensitive to digestion by phospholipase C. Two possible mechanisms are considered for the phospholipase A effect: (a) the receptor protein may be associated with a phospholipid component which is required for specific hormone binding; (b) phospholipase A may be producing detergent products that are indirectly inactivating the receptor. Examination of the effects of lysophosphatide on the receptor and assay of lipid phosphate in the receptor preparation do not support a mechanism based solely on detergent effects. Because phospholipase C, which does not produce detergent products, also inactivates the binding, we propose that the phospholipases may be digesting the phospholipid which is a requisite component of the glucocorticoid receptor.

Animals↗