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Biomedical subjects

G Litwack

Publications and source records attributed to G Litwack.

At least 109 records · Page 6Linked to original sources
PubMed1983

Rat ligandin mRNA molecular cloning and sequencing.

Recombinant plasmids containing the double-stranded cDNA sequences of mRNA for the Mr 22,000 ligandin (glutathione S-transferase B) subunit (Ya) have been constructed. The DNA sequence of an insert corresponding to the middle and 3' regions of the mRNA was determined and an amino acid sequence was proposed for the ligandin Ya subunit. The proposed sequence reveals a high content of basic amino acids (Arg and Lys) and Leu, is consistent with the amino acid composition, and predicts the correct number of peptides derived from tryptic digests reported for ligandin.

Amino Acid Sequence↗
PubMed1983

In vitro production of corticosteroid binder IB in the presence of proteolytic inhibitors.

The effect of proteolytic inhibitors on the temperature-dependent formation of corticosteroid binder IB in rat kidney cytosol was examined. Antipain increased the apparent binding of [3H]-triamcinolone acetonide in the cytosol. Leupeptin, chymostatin, soya bean trypsin inhibitor and lima bean trypsin inhibitor did not affect total binding, while L-1-tosylamide-2-phenylethyl chloromethyl ketone, N alpha-p-tosyl-L-lysine chloromethyl ketone and phenylmethylsulfonyl fluoride markedly reduced the charcoal resistant steroid binding. However, none of the inhibitors added during tissue homogenization, steroid binding or activation affected the extent of heat-dependent conversion of the [3H]-triamcinolone acetonide-receptor complexes to the IB form, which was characterized by its exclusion from DEAE-Sephadex ion exchanger. In contrast, sodium molybdate (10 mM) effectively inhibits IB formation without inhibiting protease activity of rat kidney cytosol. These observations indicate that the temperature-dependent formation of corticosteroid binder IB in vitro does not involve proteolytic transformation of unbound or steroid-bound cytosolic proteins. Addition of antipain (3 mM) to the cytosol markedly increased the radioactivity in the buffer prewash of DEAE-cellulose columns (apparent IB) only when the inhibitor was added prior to charcoal adsorption. However, a similar peak in the prewash also was obtained with receptor-free cytosol. Antipain had no effect on the rate of dissociation of performed [3H]-triamcinolone-acetonide-receptor complexes nor did it increase the amount of receptor adsorbed to hydroxylapatite. Chromatography on Sephadex G-25 and P-2 columns showed that the increased activity in the charcoal-resistant fraction in the presence of antipain is due to unbound steroid. Thus, antipain interferes with the ability of charcoal to remove unbound steroid from the cytosol.

Animals↗
PubMed1983

Comparison of corticosteroid binder IB with the alpha-chymotrypsin- and RNase-treated hepatic glucocorticoid receptors.

Rat liver and kidney cytosolic extracts contain the glucocorticoid receptor (binder II) and corticosteroid binder IB, both of which possess the steroid- and DNA-binding domains. Since it has been speculated that the smaller binder IB may be generated from binder II by proteolysis, the chymotrypsin-produced receptor fragment in rat liver cytosol has been compared with binder IB in terms of charge, size and DNA binding characteristics. The [3H]triamcinolone acetonide-receptor complex is converted to a smaller fragment by short term digestion (10 degrees C, 30 min) with 100 micrograms/ml alpha-chymotrypsin. Although the chymotrypsin fragment produced from previously heat-activated binder II and binder IB both exhibit DNA-binding capability, they differ in charge and size. Whereas the alpha-chymotrypsin-treated receptor has a Stokes radius of 30 A and elutes from DEAE-cellulose at 0.06 M potassium phosphate in a linear salt gradient, binder IB has a Stokes radius of 20 A and elutes in the buffer wash of the DEAE-cellulose column. Thus, while binder IB can be resolved from the heat-activated form of the [3H]TA-receptor on DEAE, the heat activated alpha-chymotrypsin product elutes from the anion exchange resin at the same ionic strength as intact activated binder II (i.e. at 0.05 M potassium phosphate), and the unactivated intact receptor elutes at about 0.20 M potassium phosphate. A more extended digestion with alpha-chymotrypsin (24 h, 0 degrees C) results in elimination of the DNA binding site without further reduction of the Stokes radius or change in the elution pattern from DEAE-cellulose. Furthermore, molybdate completely blocks formation of binder IB but does not inhibit the production of the receptor fragment by alpha-chymotrypsin. Treatment of the hepatic [3H]TA-receptor complex with RNase has no effect on the charge, size or DNA binding properties of the bound receptor. These results suggest that RNase does not activate the [3H]TA-receptor complex nor does it produce a IB-like component in the liver cytosol. The present results are consistent with the hypothesis that binder IB is formed in vitro by a process which may not involve proteolytic cleavage or RNase-induced modification of the glucocorticoid receptor (binder II).

Animals↗
PubMed1983

In vitro stabilization of the unoccupied glucocorticoid receptor by adenosine 5'-diphosphate.

The addition of ATP to rat liver cytosol slows the rate of heat inactivation of the unoccupied glucocorticoid receptor (25 C) and stimulates the rate of activation of the preformed glucocorticoid-receptor complex (15 C). Dose-response curves and kinetic studies show that ADP is as effective as ATP in stabilizing the unoccupied glucocorticoid receptor against heat inactivation. ATP can also be replaced by analogs with a hydrolysis-resistant alpha, beta-pyrophosphate linkage (5'-adenylyl methylenephosphonophosphate or 5'-adenylyl methylenephosphonate); however, the hydrolysis-resistant beta, gamma analog (5'-adenylyl methylenediphosophonate) is ineffective. The addition of creatine phosphate plus creatine kinase, a condition favoring ATP formation, stimulates the rate of inactivation of the unoccupied glucocorticoid receptor, and the effect is only partially overcome by ADP. A condition that favors ADP formation, the addition of creatine plus creatine kinase, has no effect on the rate of inactivation of the unoccupied receptor and does not decrease the protective effect afforded by ATP alone. Collectively, these results suggest that ATP stabilization of the steroid-binding site in vitro is due to ADP generated from the triphosphate by endogenous enzymes and is not due to phosphorylation or adenylation of the receptor by ATP. Unlike ATP stabilization of the steroid-binding site, the ATP-stimulated increase in the rate of activation of the preformed glucocorticoid-receptor complex (15 C) does not require hydrolysis of the beta, gamma-pyrophosphate bond. Dose-response curves show that both ATP and 5'-adenylyl methylenediphosophonate stimulate the rate of activation of the glucocorticoid-receptor complex. Quantitation of nucleotide levels in unfractionated rat liver cytosol by high performance liquid chromatography shows that the effective concentration of added ATP that produces an optimal response is within the physiological range reported for intact cells.

Adenosine Diphosphate↗
PubMed1983

Corticosteroid binder IB: a model of glucocorticoid receptor diversity.

Corticosteroid binder IB is a glucocorticoid receptor present in rat liver, kidney cortex and proximal colon. It is characterized by a lower molecular weight than the traditional and widely distributed glucocorticoid receptor II. The two receptors also differ in charge, affinity towards various steroids and potency of binding to DNA, nuclei and homodeoxypolymers. The in vivo and in vitro production of binder IB is not affected by protease inhibitors, nor can IB be produced by RNase digestion or proteolytic cleavage of binder II or its precursor molecule. The presumptive physiological role of corticosteroid binder IB and the implied biological significance of receptor diversity are discussed.

Animals↗
PubMed1982

Dual effects f pyridoxal 5'-phosphate on glucocorticoid-receptor complexes.

The ability of pyridoxal 5'-phosphate to inhibit DNA-cellulose binding of activated glucocorticoid-receptor complexes is pH and protein concentration dependent. At the tested pHs, all of the inhibitory activity of pyridoxal 5'- phosphate appears to be due to its ability to form a Schiff base. 2-Amino-2-(hydroxymethyl)-1,3-propanediol (100 mM) is unable to prevent or reverse the pyridoxal 5'-phosphate mediated inhibition of DNA-cellulose binding, while the same concentration of lysine is partially effective. Pyridoxal 5'-phosphate does not alter the elution profile of glucocorticoid-receptor complexes as ascertained by diethylaminoethyl (DEAE)-cellulose of DEAE-Sephadex chromatography. This observation permitted the use of these resins in detecting the previously unreported stimulation of glucocorticoid-receptor complex activation by pyridoxal 5'-phosphate. This stimulation is specific for pyridoxal 5'-phosphate and appears to be mediated via a Schiff base formation. Additionally, glucocorticoid-receptor complexes activated by pyridoxal 5'-phosphate treatment at low temperatures do not differ in size from thermally activated complexes. Thus, in vitro, pyridoxal 5'-phosphate can exert both a stimulatory effect on activation as well as an inhibitory effect on the binding of activated complexes to DNA-cellulose.

Animals↗
PubMed1982

Activation of the glucocorticoid-receptor complex.

A crucial step in the interaction of glucocorticoids with target cells is the activation step, which involves a conformational change in the cytoplasmic glucocorticoid-receptor protein complexes and facilitates their binding to the cell nucleus. Activation can be quantified by measuring the ability of glucocorticoid-receptor complexes to bind to polyanions, such as DNA-cellulose, and unactivated complexes can be separated from activated complexes by rapid ion exchange chromatography using diethylaminoethyl (DEAE)-Sephadex or DEAE-cellulose. Activation occurs in vivo under physiological conditions and the rate of activation of cytoplasmic glucocorticoid-receptor complexes can be enhanced in vitro by physical manipulations (elevated temperature, increased ionic strength, dilution). In vitro studies suggest that activation is a regulated process and a low molecular weight component termed modulator, which has been identified in rat hepatic cytosol, inhibits activation. Additional studies employing phosphatase inhibitors, such as molybdate, and purified calf intestinal alkaline phosphatase suggest that either the receptor protein or a regulatory component is dephosphorylated during activation. Results obtained with specific chemical probes suggest that activation results in the exposure of basic amino acid residues consisting minimally of lysine, arginine, and histidine. Pyridoxal 5'-phosphate, a specific probe for lysine residues, exerts dual effects on glucocorticoid-receptor complexes, since it stimulates the rate of activation and also inhibits the binding of previously activated complexes to nuclei or DNA-cellulose. The ability of 1,10-phenanthroline, a metal chelator, to inhibit the DNA-cellulose binding of activated complexes suggests that a metal ion(s) located at or near the DNA binding site may become exposed as a consequence of activation. Collectively, the results of these various experiments suggest that activation is a regulated biochemical phenomenon with physiological significance.

Alkaline Phosphatase↗
PubMed1982

Correlations between the activities of DNA polymerase alpha and the glucocorticoid receptor.

Specific inhibitors and anti-DNA polymerase alpha IgG have been utilized to probe for similarities between cytoplasmic rat hepatic glucocorticoid receptors and DNA polymerase alpha [DNA nucleotidyltransferase (DNA-directed), EC 2.7.7.7]. Rifamycin AF/013, an inhibitor of RNA and DNA polymerase activities, significantly inhibited the binding of activated [6,7-3H]-triamcinolone acetonide (TA) receptor complexes to DNA-cellulose. beta-Lapachone, an inhibitor of DNA polymerase alpha and reverse transcriptase activities, inhibited the specific binding of [6,7-3H]TA when preincubated with unbound receptors. Aphidicolin, another DNA polymerase alpha inhibitor, failed to inhibit any of the glucocorticoid-receptor functions tested. Two specific anti-DNA polymerase alpha IgGs interfered with glucocorticoid receptor functions as measured by their ability to inhibit the binding of [6,7-3H]TA to unbound receptors (85% maximal inhibition) and, to a lesser extent, to inhibit the binding of activated [6,7-3H]TA receptor complexes to DNA-cellulose (50% maximal inhibition). The anti-DNA polymerase alpha IgG and beta-lapachone failed to affect the binding of tritiated estradiol, progesterone, or 5 alpha-dihydrotestosterone to their receptors in appropriate rat target tissues or the binding of [1,2-3H]hydrocortisone to serum transcortin. The most obvious interpretation of these data is that cytoplasmic glucocorticoid receptors and DNA polymerase alpha share antigenic determinants. An alternative interpretation is that the polyclonal anti-DNA polymerase alpha antibody contains IgG molecules raised against calf thymus cytoplasmic activated glucocorticoid-receptor complexes that copurified with DNA polymerase alpha used as the antigen. Taken collectively, however, the antibody and inhibitor data suggest a relationship between DNA polymerase alpha and the glucocorticoid receptor.

Animals↗
PubMed1982

Vitamin B6 kills hepatoma cells in culture.

Data are presented which indicate that vitamin B6 (pyridoxine) can retard and eventually kill Fu5-5 rat hepatoma cells in culture. Additional studies indicate that the human kidney cell line 293-31, and a rat glial cell strain. C6, display growth characteristics similar to those of hepatoma cells when cultured in 5 mM pyridoxine-supplemented medium. However, the pyridoxine-supplemented culture medium had little effect on the growth of the human mammary cancer cell line MCF-7. The resistance of the MCF-7 cells to growth inhibition by vitamin B6 suggests that the vitamer pyridoxine must be metabolized by pyridoxal before it can act as a growth inhibitor. These findings suggest the potential use of vitamin B6 as an antineoplastic agent. Possible mechanisms by which vitamin B6 promotes this effect are presented.

Adenosine Triphosphate↗
PubMed1982

An electrophoretic characterization of the glucocorticoid response of the Fu5-5 rat hepatoma cell line.

In this study we have further characterized the response of liver-derived cells to glucocorticoid treatment using the rat hepatoma, Fu5-5. Using two-dimensional electrophoresis we have examined changes in the synthetic rates of cytosol proteins following glucocorticoid administration by using [35S]methionine. We have also demonstrated changes in the incorporation of 32P by several cytosol proteins after hormone treatment. One of these changes occurs within 1 h of hormone treatment. We were unable to detect any changes in the nuclear protein content of Fu5-5 cells after glucocorticoid treatment. We then compared the glucocorticoid-regulated changes in cytosol protein synthesis in Fu5-5 cells with those of the H35 rat hepatoma, monolayer cultures of rat hepatocytes and the response of rat liver in vivo. This examination revealed that although nearly all of the eighteen hormonally responsive proteins appeared to be present in all of the cells types, only three were responsive to glucocorticoid in more than one system. Moreover, the response patterns were often reversed in different cell types, the same protein being induced in one cell type and repressed in another.

Animals↗
PubMed1982

Monoclonal antibodies to the glucocorticoid receptor.

One thousand-fold purified unactivated rat liver glucocorticoid receptor was obtained by affinity chromatography followed by gel filtration. Splenic lymphocytes of immunized mice were fused with a nonproducer SP2 myeloma cell line to obtain hybridomas secreting antibodies against receptor. Hybridoma lines, injected into mice, developed ascites tumors which provide substantial amounts of monoclonal antibodies. A monoclonal antibody was purified from ascites fluids by chromatography on an anti-mouse IgG column. The antibody was shown to react physically with the glucocorticoid receptor.

Animals↗
PubMed1982

Pyridoxine resistance in a rat hepatoma cell line.

The natural vitamin, pyridoxine, in the millimolar range is toxic to cultured rat hepatoma cells. A pyridoxine-resistant Fu5-5 rat hepatoma cell line was established by a stepwise increase in the concentration of pyridoxine in the medium. The newly established cell line, referred to as clone 10 (Cl.10), is resistant to killing by pyridoxine in concentrations up to 5 mM. Saturation kinetics for the uptake of [3H]pyridoxine into Fu5-5 and Cl.10 cells revealed that Fu5-5 cells take up 10 times more [3H]pyridoxine than do Cl.10 cells. Whereas the Vmax value for the uptake of [3H]pyridoxine was the same for both cell lines, the apparent Km for the Cl.10 cells was 12.5 microM compared to 0.71 microM for the Fu5-5 cells. However, intracellular levels of pyridoxal 5'-phosphate were 69% higher in Cl.10 cells than in the parental line. The resistant line is neither a permeability mutant nor deficient in pyridoxal kinase. Cl.10 cells contain 37% more adenosine 5'-triphosphate than do Fu5-5 cells and have a mitochondrial volume that is 50% greater than that of the parental line. In the absence of pyridoxine in the medium, Cl.10 cells revert to parental type with respect to pyridoxine uptake but not with respect to resistance to killing. They also maintain an enlarged mitochondrial volume. Thus, increased mitochondrial volume may be related to the development of resistance to high levels of pyridoxine.

Animals↗