Amal Boctor

Role of steroids in secretion—modulating effect of triamcinolone and estradiol on protein synthesis and secretion from the rat exocrine pancreas

Following adrenalectomy of male rats, or adrenalectomy and ovariectomy of females, there was marked depletion of zymogen granules in acinar cells of the pancreas. Within 9 h after treatment with either triamcinolone or 17 beta-estradiol, complete restoration of these secretory vesicles was observed. This repletion was not inhibited by actinomycin-D. Supernatant fractions (100,000 g, 60 min) of rat pancreas, from both normal and surgically altered animals, contained proteins that bound [3H]-triamcinolone and [3H]-estradiol, suggesting that the action of these hormones is exerted directly on the pancreas. Binding of both steroid hormones required the presence of an additional coligand referred to as accessory factor. In addition, the binding proteins for [3H]-triamcinolone and [3H]-estradiol eluted in similar positions after Sephadex G-200 and CM Affi-gel Blue chromatography. It is uncertain, however, whether a single protein binds both steroid hormones since they had different binding isotherms. Scatchard analysis of binding of [3H]-estradiol yielded a single straight line of negative slope from which it was calculated that there were about 4.4 pmol of binding sites per mg protein, having an average apparent Kd of about 5 X 10(-8) M. Similar analysis of the data for [3H]-triamcinolone yielded a straight line of zero slope indicating nonsaturable binding of hormone at concentrations as high as 10 microM. Since both [14C]-L-leucine incorporation into protein and amylase secretion were affected markedly by the steroid-hormonal status of the animal, it is presumed that steroid-bound complexes in acinar cells of the pancreas modulate synthesis and secretion of protein.

Specific Binding of 3H-Estradiol to the Cytosol of Rat Pancreas and Uterus: Bound Sites In Pancreatic Extracts Do Not Translocate 3H-Estradiol to Nuclei Suggesting A Basic Difference In Mode of Action

In parallel sets of experiments, cytosol fractions from rat pancreas and uterus were incubated with 2 nM 3H-estradiol in the presence or absence of nuclei from pancreas and liver. After incubation for 1 hr at room temperature, the nuclei were removed by centrifugation and specific binding determined in the cytosol fractions as well as in the separated nuclei. The protein that binds 3H-estradiol in uterine extracts translocated the hormone to nuclei of pancreas and liver while the one in pancreas was devoid of this activity. It is presumed, therefore, that modification of transcription is not a primary action of the steroid-bound complex in pancreas.

Differential sensitivity of rat liver and rat hepatoma cells to α-amanitin

Abstract α-Amanitin, an inhibitor of RNA polymerase derived from the mushroom Amanita phalloides , was shown to be about five times more potent than actinomycin-D in inhibiting rat liver tyrosine aminotransferase induction by cortisol. Rat hepatoma cells grown in culture, however, were resistant to these inhibitory effects of α-amanitin, while actinomycin-D effectively inhibited enzyme induction in these cells. The insensitivity of the hepatoma cells could not be accounted for by: (a) specific binding of α-amanitin to fetal calf serum protein present in the tissue culture medium; (b) metabolic conversion of this bicyclic octapeptide to an active compound in rat liver which does not occur in hepatoma cells; or (c) rapid inactivation of this substance by the hepatoma cells. RNA polymerase studies with isolated nuclei indicated that inhibition of cortisol induction of rat liver tyrosine aminotransferase by α-amanitin was accompanied by an inhibition of Mn 2+ /(NH 4 ) 2 SO 4 -activated RNA polymerase. No effect on the Mg 2+ -activated polymerase was noted. Similar experiments with hepatoma cells, in conjunction with [ 14 C]-uracil and [ 3 H 5 ]-orotic acid uptake studies, indicated that the RNA polymerase of these cells was sensitive to α-amanitin only when the cell membrane was broken. The two major conclusions from these data are: (1) hepatoma cells are insensitive to α-amanitin because they are impermeable to this substance, and (2) the correlation between inhibition of the Mn 2+ /(NH 4 ) 2 SO 4 -activated RNA polymerase and inhibition of tyrosine aminotransferase induction by cortisol strongly suggests that modification of transcription by this hormone is the primary stimulus for increased synthesis of this enzyme.

Specific binding of [3H]-estradiol to the cytosol of rat pancreas: Alteration of the apparent number of binding sites by an endogenous factor and oligopeptide derivatives

Abstract The supernatant fraction of rat pancreas contains an estrogen-binding protein that differs in several ways from the one found in uterus: (1) binding of 17β-[ 3 H]-estradiol in pancreas was non-saturable at hormone concentrations ranging from 2–50 nM; (2) diethylstilbestrol (as well as 17α-estradiol) was only about half as effective as unlabeled 17β-estradiol in competing for these specific binding sites; and (3) the binding reaction in pancreas requires the presence of a coligand, referred to as accessory factor. Accessory factor is an endogenous substance isolated from rat pancreas that is required for specific binding of [ 3 H]-estradiol to a soluble protein present in this tissue. The activity of this factor can be mimicked by the synthetic oligopeptide N -benzoyl- l -phenylalanyl- l -valyl- l -argininyl- p -nitroanilide, or the simpler derivative N -benzoyl- l -argininyl- p -nitroanilide. Removal of endogenous accessory factor by CM Affi-gel Blue chromatography renders the estrogen-binding protein incompetent. Using such preparations, the capacity to reactivate binding of [ 3 H]-estradiol by partially purified accessory factor and synthetic oligopeptide was compared. When specific binding of [ 3 H]-estradiol was determined at concentrations ranging from 2–50 nM in the presence of limiting amounts of accessory factor, a saturable isotherm was obtained. In the presence of increasing amounts of accessory factor, specific binding was nonsaturable as was observed with the initial cytosol. Scatchard analysis indicated: (a) that the number of binding sites for [ 3 H]-estradiol increased as a function of accessory factor concentration; and (b) the affinity of these sites for [ 3 H]-estradiol declined concomitantly. As a working hypothesis it is suggested that binding of [ 3 H]-estradiol in the presence of limiting amounts of accessory factor results in a ternary complex (binding protein: [ 3 H]-estradiol: and accessory factor). Upon further addition of factor a supramolecular structure seems to form that is nonsaturable with respect to binding of [ 3 H]-estradiol at concentrations as high as 50 nM. One possible function of this binding system is that the coligand (accessory factor) may enable the cell to concentrate steroid, and depending on the extent of steroid binding, regulate the intensity of the reaction in which the binding complex participates.

Tyrosine aminotransferase converting factor kinetic properties, cellular localization, and tissue distribution

Utilizing a more rapid procedure for determining tyrosine aminotransferase-converting factor activity, the kinetic properties of this factor were characterized further. Tyrosine aminotransferase-converting factor is a heat-labile substance present in the particulate fraction of rat liver that converts tyrosine aminotransferase form III to I at 4 degrees C. Analysis of the distribution of marker enzymes for mitochondria and lysosomes, and of converting factor, following differential and discontinuous sucrose gradient centrifugation indicated that this factor was associated with light lysosomes. The activity of converting factor was not altered following administration of cortisol. Converting factor activity, equivalent to that in liver, was also observed in particulate fractions from kidney and spleen, and to a lesser extent, in pancreas and salivary gland. No detectable activity was observed in brain, heart, small intestine, skeletal muscle, red blood cells, serum, or plasma. The presence of converting factor activity in kidney and spleen suggests that other proteins are substrates for this factor since tyrosine aminotransferase is virtually absent from these tissues. Alternatively, the absence of converting factor from other tissues need not indicate they are devoid of converting factor-like activity merely, that such activity in these tissues has different specificities and does not utilize tyrosine aminotransferase as a substrate.

Endogenous factor in rat liver that converts tyrosine aminotransferase form III to form I: a rapid assay of this converting factor.

Abstract After induction by cortisol, tyrosine aminotransferase ( l -tyrosine:2-oxoglutarate aminotransferase, EC 2.6.1.5) present in rat liver homogenates can be resolved into three peaks of activity by CM-Sephadex chromatography. Based on differential elution of these forms by a linear KCl gradient, a three-tube assay was developed that quantitates the amount of form III relative to total enzyme. The assay was used to determine the presence of a factor in the liver that converts tyrosine aminotransferase form III to form I. Definitive evidence for the liberation of such a factor is presented.

Induction of tyrosine aminotransferase by D-glucosamine in rat hepatoma cell culture

Abstract Tyrosine aminotransferase (TA) was induced 5–8 fold by D-glucosamine (0.025M) in rat hepatoma cells grown in culture. This effect was observed only in a D-glucose deficient incubation medium. Under these conditions D-glucosamine cannot replace D-glucose as a carbon source as evidenced by the marked reduction in protein and DNA in these cultures over a 72 hour period. Although total protein per culture decreased, there was a net increase in TA which was sensitive to actinomycin-D and cycloheximide treatment. N-acetyglucosamine was without effect under these conditions.

Isolation of citroylformic acid-γ-lactone from a commercial batch of oxaloacetic acid: Inhibition of apotyrosine aminotransferase conversion to holoenzyme by this substance

Abstract Citroylformic acid-γ-lactone (CFA, 1-keto-2,4-dihydroxy-4-carboxyadipenoic acid(2–3)-1,4-lactone), isolated from a commercial batch of oxaloacetate, inhibited conversion of rat liver apotyrosine aminotransferase (EC 2.6.1.5) to holoenzyme. Using partially purified enzyme, the K i was determined to be less than 0.7 m m . A more definitive K i was difficult to obtain because at pH 7 CFA had a half-life of about 2 hr. Inhibition of the enzyme by CFA was stereospecific and reversible; the S (−) stereoisomer was approximately 10 times more inhibitory than its R (+) antipode, and over 90% of inhibited enzyme was recoverable after overnight dialysis. Preineubation of apotyrosine aminotransferase with its coenzyme (pyridoxal phosphate) prevented inhibition by CFA, and a substantial fraction of enzyme that had been inhibited by CFA could be readily reactivated by addition of high concentrations of pyridoxal phosphate. Studies with inhibitor analogs indicated that both a partially unsaturated lactone ring and a stereospecific carboxymethyl group are required for maximal inhibitory activity. The sodium salts of citroylformic acid and oxalopyruvic acid, formed by the hydrolysis of their respective lactones, were not inhibitory; 1-keto-2,4-dihydroxy-4-carboxyadipic acid-γ-lactone and little inhibitory activity, and 1-keto-2,4-dihydroxyglutarenoic acid-γ-lactone and 1-keto-2,4-dihydroxybutene-γ-lactone were somewhat better inhibitors than the R (+) stereoisomer of CFA. The possibility that CFA is a naturally occurring biological substance is discussed.

Post-transcriptional inhibition of protein synthesis by rifampicin in rat hepatoma cells☆

RIFAMPICIN (rifampin) has been shown to inhibit induction of tyrosine aminotransferase by cortisol in rat hepatoma cells (Reuber H-35) grown in culture but not in rat liver. This effect was independent of any action on RNA polymerase since no change in either the Mg++- or Mn++/(NH4)2SO4-activated enzymes of hepatoma cells was noted. Since a 30% decrease in L[14C] leucine incorporation into protein of hepatoma cells was observed in the presence of rifampicin (166 μg/ml), it is likely that inhibition of tyrosine aminotransferase induction occurred at a site related to translation rather than to transcription.