Albert Grossman

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.

Problems interpreting the significance of multiple form patterns of rat liver tyrosine aminotransferase

Summary Cortisol induced tyrosine aminotransferase (L-tyrosine: 2-oxoglutarate aminotransferase, EC 2.6.1.5) of rat liver cytosol can be resolved into several peaks of activity by CM-Sephadex chromatography. These separable activities have been considered distinct molecular species and are referred to as multiple forms of this enzyme. The present study demonstrates that a number of in vitro manipulations considerably alter the pattern of multiple forms of tyrosine aminotransferase. These include homogenization of the liver in buffers of varying ionic strength and pH, freezing and thawing, and sonication. Based on these findings we conclude that one must proceed cautiously when attempting to relate multiple form patterns observed in vitro to the form(s) of the enzyme in vivo .

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.

Site-specific antibodies to the DNA-binding domain of estrogen receptor distinguish this protein from the 3H-estradiol-binding protein in pancreas

The estradiol-binding protein (EBP) in extracts of rat and rabbit pancreata was characterized by sucrose density gradient analysis, immunoaffinity adsorption and Western blot (immunoblot) analysis using polyclonal antibodies raised against EBP. Rat pancreatic extracts labeled with 3H-estradiol contained a readily resolvable peak of steroid-binding activity that sedimented as a 4S complex on sucrose density gradients in the presence or absence of 0.4 M KCl. Estrogen receptor (ER) from calf uterine cytosols sedimented as a 4S complex on gradients containing 0.4 M KCl and as an 8S entity on gradients without KCl. Incubation of cytosol fractions from rat pancreas and calf uterus with benzoyl-DL-arginyl-p-nitroanilide (BAN) increased specific binding of 3H-estradiol to EBP but not to ER. Furthermore, two distinct site-specific antibodies to the DNA-binding domain of ER caused a marked increase in sedimentation rate of 3H-estradiol-labeled ER while normal rabbit serum and antibodies against EBP were ineffective in this regard. These data suggest that a significant portion, if not all, of the DNA-binding domain of ER is absent from EBP. Examination of the amino acid sequence of the DNA-binding domain of ER revealed a region of 10 amino acids that is significantly homologous to somatostatin, a tetradecapeptide that is a co-ligand in the binding of 3H-estradiol to EBP. Based on this observation, a possible mode of action of EBP in pancreatic acinar cells is proposed.

Information transfer in biological systems: targeting of proteins to specific organelles or to the extracellular environment (Secretion)

Orderliness is the salient characteristic of living systems. Cells are intolerant of disorder. They express this by rapidly eliminating or degrading out-of-place molecules. When cells are broken apart and their constituent organelles separated and analysed, the same types of macromolecules are always associated with the same subcellular structures. One finds, for example, the same proteins in mitochondria time after time, and these differ from the sets of proteins found in nuclei, secretory granules, or plasma membranes. The information necessary to target each protein to its appropriate intracellular destination is determined primarily by the gene for that protein. Encoded within the DNA structure of genes are signals that specify where each protein molecule belongs. Thus, it is the transfer of information from one macromolecule to another that maintains the integrity and orderliness of living cells.

Evidence that somatostatin (SRIF14) is the primary coligand in pancreas required for specific binding of [3H]estradiol in pancreatic tissue: demonstration that [3H]estradiol and [125I]SRIF14 form complexes of varying size with a specific binding protein

There is present in rat pancreas a protein that requires an accessory factor in order to bind [3H]estradiol. To identify this accessory factor 874g of dog pancreas were acid extracted, and following selective filtration and dialysis, the low molecular weight constituents (less than 10,000) were concentrated by lyophilization. Samples of this lyophilizate were fractionated by high performance liquid chromatography (HPLC) and eluate fractions analyzed for their capacity to enhance binding of [3H]estradiol to a protein fraction from rat pancreas that had been purified relatively free of endogenous accessory factor. Such enhancement of [3H]estradiol-binding activity eluted predominantly in one peak that coincided with the elution profile of pure somatostatin (SRIF14). Analysis of eluate fractions for somatostatin-like immunoreactive material (SLIM) indicated coincidence of SLIM with the factor that enhanced binding of [3H]estradiol. It appears likely that accessory factor in pancreas is primarily somatostatin (SRIF14). Following incubation of [125I]SRIF14 and [3H]estradiol with a partially purified binding-protein fraction from rat pancreas, a complex containing labeled [125I] and [3H] was separated by Sephadex G-200 column chromatography. In the presence of 25 microM SRIF14, which activates [3H]estradiol-binding maximally in the presence of 10 nM steroid, a protein peak containing both radiolabeled ligands eluted in the void volume indicating an apparent molecular size in excess of 200,000 Daltons. At a concentration of 1 microM SRIF14, a complex eluted at a position corresponding to an apparent Mr of 120,000. Evidently, the steroid and polypeptide mutually enhance binding to this pancreatic protein, and depending on their concentrations form structures of widely varying sizes.

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.