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Biomedical subjects

G A Puca

Publications and source records attributed to G A Puca.

At least 55 records · Page 3Linked to original sources
PubMed1977

Estrogen binding proteins of calf uterus. Molecular and functional characterization of the receptor transforming factor: A Ca2+-activated protease.

It was previously shown that calf uterus cytosol contains a Ca2+-activated receptor transforming factor (RTF) which irreversibly converts the larger molecular states of estrogen receptor (5.3 to 8.6 S, depending on ionic strength) into a smaller, salt-stable form (4.5 S, independent of ionic strength). We now describe a method for rapid and reliable separation of precursor and RTF-transformed receptor forms, which takes advantage of a difference in isoelectric point between the two: the more acidic precursor (isoelectric point, 6.2) is still retained by DEAE-cellulose under conditions (0.12 M KCl, pH 8.3) which produce release from cellulose of the less acidic transformed form (isoelectric point, 6.6 to 6.8). Based on this method of separation, RTF activity can be assayed easily and we could thus progress in the purification and physical and functional characterization of this factor, RTF has been purified about 100-fold. Molecular properties, as assayed by methods suited to partially purified preparations, are as follows: sedimentation coefficient, 6.4 S; Stokes radius, 45 A; molecular weight, 115,000; isoelectric point, 4.9. The Michaelis constant, expressed as moles/liter of estradiol binding sites, is 1.25 X 10(8), at pH 7.5 and 4 degrees, pH 8.5 is optimum for activity. RTF attacks native casein (Km, 1.25 X 10(-5) mol/liter at pH 7.5 and 22 degrees) but not hemoglobin, ovalbumin, or albumin. N-Benzoylarginine methyl ester is a competitive inhibitor of RTF-induced receptor transformation, while L-leucylglycylglycine and N-benzoyltyrosinamide are not. RTF activity is protected by -SH compounds. RTF activity is Ca2+-dependent. Ca2+ starts an activation-inactivation cycle of RTF, with permanent loss of transforming activity which proceeds at a particularly fast rate in the absence of substrate. Mg2+ is inactive, while Sr2+ and Mn2+ may in part substitute for Ca2+. RTF is present in both endometrium and myometrium. RTF is not a lysosomal hydrolase, as shown by its alkaline pH optimum (8.5) and exclusive location in cytosol, nor is it trypsin or a protease of the trypsin group. Also, it is distinct from known proteases of human uterus. The functional significance of this Ca2+-activated protease of cytosol with alkaline pH optimum and high affinity for the larger native form of receptor is still unknown.

Animals↗
PubMed1977

Estradiol receptor of calf uterus: interactions with heparin-agarose and purification.

Heparin attached covalently to agarose beads binds the "native" form of the estradiol receptor with very high affinity. Chondroitin sulfate does not bind to the receptor. When the receptor is complexed with hormone, the affinity is at least 10 times higher. Only the "native" and not the "nuclear" or the "derived" (i.e., after activation by a calcium-dependent enzyme) forms of the estradiol receptor interact with heparin. The "native" estradiol-receptor complex is purified to homogeneity after chromatography on columns of heparin-agarose, Sephadex G-200, and DEAE-cellulose, followed by two more Sephadex G-200 columns. The purified molecule is a single polypeptide of molecular weight 69,000 by polyacrylamide gel electrophoresis in sodium dodecyl sulphate. The sedimentation coefficient on sucrose gradients is 4.3 S, the Stokes radius from gel filtration is 36.5 A, and the isoelectric point is 6.4. The purified [3H]estradiol-receptor complex exchanges the radioactive hormone with estradiol or other estrogenic steroids, but not with testosterone, 5alpha-dihydrotestosterone, or progesterone.

Animals↗
PubMed1976

Guanylate cyclase. Existence of different forms and their regulation by nucleotides in calf uterus.

The activity of calf uterus guanylate cyclase (EC 4.6.1.2) exists in at least two and most probably three distinct forms. The cytosolic enzyme exhibits hyperbolic substrate curves with respect to GTP and Mn2+, while the particulate cyclases (nuclear and microsomal)display sigmoidal (GTP) and hyperbolic (Mn2+) relationships. The Hill coefficient for the GTP dependence is 0.9 for the cytosolic, 1.5 for the nuclear, and 1.4 for the microsomal enzyme. The cytosolic enzyme has a Km for GTP of 70 muM while half maximal velocity occurs at 90 and 100 muM GTP for the nuclear and microsomal enzymes, respectively. The Ka for Mn2+ is 0.57, 0.71 or 0.75 mM for the cytosolic, nuclear, or microsomal enzyme, respectively.

Adenosine Triphosphate↗
PubMed1976

Estrogen binding proteins of calf uterus. Inhibition of aggregation and dissociation of receptor by chemical perturbation with NaSCN.

Sodium thiocyanate up to 0.5 M is compatible with a stable estradiol-t-receptor complex during sucrose gradient centrifugation; however, the maximum permissible concentration in 0.1 M during Sephadex G-100 and G-200 chromatography. When NaSCN 0.1 M is added to low-salt cytosol (approximately 7 mg of protein/ml); (1) age-dependent aggregation of receptor is inhibited; (2) peaks of estrogen-binding activity in sucrose gradients and on Sephadex chromatography are sharp; (3) instead of the usual larger molecular states ("8S") found in low salt, most of estrogen receptor is under the following form: 4.1S; Stokes radius, 36 A; mol wt 61 000; flfo, 1.25; homogeneous at electrofocusing, with isoelectric point at 6.0. When cytosol containing NaSCN 0.1 M is diluted down to 2-3 mg of protein/ml or, only for sucrose gradients, NaSCN concentration is increased to 0.4-0.5 M, the 61000 dalton species decreases, being substituted, without loss of bound estradiol-t, by the following estrogen-binding entity: 28S; Stokes radius, 28 A; mol wt 32 000; flfo, 1.44. In the presence of NaSCN, KCl up to 0.4 M does not affect in a significant manner the molecular properties of the above forms. When NaSCN is dialyzed out, most receptor reverts to a 8-9S state. When cytosol is preincubated with Ca2+ (4 mM) and KCl (0.4 M) before addition of NaSCN, the above picture is modified only in the following aspects: (1) Sephadex chromatography peaks are broader and slightly but reproducibly shifted toward higher elution volumes; (2) the electrofocusing pattern consists of a two-peak heterogeneous band shifted toward higher pH (isoelectric points 6.4 and 6.6); (3) upon dialysis of NaSCN there is little or no reversion to faster sedimenting states. These modifications appear to depend on limited proteolytic attack of receptor by Ca2+ -activated receptor transforming factor (RTF), not on binding of Ca2+ to receptor. Present data suggest that the 4.1S entity is a dimer resulting from side-by-side pairing of 2.8S subunits. Molecular dimension of larger receptor forms purified from cytosol are consistent with the hypothesis that under native conditions in vivo dimers are coupled end-by-end into tetrameric structures with two stronger (between subunits) and two weaker (between dimers) bonding regions, and that tetramers may further self-associate. While NaSCN reversibly releases native dimers and subunits by direct impairment of intersubunit bonds, Ca2+ activated RTF irreversibly and specifically releases slightly modified, about 60000 mol wt dimers, by preferential proteolytic attack of the weaker bonding regions and indirect destruction of involved bonds. In vivo, this effect of RTF may be instrumental in mobilization and nuclear penetration of receptor-estradiol complex. Heteroassociation of receptor with other proteins of cytosol is not excluded by the above hypothesis.

Animals↗
PubMed1975

Interaction of the estradiol receptor from calf uterus with its nuclear acceptor sites.

The specific interaction between 17 beta-estradiol-receptor complex and nuclear acceptors was analyzed by immobilizing various nuclear proteins to CNBr-activated agarose. The specific, high affinity sites identified in a fraction of basic proteins that can be solubilized from purified nuclei of calf uterus (Puca, G.A., Sica, V., and Nola. E (1974) Proc. Natl. Acad. Sci. U.S.A. 71, 979-983) were chromatographed on Sephadex G-100 columns. Elution of the acceptor activity depends on the pH and ionic strength of the buffer used. With 5 mM HCl, however, a peak of acceptor activity with a molecular weight of about 70,000 was partially dissociated from the other basic nuclear proteins. The high affinity binding of the receptor to the acceptor proteins was estradiol-, but not progesterone-, cortisone-, or testosterone-dependent; it was very sensitive to ionic strength and showed a physiological pH optimum. Low affinity binding, such as that seen between receptor and histone, showed no estradiol dependence and little ionic strength and pH sensitivity. Native or heat-denatured DNA strongly modified the receptor-acceptor interaction, reducing the number of binding sites of acceptor for the receptor without changing the high affinity of the interaction. Heating of the acceptor protein before its covalent linkage to agarose considerably increased the affinity of the resulting agarose derivative. Free sulfhydryl groups of the receptor but not of the acceptor molecule play an important role in the acceptor-receptor interaction. When receptor and acceptor preparations were incubated in solution, the resulting complex was included on a Sephadex G-100 column and it eluted from DEAE-cellulose columns at lower ionic strength than the receptor alone. Even though not absolutely specific, these two properties allowed determination of the molecular weight (85,000) of the acceptor protein at neutral pH and more nearly physiological ionic strength. The apparent KD of the acceptor-receptor interaction was determined to be 2 x 10(-10) M at O degrees. Apparently similar, high affinity binding sites for estradiol receptors are also present in nuclei of other tissues.

Animals↗
PubMed1974

Identification of a high affinity nuclear acceptor site for estrogen receptor of calf uterus.

By means of affinity chromatography, specific nuclear acceptor sites for estradiol receptors are identified in a fraction that can be solubilized from purified nuclei with 2 M NaCl. Interaction between these acceptor sites and crude or partially purified estradiol receptor shows a high association constant (over 10(9) M). Receptor-acceptor interaction is dependent on physiological concentrations of 17beta-estradiol; it is disrupted by high ionic strength. The nuclear acceptor sites appear to be protein in nature and exist in 5- to 10-fold excess over the estrogen binding sites present in the cytosol. Single- or double-stranded DNA does not bind estrogen-receptor complexes. Acceptor sites appear to be associated with basic nuclear proteins as judged by hydroxyapatite chromatography. The nuclear acceptor sites probably represent less than 0.1% of the purified basic proteins from the nucleus.

Animals↗
PubMed1969

Purification of an estrophilic protein from calf uterus.

A method has been devised for purifying the estrogen-binding protein ("uptake receptor") of uterine cytosol which circumvents the marked tendency of this receptor to aggregate during attempted isolation. Ammonium sulfate precipitation of the estradiol-receptor complex of calf uterine cytosol in the presence of calcium ions yields both an 8S complex and its 4S subunit which are stable during further purification by gel filtration and ion exchange chromatography. The major product is the 4S complex which does not revert to 8S in the absence of salt and which has been purified about 2500-fold. The approximate isoelectric points of these partially purified 8S and 4S complexes are 5.8 and 6.4, respectively.

Animals↗