Hormone storage granules in the beef anterior pituitary. I. Isolation, ultrastructure, and some biochemical properties.

A method is described for isolation of relatively large quantities of large and small hormone storage granules from the beef adenohypophysis. The hormone storage granules are highly purified, as indicated by ultrastructural and biochemical criteria. The average size of large granules is 400 mmicro and of small granules is 220 mmicro. The large granules contain growth hormone and prolactin; the small granules contain high concentrations of follicle-stimulating, luteinizing, and thyroid-stimulating hormones. An alkaline protease with a pH optimum of 8.3 is associated with the small granule fraction.

Isolation of corticotropin-beta-lipotropin common precursor from ovine pituitary glands.

Ovine corticotropin-beta-lipotropin common precursor was purified to homogeneity from commercial frozen ovine pituitary glands. A crude preparation was obtained following a procedure published elsewhere (Lee, T.H. and Lee, M.S. (1977) Biochemistry 16, 2824-2829) and was purified by gel filtration on Sephadex G-200 in the presence of 0.5% SDS and 0.1% 2-mercaptoethanol, and under an atmosphere of nitrogen. The gel filtration was repeated once. The partially purified preparation obtained from the second Sephadex G-200 gel filtration was further fractionated by preparative SDS-acrylamide gel electrophoresis, using immunoprecipitated and electrophoretically purified [125I]corticotropin-beta-lipotropin common precursor as a marker. The preparation was judged homogeneous by the appearance of a single protein band in analytical SDS-acrylamide gel electrophoresis, which exhibited both corticotropin and beta-lipotropin immunoreactivities, and a single symmetrical peak in high-pressure liquid chromatography on a reverse phase C18 column. The isolated ovine corticotropin-beta-lipotropin common precursor possessed specific activities of 116 micrograms of immunoreactive corticotropin and 210 micrograms of immunoreactive beta-lipotropin per mg of protein, equivalent to 89 and 62% of theoretical values, respectively. The amino acid composition of the homogeneous preparation was determined.

Characterization of a macromolecular aggregate of ovine pituitary corticotropin-beta-lipotropin common precursor.

A macromolecular aggregate of corticotropin-beta-lipotropin common precursor had been observed in ovine pituitary preparations as an excluded fraction of Sephadex G-200 gel filtration. This fraction could not penetrate a 10% gel during sodium dodecyl sulfate-polyacrylamide gel electrophoresis, when 2-mercaptoethanol or other disulfide-cleaving agents were not present in the buffer used to solubilize the protein preparation prior to the electrophoresis. On a 4.6% gel (acrylamide:bisacrylamide, 20:1), the material migrated as a diffuse band to a position between those of beta-galactosidase (Mr 130 000) and myosin (Mr 200 000). Both observations were consistent with an apparent Mr greatly in excess of that of the corticotropin-beta-lipotropin common precursor reported by many investigators. Neither 5% SDS nor 1% Triton X-100 could dissociate the macromolecular aggregate, but 2-mercaptoethanol and urea, either alone or in combination, were able to dissociate it to two main protein components, one of which was identified as corticotropin-beta-lipotropin with an apparent Mr of 34 000. The fact that urea alone could dissociate this macromolecular aggregate led us to believe that it might be a non-covalent aggregate and that 2-mercaptoethanol probably did not achieve the dissociation through the cleavage of an interchain disulfide bond but by bringing about conformational changes as a result of reduction of intrachain disulfide bonds so that aggregation became unfavorable. Moreover, the dissociation by urea or by 2-mercaptoethanol was found to be irreversible. The origin of the macromolecular aggregate of corticotropin-beta-lipotropin common precursor remains obscure.

Distribution of immunoreactivities for adenohypophysial hormones in the pituitary gland of the polypteriform fish, Polypterus endlicheri.

Polypteriform fish constitutes the most primitive living descendent of the ancient bony fish. In polypteriform fish, only proopiomelanocortin (POMC) has been identified so far in the adenohypophysis, which is surprising in view of their evolutionary importance. In the present study, distribution of immunoreactive adenohypophysial hormones was examined in juvenile individuals of Polypterus endlicheri. Antisera to tetrapod and fish adenohypophysial hormones were used as immunostaining probes. Adrenocorticotropin (ACTH)-like cells were detected by antisera to salmon POMC N-terminal peptide, porcine ACTH and mammalian alpha-melanotropin (MSH), and were distributed in the rostral pars distalis in close proximity to the hypophysial duct. MSH-like cells were found in the pars intermedia, and were stained by anti-salmon N-Ac-beta-endorphin II as well as anti-mammalian alpha-MSH and anti-salmon POMC-N terminal peptide. Prolactin (PRL)-like cells were detected only after application of anti-sturgeon PRL, and were distributed in the rostral pars distalis, where PRL-positive material was found in columnar mucinous cells lining the diverticuli of the hypophysial duct. Growth hormone (GH)-like cells were stained with antisera to sturgeon GH, human GH, salmon GH and blue shark GH, and were distributed in the proximal pars distalis. Somatolactin (SL)-like cells were stained with anti-salmon SL, and were distributed in the pars intermedia. Two types of glycoprotein hormone-positive cells were detected in the proximal pars distalis. Although both types of cells were stained with several antisera to glycoprotein hormones, such as sturgeon LHbeta and salmon LHbeta, it was difficult to know which types of cells produce LH, FSH, or TSH. Thus, the present study revealed seven types of adenohypophysial hormone-like cells in the Polypterus pituitary gland, which may provide the morphological basis for better understanding on evolution of the pituitary gland and the adenohypophysial hormones in vertebrates.

Characterization and purification of iodinated bovine thyrotropin by high performance liquid chromatography.

Reversed-phase (RP) HPLC, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and gel permeation chromatography have been used to study the incorporation of 125I into bovine (b) TSH by the lactoperoxidase-catalyzed radioiodination procedure. Two preparations of [125I]bTSH were studied, being freshly iodinated bTSH and tracer purified from this preparation by the method of receptor adsorption. It is demonstrated with these methods that both the alpha- and beta-subunits of bTSH are labeled with 125I, and that the tracer purified by receptor adsorption retains this incorporation pattern. However, the implied theoretical specific activity of (at least) 2 I atoms per TSH molecule (or approximately 140 muCi/micrograms) suggested by this result was not achieved, with observed tracer specific activity being 30-60 muCi/micrograms, indicating that hormone molecules with varying extents of labeling must exist. Evidence to support this was provided by comparison of the MIT/DIT ratios for the 2 tracer preparations. Receptor adsorption decreased the MIT/DIT ratio from 75:25 in the freshly iodinated bTSH to 93:7, indicating the selection of particular iodinated species. Tryptic mapping by RP-HPLC was used to study both tracer preparations, and it is shown that at least 14 iodine-containing tryptic peptides may be resolved for each preparation, which is greater than the theoretical maximum of 13 peptides if every tyrosine was labeled and tryptic cleavage occurred at all possible lysine and arginine residues. Tracer heterogeneity was also studied by purification using RP-HPLC. Selection of peak fractions demonstrated that intact [125I]bTSH may be recovered from RP-HPLC which in TSH radioreceptor assay exhibit increased assay sensitivity, increased saturable binding, and decreased nonsaturable binding.

Renotropic activity in ovine luteinizing hormone isoform(s).

Renotropic activity was previously demonstrated in an ovine LH preparation. This preparation was further purified with a series of chromatographic steps, and the fractions were assayed for renotropic activity in vivo by their ability to stimulate [3H]thymidine incorporation into renal DNA of castrated hypophysectomized male rats. A purified preparation could be dissociated by acid treatment into two major constituent subunits, designated alpha and beta, each of which was composed of three microheterogeneous components (subunits alpha 1-3 and beta 1-3) by reverse phase HPLC. Peptide mapping, including amino acid analyses and partial sequencing of the purified peptides, showed that 1) subunits alpha 3 and beta 3 possess the full length of the polypeptide chains, with the same amino acid sequences as those of the corresponding LH subunits alpha and beta, respectively; and 2) subunits alpha 1 and alpha 2 are complexes of three polypeptides which are missing several N-terminal residues from subunit alpha 3. Conversely, subunits beta 1 and beta 2 lack the C-terminal two residues and one residue, respectively, of subunit beta 3. Renotropic activity was not detected in any of the dissociated subunits alone, but association of alpha 1-3 with beta 1-3 reconstituted the hormonal activity with different potencies. In particular, combination of subunits alpha 3 and beta 3 (alpha 3.beta 3) yielded a potent renotropic activity with weak gonadotropic activity. The carbohydrate composition of the purified preparation exhibiting renotropic activity differed from that of a reference oLH preparation, which possessed greater gonadotropic activity but was devoid of renotropic activity. Furthermore, renotropic activity was decreased after removal of sialic acid by treatment with neuraminidase. Thus, the oligosaccharide moieties as well as the amino acid sequences of the subunits may play an important role in the expression of renotropic activity in vivo, these effects over and above those arising from differential metabolic clearance. We conclude that pituitary renotropin represents a novel activity of a LH- isoform(s) and that the posttranslational (or the artificial, i.e. during preparation) modification of the constituent LH subunits may be responsible for modulation of renotropic activity as well as the intrinsic gonadotropic activity.

Purification and characterization of an incompletely glycosylated form of human chorionic gonadotropin from human placenta.

A small form of hCG (SP-hCG) was purified from an acetone powder preparation of human first trimester placenta by repeated gel filtration and ion exchange chromatography on a Q-Sepharose or FPLC Mono Q column. The estimated mol wt (Mr) of the small hCG by gel filtration is 43K compared to 58K for authentic hCG. The pI of SP-hCG is 10.0, suggesting deficiency of sialic acids. SP-hCG dissociates into subunits when treated with 6 M guanidine-HCl or analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Two beta-subunits of SP-hCG were found with estimated Mr of 23K and 20K. Both are distinctly smaller than authentic hCG beta. A single alpha-subunit was found, with an estimated Mr of 21 K. The immunoactivity (8,900-10,000 IU/mg) of highly purified SP-hCG was comparable to that of reference hCG (CR119) determined by a RIA method using anti-hCG antibodies. The hCG/LH receptor-binding activity of SP-hCG is equivalent to that of reference hCG (CR119). Its biological activity is lower than that of reference hCG (approximately 30% or more) assayed by the in vitro stimulation of rat Leydig cells to produce testosterone and cAMP. A high dose is required to attain the same level of stimulation as reference hCG. The amino acid composition of SP-hCG is similar to that of reference hCG, whereas its hexsamine content is significantly lower. Its glucosamine content is about half that in reference hCG, while it completely lacks galactosamine. These findings suggest that SP-hCG is deficient in O-linked oligosaccharide chain in the beta-subunit, and that the N-linked oligosaccharide chains of both subunits are shortened. SP-hCG is one of the principal forms of the hormone present in first trimester placenta and may be a key intermediate in the posttranslational biosynthesis of hCG. Although it lacks O-linked sugar chains and shortened N-linked sugar chains, it possesses substantial biological activity. To have full biological activity, the hCG molecule must contain the complete complement of sugar chains.

On the isolation of human pituitary hormones.

Six human pituitary hormones can be obtained from a single batch of human pituitary glands by the procedure described. Starting from an acetone powder of the glands, the hormones are segregated into three fractions; one containing GH and PRL, a second containing the glycoprotein hormones, and the third fraction containing smaller peptides including ACTH and MSH. Clinical grade GH is extracted from the residue at alkaline pH, followed by a series of ammonium sulfate and pH fractionations. Further chromatography on G-100 yields a highly purified GH preparation. FSH is purified by chromatography on carboxymethyl cellulose at pH 5.4 followed by gel filtration on G-100 and fractionation on sulfopropyl Sephadex C-50 at pH 5.4. LH and TSH are separated by DEAE-cellulose chromatography at pH 9.5. The GH and glycoprotein hormones are recovered in a highly purified form and good yields. The GH is obtained in an active form which is easily soluble and well suited for clinical use. A large part of the PRL is also saved during the extraction procedure.

Human plasma beta-endorphin-like peptides: a rapid, high recovery extraction technique and validation of radioimmunoassay.

This is a report of the development, calibration, and validation of a series of techniques required to measure beta-endorphin (beta-END)-like immunoreactivity in human plasma, including sieve and affinity chromatography. The RIA, which uses the antibody Brenda, is very sensitive (IC50 = 5-15 fmol/tube at a final concentration of 1:40,000). The extraction process, which uses the Sep-Pak C18 cartridge (Waters Associates, Inc.), is simple and rapid and has a recovery rate of more than 90%. It extracts proopiomelanocortin, beta-lipotropin, and beta-END. Physiological validation was provided by the measurement of beta-END-like immunoreactivity in a pool of plasma of normal humans (2.25 fmol/ml plasma), two pregnant women at term (9.5 and 10.75 fmol/ml), and one patient with Nelson's disease (2 pmol/ml plasma).

Rat placental mRNA directs the synthesis of a polypeptide immunologically related to alpha-subunit of glycoprotein hormones.

In order to investigate the existence of a chorionic gonadotropin (CG) in the rat, placental mRNA was prepared from either the foetal disc or the maternal site of implantation in pregnant rats and translated in a wheat-germ cell-free translation system in the presence of 35S-labeled methionine and cysteine. Sodium dodecylsulphate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of the radioactive material immunoprecipitated using antiserum specific to native rat alpha-subunit allowed us to isolate in translation products from both sources (foetal disc and maternal site of implantation) a single polypeptide of 17 kDa having the electrophoretic properties of the rat pituitary alpha-precursor. This polypeptide was absent in media derived from translation of mRNA extracted from uterine horn of non-pregnant female rats, and was approximately 10 times more abundant when RNA was derived from the maternal part (about 0.25% of cpm in total proteins) rather than the foetal part (0.026%) of the placenta. Immunoprecipitation was prevented in the presence of an excess of rat (but not ovine) alpha-subunit. Antisera directed against denatured bovine alpha-subunit, which cross-react with the rat pituitary alpha-precursor, did not bind the placental peptide. These results suggest that this rat placental polypeptide and the rat alpha-subunit of pituitary glycoprotein hormones have important differences in their primary structure, but share discrete structurally and/or conformationally related regions in their polypeptide chains. The possibility that these partial homologies account for gonadotropin-like activity of a presumed rat CG remains to be ascertained.