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Three neurophysins have been isolated from acetone-dried sheep posterior lobe powder and have been named neurophysins I, II and III. Neurophysins I, II and III account for approximately 15, 15 and 70%, respectively of the total neurohypophysial hormone-binding proteins present in the sheep posterior pituitary lobe. All the neurophysins bind oxytocin and [8-arginine]-vasopressin. Neurophysins I and II each were found to contain one molecule of histidine/molecule of protein but to lack methionine, whereas neurophysin III contained no histidine but had one molecule of methionine/molecule of protein. The molecular weights of sheep neurophysins I, II and III were 11348, 9803 and 10377, respectively, as determined by amino acid analysis. An approximate value of 10000 was estimated for the apparent molecular weight of the neurophysins by gel exclusion chromatography on Sephadex G-75. Solid amorphous complexes were formed between the neurophysins and oxytocin as well as with [8-arginine]-vasopressin. In each case one molecule of neurophysin complexed with one molecule of hormone. The stoichiometry of the sheep neurophysins relative to the neurohypophysial hormones is discussed.
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The polypeptide hormones of the posterior pituitary gland, oxytocin and vasopressin, can be precipitated out of solution by the addition of sodium chloride to an extract of the gland. The hormones are salted-out complexed to the soluble proteins known as neurophysins and this protein-hormone complex has been found to possess the pressor and oxytocic activities in ratios similar to those found in the fresh gland (van Dyke, Chow, Greep & Rothen 1942). Neurophysins have been isolated from the pig (Uttenthal & Hope, 1970), ox (Rauch, Hollenberg & Hope, 1969) and cod (Pickering, 1968). In the present paper the proteins precipitated from the posterior lobe of the human pituitary gland were investigated by gel exclusion chromatography and starch gel electrophoresis, and their ability to bind oxytocin and vasopressin was determined.
Acetone-dried posterior pituitary lobes (2·4 g) were extracted for 24 h at 4 °C in 0·1 m-hydrochloric acid (100 ml),
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Three neurophysins have been isolated and biochemically characterized from the ox (Rauch, Hollenberg & Hope, 1969), pig (Uttenthal & Hope, 1970) and sheep (Watkins, 1972) while the presence of a neurophysin has been demonstrated in cod pituitary glands (Pickering, 1968).
By means of immunological and biosynthesis techniques, Norstrom, Sjöstrand, Livett, Uttenthal & Hope, (1971) have tentatively identified one neurophysin in the rat posterior pituitary gland. In a recent extension of this work, Burford, Jones & Pickering (1971) using polyacrylamide electrophoresis have been able to resolve the major rat posterior pituitary gland protein into two major components one of which was tentatively described as an oxytocin-neurophysin and the other as a vasopressin—neurophysin.
In this present investigation, the neurophysins of the rat were fractionated using procedures previously established for the purification of the ox, pig and sheep neurophysins. The protein-hormone complex was prepared in a yield of 26·9 mg from an
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SUMMARY
Acetone-dried rat posterior pituitary glands were extracted with 0·1mhydrochloric acid and the soluble proteins fractionated by Sephadex gel exclusion and ion exchange chromatography. Three proteins had chromatographic and biochemical properties expected of neurophysins and also cross-reacted immunologically (as demonstrated by microimmuno-diffusion and -electrophoresis in agarose) with an anti-serum raised against porcine neurophysin-II. The three proteins were named neurophysin-I, -II and -III in order of their electrophoretic mobility on starch-gel and accounted for approximately 8, 70 and 22% respectively of the total neurophysin present in the gland. The assignment of the major rat posterior pituitary lobe protein as a neurophysin (neurophysin-II) is confirmed by its ability to bind oxytocin and [8-arginine]-vasopressin. A minimum molecular weight of 10056 was calculated for neurophysin-II from its amino acid analysis and based upon the presence of one molecule of methionine and one molecule of tyrosine per molecule of protein. The stoicheiometry of the neurophysins relative to the neurohypophysial hormones is discussed.
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A method has been described for the purification of ovine placental lactogen (oPL) involving the use of freshly obtained sheep foetal cotyledons. Tissue was extracted with 0·1 m-ammonium bicarbonate and the supernatant fraction, adjusted to pH 7, was brought to 60% saturation with ammonium sulphate. The resulting precipitate was then subjected to a sequence of chromatographic steps using columns of Sephadex G-100 and carboxymethylcellulose. During each stage of the purification, the lactogenic activity was monitored with a pregnant rabbit mammary gland radioreceptor assay. The yield of oPL corresponded to 8 mg/kg wet foetal tissue and the oPL possessed lactogenic activity equivalent to 1 mg ovine prolactin/mg protein and GH-like activity equivalent to 0·8 mg human GH/mg protein. The biological activity of oPL was confirmed using a rabbit intraductal mammary gland assay in vivo.
After polyacrylamide gel electrophoresis at pH 8·9, oPL was resolved into one major band (isoelectric point 8·2–8·4) and four minor components, which were thought to be deamidation products of oPL. Microimmunoelectrophoresis and immunodiffusion studies confirmed that the preparation of oPL was free from serum protein contaminants.
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[35S]Cysteine was injected intracisternally into guinea-pigs and 5 h later the soluble proteins of the neural lobe were subjected to electrophoresis on starch and polyacrylamide. In both systems approximately 80% of the total radioactivity was recovered in a single peak with an electrophoretic mobility corresponding to the position of the major protein component. The proteins present in the guinea-pig neural lobe were extracted in 0·1 m-HCl. Fractionation of the protein—neurohypophysial hormone complex on Sephadex G-75 resulted in the isolation of a protein which cross-reacted immunologically with an antiserum raised against porcine neurophysin II. The protein designated a neurophysin contained one molecule of lysine, isoleucine and tyrosine per molecule of protein. It contained no histidine or methionine and its minimum molecular weight was estimated as 8436 by amino acid analysis.
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Short-term incubation of human placental tissue in Krebs–Ringer bicarbonate buffered media with various concentrations of K+ and Ca2+ showed a graded response in human placental lactogen (HPL) release at different Ca2+ concentrations, but no effect at increased K+ concentration. Media with high Ca2+ caused an inhibition of release, while Ca2+-free media caused a stimulation in HPL release. High concentrations of Mg2+ inhibited release minimally, while Ba2+ had no effect. There was no change in HPL release when Na+ concentration was increased. La3+-Locke's solution markedly inhibited release of HPL but the significance of this effect is unknown. These results suggest that Ca2+ is not required for HPL secretion from placental tissue. It seems that HPL secretion in vitro does not follow the usual pattern where a physiological stimulus or high K+ concentration causes inward movement of calcium which couples stimulation to secretion.
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The rate of clearance from the circulation and uptake into tissues of radioactive label was studied after i.v. injection of 125I-labelled human placental lactogen (HPL) into rats at various stages of pregnancy. The half-life was obtained for the disappearance of the trichloroacetic acid-precipitable material from the plasma. The half-life, t ½(S), calculated over the first 5 min after injection of the hormone was 5·4 ± 1·1 (s.d.) min, while a half-life, t ½(L), of 27·9 ± 2·3 min was obtained from the decay period of 15–35 min.
In the non-pregnant and pregnant rat the highest ratio of the radioactivity in an organ to that in the blood was 12–14:1 in the kidney. That the kidney is mainly involved in the uptake of exogenous HPL is further confirmed by the application of the histochemical immunoperoxidase technique. Human placental lactogen was localized in the cells of the proximal tubules of the cortex and to a lesser extent in the tubular lumen and the tubules of the medulla region.
Uptake of HPL in vivo also occurs in the mammary gland tissue of the post-partum rat and reaches a maximum uptake between 15 and 30 min after injection of the hormone. Furthermore, specific uptake of HPL was observed on the alveolar cell membranes after the incubation of paraffin-embedded sections of formalin-fixed mammary gland and subsequent treatment by the peroxidase-labelled antibody method.
These findings support the work of others who have demonstrated the presence of specific membrane receptors in the mammary gland for hormones with prolactin-like activity.
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The rise in concentrations of FSH and LH in serum seen 24 h after castration was suppressed by the administration of an extract of bull seminal plasma or testosterone propionate at the time of castration. Whereas testosterone propionate preferentially suppressed LH, the seminal plasma extract suppressed FSH and LH equally. Small doses of bull seminal plasma extract and testosterone, that had little effect separately, acted synergistically to suppress levels of FSH and LH to those found in intact animals, while combinations of larger doses had little further effect. This selective interaction suggests how inhibin and testosterone might together regulate concentrations of FSH and LH in the blood of the male rat.
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Pituitary glands were collected from a selection of 22 domestic and exotic mammalian species. The soluble proteins extracted from the neurohypophyses were characterized by horizontal starch-gel electrophoresis at pH 8·1. Those species which were closely related phylogenetically, e.g. fallow deer and muntjac deer, pig and hippopotamus, dog and coatimundi, and members of the primates, had similar and in some cases identical protein profiles. The ability of proteins extracted from the starch-gel to cross-react immunologically with an antiserum raised against porcine neurophysin-II was determined by microimmunodiffusion. Using this technique for the identification of neurophysins in conjunction with osmotic stimulation experiments, it was found that the number of major neurophysins present in the mammalian neurohypophyses studied varied from one in the guinea-pig and hedgehog to four in man. The concept of multiple neurophysins is discussed.
