Occurrence of neurophysin-like proteins in the mammalian hypothalamoneurohypophysical system

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Short Communications Occurrence of neurophysin-like proteins in the mammalian hypothalamoneurohypophysial system The pressor and oxytocic activities associated with the posterior pituitary lobe can be precipitated out of solution1 on the addition of sodium chloride 7. The polypeptide hormones are complexed to a protein termed neurophysin which was originally thought by van Dyke e t al. s to be apparently homogenous with a molecular weight of 30,000. However, recent work s has shown that bovine neurophysin can be fractionated into 3 major proteins with a minimum molecular weight of approximately 10,000. Differential centrifugation studies on homogenates from the ox posterior pituitary has demonstrated that the major neurophysins are associated with the hormones in vesicles of the neurohypophysial secretory axons and that the population of the vesicles associated with oxytocin differs from those containing vasopressin2. It is generally considered that the synthesis of oxytocin and vasopressin occurs predominantly in the perikaryon of the paraventricular and supraoptic nuclei respectively. Synthesis might also occur within the axons leading from the hypothalamic nuclei to the posterior pituitary lobe. On the other hand, the origin of the neurophysins has yet to be determined, although this site might be expected to be similar to that of the hormones. Hawker e t al. 5 recently indicated the presence of a protein in the bovine hypothalamus which binds oxytocin but its identification as a neurophysin was not established. In this present work, posterior pituitary glands, pituitary stalks and the hypothalami of oxen, pigs and sheep were extracted so as to isolate the neurophysins. The presence of neurophysins was demonstrated by comparative starch-gel electrophoresis against authentic samples of neurophysin from the various species. The posterior pituitary lobes, pituitary stalks and hypothalami from 15 oxen, 20 pigs and 30 sheep were dissected within 45 min of death and ground to a powder in cold acetone. The powder was extracted in 0.1 N hydrochloric acid (final pH 1.5) at 4°C for 24 h. After centrifugation (3,000 rev./min for 10 min) the supernatant was adjusted to pH 7 with 1 N sodium hydroxide, the resulting precipitate discarded and then the pH of the supernatant further adjusted to 3.9 with 1 N hydrochloric acid. Sodium chloride was added to a final concentration of 10~ (w/v)and the precipitate which formed after 18 h at 4°C was collected and dialysed in 18/32 Visking membranes against distilled water (3 x 5 L at 4°C). The contents of the dialysis bags were freezedried and chromatographed in 0.1 N formic acid on a column (140 c m x 2 cm) of Sephadex G-75. This column had been previously calibrated with authentic samples of porcine s, bovine9 and ovine10 neurophysins isolated from the neural lobes of these species. Fractions possessing an elution volume similar to that of the standard neurophysins were pooled, freeze-dried and the proteins subjected to electrophoresis on Brain Research,

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S t t O R I COMMUNICATIONS

starch-gel. The borate-lithium hydroxide buffer system of Ferguson and Wallace 4, pH 8.1, was found to give the greatest resolution of the neurophysins from the 3 species. The presence of the neurophysins in the various protein fractions was supported by comparing their electrophoretic pattern on starch gel with the mobility of the standard proteins. With the hypothalamic extracts, a second chromatography on Sephadex G-75 was carried out in order to concentrate the neurophysin fractions. In this step only the 3 peak tubes were pooled and freeze-dried and the presence of the neurophysin-like proteins revealed by electrophoresis (Fig. 1, channel 5). SHEEP

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Brain Research, 32 (1971) 441 444

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SHORT COMMUNICATIONS

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Fig. 1. Diagrammatic representation of horizontal starch-gel electrophoresis of protein fractions at pH 8.1 using a starch concentration of 13.5 %. Protein samples (0.8 rag) were dissolved in gel buffer (20 #1), applied to wicks of Whatman No. 1 filter paper and inserted into slots cut in the starch-gel. Electrophoresis was carried out at a constant voltage gradient of 28 V/cm for 1.5 h. Proteins were detected by staining the gel with nigrosine (0.05 % w/v) in water-acetic acid-methanol (5:5:1). 1, Fresh or acetone-dried posterior pituitary lobes extracted in either 0.1 N HCI or electrophoresis buffer pH 8.1. 2, Posterior lobe post G-75 Sephadex chromatography. 3, Pituitary stalks after chromatography. 4, Hypothalamic extract after one chromatography. 5, Hypothalamic extract after second chromatography, e , Electrophoretic mobility of serum albumin. N-I, -II and -III represent the position of authentic samples of neurophysins-I, -II and -III.

After extraction of the pituitary stalks o f the 3 species, the only proteins which were visible on electrophoresis were those possessing a mobility identical to the neurophysins. With the bovine hypothalamic extracts, the major proteins present were also neurophysin-like in mobility on starch-gel. On the other hand, in the case of the hypothalamic extract from the pig, only the major neurophysins - - porcine N-I (which account for 44% of the total binding proteins 9) and porcine N - I I (39%) - were visible. The third neurophysin, N-III, being only a minor component (8 %), was much more difficult to visualise. Fractionation of sheep posterior pituitary lobes also yields 3 neurophysins: approx. N-I (15 %); N - I I (15 %) and N - I I I (70 %)10; but with the hypothalamic extracts only the major component N - I I I and one of the minor species N - I were visible. The results presented here support the concept that the neurophysins are present in the mammalian hypothalamus and thus suggest that synthesis could occur in this region of the brain in association with the neurohypophysial hormones. Under the experimental conditions employed, the minor neurophysins porcine N - I I I and ovine N - I I were not readily detected in the hypothalamic extracts and this finding suggests Brain Research, 32 (1971) 441 A.A.'!

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that synthesis of these proteins may occur to a limited extent within the neurosecretory axons of the pituitary stalk. This work was supported by a grant from the New Zealand Medical Research Council. Postgraduate School of Obstetrics and Gynaecology, University of Auckland, Auckland (New Zealand)

W. B. WATKINS H. K. ELLIS

1 ACHER, R., AND FROMAGEOT,C., The relationship of oxytocin and vasopressin to active proteins of posterior pituitary origin. In H. HEELER(Ed.), The Neurohypophysis, Butterworths, London, 1957, pp. 39-50. 2 DEAN, C. R., HOPE, D. B., AND KA;~I~, J., Evidence for the storage of oxytocin with neurophysin-I and of vasopressin with neurophysin-II in separate neurosecretory granules, Brit. J. Pharmacol., 34 (1968) 192P-193P. 3 VANDYKE, H. l . , CHOW, B. F., GREEP, R. O., AND ROTHEN,A., The isolation of a protein from the pars neuralis of the ox pituitary with constant oxytocic pressor and diuresis - - inhibiting activities, J. Pharmacol. exp. Ther., 74 (1942) 190-209. 4 FERGUSON,K. A., AND WALLACE,A. L. C,, Starch-gel electrophoresis of anterior pituitary hormones, Nature (Lond.), 190 (1961) 629-630. 5 HAWKER, R. W., NORTH, W. G., AND ZERNER,B., The pharmacology of a new oxytocic principle from ox hypothalamus, Brit. J. Pharmacol., 35 (1969) 175-186. 6 HOPE, D. B., WATKINS, W. B., AND HOELENBERG,M. D., unpublished work. 7 KAMM,O., ALDRICH,T. B., GROTE,I. W., ROWE, L. W., AND BUGBEE,E. P., The active principles of the posterior lobe of the pituitary gland. 1. The demonstration of the presence of two active principles. 2. The separation of the two principles and their concentration in the form of potent solid preparations, J. Amer. chem. Soc., 50 (1928) 573-601. 8 RAUCH, R., HOLLENBERG,M. D., AND HOPE, D. B., Isolation of a third bovine neurophysin, Biochem. J., 115 (1969) 473-479. 9 UTTENTHAL,L. O., AND HOPE, D. B., The isolation of three neurophysins from porcine posterior pituitary lobes, Biochem. J., 116 (1970) 899-909. 10 WATKINS, W. B., Unpublished results. (Accepted June 24th, 1971)

Brain Research, 32 (1971) 441-444