J o w i i u l ol
Nriil.ocliPriiiarl.~.1975.
Vol 24. pp. 1375 1276 Pergamon Prcss. Printed in Great Britain.
SHORT COMMUNICATION Polyacrylamide-gel electrophoresis of bovine neurophysins (Received 31 October 1974. Accepted 12 December 1974)
IN THE neural lobe of the mammalian pituitary gland the hormones vasopressin and oxytocin are stored in subcellular vesicles called neurosecretory granules. Besides hormones the vesicles also contain proteins of low molecular weight, the neurophysins (DEAN& HOPE,1967).The neurophysins have been shown to bind the hormones in vitro with binding occurring optimally at pH 5.8 (GINSBURG & IRELAND, 1963, 1964). These proteins have been isolated from extracts of whole bovine posterior putuitaries (HOLLENBERG & HOPE, 1968; RAUCHet al., 1969) and also from purified neurosecretory granules (DEAN& HOPE, 1967). Three major components, neurophysin I, I1 and C, all bind oxytocin and vasopressin. Gel electrophoresis of such extracts has, however, shown the presence in the granules of other proteins. These components, which constitute a small fraction of the soluble proteins in the neurosecrctory granules are relatively rich in cystine (RAUCHor al., 1969). In the present experiments we have used polyacrylamide-gel electrophoresis to demonstrate the presence of these proteins in extracts from freshly prepared neurosecretory granules. By means of affinity chromatography it could be shown that the proteins bind to agarose-vasopressin.
taken as an indication of the presence of neurophysins. When the material not binding to the column had been eluted the pH of the buffer was changed to 8.1 and elution was continued until a peak of disulphide-rich material had been recovered. The corresponding fractions were pooled, dialysed and evaporated down to a small volume, which was taken up in 5 mM-Tris-HCL, pH 7.5 and used for polyacrylamide-gel electrophoresis. Polyacrylamide-gel electrophoresis was performed in 5.5%gels (VILHARDT& HOPE,1974) using the buffer system described by CLARKE(1964). The gels were fixed and stained in 7.5%(v/v) acetic acid saturated with Amido Black.
RESULTS AND DISCUSSION The preparation of neurosecretory vesicles used in the present experiments has been shown to possess a high degree of purity (DEAN& HOPE,1967) with only minimal contamination by mitochondria and lysosomes (VILHARDT, BAKI.R& HOPI., to be published). When extracts of such preparations were subjected to polyacrylamide-gel electrophoresis (Fig. la) three major proteins were identified as neurophysins I, C and I1 by comparison of their mobilities with those of purified bovine neurophysin standards. MATERIAL AND METHODS Besides these well known proteins the presence of four Fresh bovine pituitary glands were obtained from the other components could be demonstrated on the polyacryllocal slaughter house. The isolated neural lobes were amide-gels. These bands are very weak and can only be homogenized in ice-cold 0.3 M-sucrose and the homogenate visualized when abundant amounts of protein are electrowas centrifuged at XOOg,, for 15min. The supernatant phoresed. Three of the proteins have mobilities intermewas then centrifuged at lOO,OOOg,, for 60min and the diate to those of neurophysins I1 and C, whereas the fourth pellet was extracted with 0.1 M-HCL for 6 h. After centrifu- band migrates slightly ahead of neurophysin I, but not gation at lOO,OOOg., for 60min the supernatant was a t the electrophoretic front (as determined by Bromoadjusted to pH 5.8 with NH,OH and used for affinity chro- phenol Blue). The electrophoretic pattern was similar to the one observed by RAUCHet al. (1969) on starch-gel ma tography. Neurosecretory vesicles were prepared by the method electrophoresis of extracts of purified neurosecretory & HOPE granules. of DEAN& HOPE(1967) as modified by VILHARDT When portions of extracts of neural lobe homogenates (1974). Fraction 111 obtained by differential centrifugation of a homogenate of bovine neural lobes was resuspended were electrophoresed on polyacrylamide-gels a number of and centrifuged at I IO,OOOg., for 60min on a discon- proteins were observed in addition to the neurophysins tinuous density gradient ranging from 1.3 to 2 0 M-SUCTOSe. (Fig. lb). The major band observed between neurophysins The material recovered at densities higher than 1.2 g/ml I1 and C was identified as serum albumin (addition to consisted of neurosecretory vesicles (band C ; VILHARDT the extract of purified bovine serum albumin increased & HOPE, 1974). The soluble content of the vesicles was the intensity of this band on electrophoresis). Portions extracted as described above. of the extract were subjected to affinity chromatography. Affinity chromatography was performed as described by The material binding to the column at pH 5.8 was eluted ROBINSON& WALKER (1974). (8-Lysine)-vasopressin (LVP) and subjected to polyacrylamide-gel electrophoresis was coupled to a bed of agarose (CNBr-Sepharose, Phar- (Figs. lc, 2). The migration pattern found was indismacia) in such a way that the ability of the hormone tinguishable from that observed when extracts of neurosecto bind neurophysin was retained. After application of retory granules were electrophoresed, showing that the the samples to the top of a column of agarose-LVP, elution four minor components were bound to agarose-vasopressin was started with 100mM-acetate buffer at pH 5.8. The at pH 5.8. It is unlikely that the presence of these minor effluent was monitored for proteins containing disulphide components reflects a contamination with other proteins et al. (1964). displaying electrophoretic mobilities similar to those of bonds using the method described by KARUSH The demonstration of disulphide bonds in the effluent was the neurophysins. The only major protein in the extract
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or precursors, aggregates or breakdown products of the major neurophysins. The results suggest that bovine neurosecretory granules do not contain any proteins soluble in acid, which cannot be classified a s neurophysins. Acknowledgements-Hans Vilhardt was supported by Nordisk Insulinfond and Kong Christian Den Tiendes Fond.
Soluble pmteins.Bovine NSG
L-
FIG. 2. Extract of neural lobe tissue bound by affinity chromatography. The bound material was electrophoresed on a 5 5 % polyacrylamide-gel followed by staining by Amido Black. The gel was scanned by densitometry (Vitatron Universal Photometer, Holland). The anode is to the left. The bands corresponding to neurophysins I, C and I1 are shown and arrows indicate the positions of the other proteins.
which migrates close to the neurophysins is serum albumin; this protein does not bind to agarose-LVP. According to the present results bovine neurosecretory vesicles contain at least seven neurophysins, four of which, however, constitute a small proportion of the soluble protein of the vesicles. The significance of these minor components is obscure. They could be individual neurophysins,
Department of Pharmacology, University of’Oxjord, O.xjord, OX1 3QT. England
H. VILHARDT I. C. A. F. ROBINSON
REFERENCES CLARKE J. T. (1964) Ann. N . Y. Acad. Sci. 121, 428-436. DEANC. R. & HOPE D. B. (1967) Biochem. J . 104, 10821088. GINSBURG M. & IRELAND M. (1963) J . Physiol., Lond. 169, 15P-16P. GINSBURG M. & IRELAND M. (1964) J . Endocr. 30, 131-145. HOLLENBERG M. D. & HOPE D. B. (1968) Biochem. J . 106, 557-564. KARUSHF., KLINMANN. R. & MARKSR. (1964) Analyt. Biochem. 9, 10&114. M. D. & HOPE D. B. (1969) BioRAUCHR., HOLLENBERG chem. J . 115, 473-479. I. C. A. F. & WALKERJ. M. (1974) J . Endocr.. ROBINSON 63. 29p. VILHARDT H. & HOPE D. B. (1974) Biochem. J . 143, 181190.
FIG.1. (a) Extract of purified neurosecretory vesicles. (b) Extract of homogenate of bovine neural lobes. (c) Extract of neural lobe tissue bound by affinity chromatography. The samples were electrophoresed on 5.5 % polyacrylamide-gels followed by staining with Amido Black. The anode is at the bottom of the picture. The bands corresponding to neurophysins I, C and I1 are labelled and arrows indicate the positions of the other proteins.
Nriil.ocliPriiiarl.~.1975.
Vol 24. pp. 1375 1276 Pergamon Prcss. Printed in Great Britain.
SHORT COMMUNICATION Polyacrylamide-gel electrophoresis of bovine neurophysins (Received 31 October 1974. Accepted 12 December 1974)
IN THE neural lobe of the mammalian pituitary gland the hormones vasopressin and oxytocin are stored in subcellular vesicles called neurosecretory granules. Besides hormones the vesicles also contain proteins of low molecular weight, the neurophysins (DEAN& HOPE,1967).The neurophysins have been shown to bind the hormones in vitro with binding occurring optimally at pH 5.8 (GINSBURG & IRELAND, 1963, 1964). These proteins have been isolated from extracts of whole bovine posterior putuitaries (HOLLENBERG & HOPE, 1968; RAUCHet al., 1969) and also from purified neurosecretory granules (DEAN& HOPE, 1967). Three major components, neurophysin I, I1 and C, all bind oxytocin and vasopressin. Gel electrophoresis of such extracts has, however, shown the presence in the granules of other proteins. These components, which constitute a small fraction of the soluble proteins in the neurosecrctory granules are relatively rich in cystine (RAUCHor al., 1969). In the present experiments we have used polyacrylamide-gel electrophoresis to demonstrate the presence of these proteins in extracts from freshly prepared neurosecretory granules. By means of affinity chromatography it could be shown that the proteins bind to agarose-vasopressin.
taken as an indication of the presence of neurophysins. When the material not binding to the column had been eluted the pH of the buffer was changed to 8.1 and elution was continued until a peak of disulphide-rich material had been recovered. The corresponding fractions were pooled, dialysed and evaporated down to a small volume, which was taken up in 5 mM-Tris-HCL, pH 7.5 and used for polyacrylamide-gel electrophoresis. Polyacrylamide-gel electrophoresis was performed in 5.5%gels (VILHARDT& HOPE,1974) using the buffer system described by CLARKE(1964). The gels were fixed and stained in 7.5%(v/v) acetic acid saturated with Amido Black.
RESULTS AND DISCUSSION The preparation of neurosecretory vesicles used in the present experiments has been shown to possess a high degree of purity (DEAN& HOPE,1967) with only minimal contamination by mitochondria and lysosomes (VILHARDT, BAKI.R& HOPI., to be published). When extracts of such preparations were subjected to polyacrylamide-gel electrophoresis (Fig. la) three major proteins were identified as neurophysins I, C and I1 by comparison of their mobilities with those of purified bovine neurophysin standards. MATERIAL AND METHODS Besides these well known proteins the presence of four Fresh bovine pituitary glands were obtained from the other components could be demonstrated on the polyacryllocal slaughter house. The isolated neural lobes were amide-gels. These bands are very weak and can only be homogenized in ice-cold 0.3 M-sucrose and the homogenate visualized when abundant amounts of protein are electrowas centrifuged at XOOg,, for 15min. The supernatant phoresed. Three of the proteins have mobilities intermewas then centrifuged at lOO,OOOg,, for 60min and the diate to those of neurophysins I1 and C, whereas the fourth pellet was extracted with 0.1 M-HCL for 6 h. After centrifu- band migrates slightly ahead of neurophysin I, but not gation at lOO,OOOg., for 60min the supernatant was a t the electrophoretic front (as determined by Bromoadjusted to pH 5.8 with NH,OH and used for affinity chro- phenol Blue). The electrophoretic pattern was similar to the one observed by RAUCHet al. (1969) on starch-gel ma tography. Neurosecretory vesicles were prepared by the method electrophoresis of extracts of purified neurosecretory & HOPE granules. of DEAN& HOPE(1967) as modified by VILHARDT When portions of extracts of neural lobe homogenates (1974). Fraction 111 obtained by differential centrifugation of a homogenate of bovine neural lobes was resuspended were electrophoresed on polyacrylamide-gels a number of and centrifuged at I IO,OOOg., for 60min on a discon- proteins were observed in addition to the neurophysins tinuous density gradient ranging from 1.3 to 2 0 M-SUCTOSe. (Fig. lb). The major band observed between neurophysins The material recovered at densities higher than 1.2 g/ml I1 and C was identified as serum albumin (addition to consisted of neurosecretory vesicles (band C ; VILHARDT the extract of purified bovine serum albumin increased & HOPE, 1974). The soluble content of the vesicles was the intensity of this band on electrophoresis). Portions extracted as described above. of the extract were subjected to affinity chromatography. Affinity chromatography was performed as described by The material binding to the column at pH 5.8 was eluted ROBINSON& WALKER (1974). (8-Lysine)-vasopressin (LVP) and subjected to polyacrylamide-gel electrophoresis was coupled to a bed of agarose (CNBr-Sepharose, Phar- (Figs. lc, 2). The migration pattern found was indismacia) in such a way that the ability of the hormone tinguishable from that observed when extracts of neurosecto bind neurophysin was retained. After application of retory granules were electrophoresed, showing that the the samples to the top of a column of agarose-LVP, elution four minor components were bound to agarose-vasopressin was started with 100mM-acetate buffer at pH 5.8. The at pH 5.8. It is unlikely that the presence of these minor effluent was monitored for proteins containing disulphide components reflects a contamination with other proteins et al. (1964). displaying electrophoretic mobilities similar to those of bonds using the method described by KARUSH The demonstration of disulphide bonds in the effluent was the neurophysins. The only major protein in the extract
1275
1216
Short communication I
or precursors, aggregates or breakdown products of the major neurophysins. The results suggest that bovine neurosecretory granules do not contain any proteins soluble in acid, which cannot be classified a s neurophysins. Acknowledgements-Hans Vilhardt was supported by Nordisk Insulinfond and Kong Christian Den Tiendes Fond.
Soluble pmteins.Bovine NSG
L-
FIG. 2. Extract of neural lobe tissue bound by affinity chromatography. The bound material was electrophoresed on a 5 5 % polyacrylamide-gel followed by staining by Amido Black. The gel was scanned by densitometry (Vitatron Universal Photometer, Holland). The anode is to the left. The bands corresponding to neurophysins I, C and I1 are shown and arrows indicate the positions of the other proteins.
which migrates close to the neurophysins is serum albumin; this protein does not bind to agarose-LVP. According to the present results bovine neurosecretory vesicles contain at least seven neurophysins, four of which, however, constitute a small proportion of the soluble protein of the vesicles. The significance of these minor components is obscure. They could be individual neurophysins,
Department of Pharmacology, University of’Oxjord, O.xjord, OX1 3QT. England
H. VILHARDT I. C. A. F. ROBINSON
REFERENCES CLARKE J. T. (1964) Ann. N . Y. Acad. Sci. 121, 428-436. DEANC. R. & HOPE D. B. (1967) Biochem. J . 104, 10821088. GINSBURG M. & IRELAND M. (1963) J . Physiol., Lond. 169, 15P-16P. GINSBURG M. & IRELAND M. (1964) J . Endocr. 30, 131-145. HOLLENBERG M. D. & HOPE D. B. (1968) Biochem. J . 106, 557-564. KARUSHF., KLINMANN. R. & MARKSR. (1964) Analyt. Biochem. 9, 10&114. M. D. & HOPE D. B. (1969) BioRAUCHR., HOLLENBERG chem. J . 115, 473-479. I. C. A. F. & WALKERJ. M. (1974) J . Endocr.. ROBINSON 63. 29p. VILHARDT H. & HOPE D. B. (1974) Biochem. J . 143, 181190.
FIG.1. (a) Extract of purified neurosecretory vesicles. (b) Extract of homogenate of bovine neural lobes. (c) Extract of neural lobe tissue bound by affinity chromatography. The samples were electrophoresed on 5.5 % polyacrylamide-gels followed by staining with Amido Black. The anode is at the bottom of the picture. The bands corresponding to neurophysins I, C and I1 are labelled and arrows indicate the positions of the other proteins.
