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Distribution of enkephalin-containing peptides within bovine chromaffin granules
Nemyqrides (1987)9,263-261 0 LongmanGroupUK Ltd 1987
DISTRIBUTION OF ENKEPHALIN-CONTAINING PEPTIDES WITHIN BOVINE CHROMAFFIN GRANULES Vivian Y.H. Hook and Dane Liston* Department of Biochemistry, Uniformed Services University of the Health Sciences, Bethesda, Maryland 20814-5799 (reprint requests to VH) and *Institute for Advanced Biomedical Research, The Oregon Health Sciences University, Portland, Oregon, 97201 ABSTRACT The distribution of large enkephalin-containing peptides (ECP's) between soluble and membrane components of bovine chromaffin granules was examined by immunoblotting with synenkephalin antiserum which recognizes the NH2-terminus of proenkephalin. Immunoblots showed that the 23.3 and 18.2 kilodalton ECP's were present in both soluble and membrane granule compartments but the 12.6 kilodalton ECP was present only in the soluble fraction, These results suggest that the larger ECP's may be preferentially associated with the granule membrane and may be redistributed to the soluble granule compartment upon proteolytic processing. INTRODUCTIUN The enkephalin peptides are initially synthesized as a large precursor (l-3) which must undergo proteolytic processing to form the small biologically active peptides. The bovine adrenal medulla contains the large 23.3, 18.2, 12.6 and 8.6 kilodalton enkephalin-containing peptides (ECP's) (4-6) which are thought to be intermediates in the sequence of proteolytic steps that convert proenkephalin to its smaller products. All of these ECP's contain the amino-terminus of proenkephalin and, thus, can be identified by the synenkephalin antiserum which recognizes the NH2-terminal synenkephalin fragment (4,7). In this report, immunoblots of bovine adrenal medulla chromaffin granule membrane and soluble fractions revealed that the ECP's are differentially distributed between these two granule compartments.
263
MATERIALS
AND METHODS
Chromaffin granules from one bovine Preparation of Chromaffin Granules. adrenal medulla were purified by a modification of the method described previously (8). The medulla was dissected from-a fresh adrenal gland (obtained fresh from Mount Airy, MD) and
) in 1.5 mm thick slab gels (15OV, 5-6 Proteins were then electrophoretically transferred to nitrocelluhours). lose paper in a transblot cell (Hoefer). Immunoblotting was performing as described previously (10). Membranes were blocked with 10% fetal calf serum in phosphate-buffered saline (PBS) pH 7.4 at room temperature and incubated for 2 hours (with rocking) with synenkephalin antiserum (final dilution 1:750 in IO% fetal calf serum/PBS pH 7.4). Antisynenkephalin complexes were visualized with peroxidase-conjugated goat anti-rabbit immunoglobulins (Boehringer Mannheim). RESULTS AND DISCUSSION Immunoblot analysis of chromaffin granule proteins with the synenkephalin antiserum that recognizes the amino-terminal region of proenkephalin (7) revealed that enkephalin-containing peptides (ECP's) are differentially distributed between membrane and soluble components of the chromaffin granule (Figure 1). The synenkephalin antiserum recognized four immunoreactive protein bands in chromaffin granules with apparent molecular weights of 25, 23, 19, and 14 kilodaltons.
264
Characterization of these proteins (4) have shown that they correspond to the 23.3, possible glycosylated 18.2, non-glycosylated 18.2, and 12.6 kilodalton proenkephalin-derived intermediates. The soluble granule component contained the 23.3, 18.2, and 12.6 kioldalton ECP's while only the 23.3 and 18.2 kilodalton species were present in the membrane component. A recent study has shown that an antiersum directed towards residues 95-117 of proenkephalin also detects a 23.3 kilodalton (27 kd, apparent Mr) in the membrane component of chromaffin granules (11). Our studies show that some 18.2 kilodalton species is also associated with the granule membranes. These findings suggest that the larger ECP's are preferentially associated with the granule membrane and that upon proteolytic cleavage they may be transferred to the soluble compartment.
-45K - 31K
- 21K -14K
Figure 1. Enkephalin-Containing Peptides in Membrane and Soluble Components of Chromaffin Granules Fraction 4 from the sucrose gradient was separated into soluble and membrane fractions. Immunoblots with synenkephalin antiserum of purified chromaffin granules (30 IJ~of fraction 4, lane l), soluble granule fraction (from 30 II~fraction 4, lane 2) and membrane granule fraction (from 200 l.11 of fraction 4, lane 3) are shown. Immunoblots of fractions 3 and 5 showed identical results. Immunoreactive bands A, R, C, and D show apparent molecular weights of 26, 23, 19, and 14 kilodaltons.
265
The 8.6 kilodalton proenkephalin derived product (4,11), was detected by the synenkephalin antiersum only when intact granules (in 0.32 M sucrose) were allowed to remain at 4o C for 12 hours before lysis and denaturation by sodium dodecyl sulfate (unpublished observations). Processing of proenkephalin in isolated chromaffin granules has been observed (12). Thus, the presence of the 8.6 kilodalton proenkephalin derived product found in other experiments (4,ll) may depend on the period of time for granule preparation and isolation. Secretory granule membrane localization of prohormones, and not their smaller peptide products has also been found for proopiomelanocortin (POMC) in pituitary pars intermedia (13) and for proglucagon, prosomatostatin, and proinsulin in anglerfish islet cells (14). This membrane association has been suggested to represent a general mechanism for proper intracellular sorting and targeting of prohormones to their secretory granule destination (13,14). Within the granule, the membrane association may also be important for correct orientation between prohormone substrate and processing enzyme to enhance and promote proteolytic activity. This would be important if the processing enzyme also had a particular distribution within the granule. Indeed, several processing enzyme activities (15, 16) have been found to reside with membrane as well as soluble components of secretory granules. Association of large peptide precursors with the granule membrane implies that they are not released to the extracellular environment upon exocytosis. Presumably, only the soluble components are released and membrane-bound proteins become assimilated with the plasma membrane; This may be a mechanism to insure that only the biologically active peptide products and not their inactive precursors are released. Localization of precursors to the membrane may be important for proper processing and release of neuroendocrine peptides. REFERENCES 1.
2.
3.
4.
5.
Comb. M., Seeburg, P.H., Adelman, J., Eiden, I_. and Herbert E. (1982) Primary structure of the human Met- and Leu-enkephalin precursor and its mRNA. Nature 295, 663-666. Gubler, U., Seeburg, P., Hoffman, R.J., Gage, L.P., and Udenfriend S. (1982) Molecular cloning establishes proenkephalin as a precursor of enkephalin-containing peptides. Nature 295, 206-208. Noda, M., Furutani, Y., Takahashi,Toyosato, M., Hirose, T., Inayama, S., Nakanishi, S., and Numa S. (1982) Cloning and sequence Nature 295, 202-206. analysis of cDNA for bovine preproenkephalin. Patey, G., Ciston, D., and Rossier, J. (1984) Characterization of new enkephalin-containing peptides in the adrenal medulla by immunoblotting. FERS Letters 172, 303-308. Kilpatrick, D.L., Jones, R.N., Lewis, R.V., Stern, A.S. Kojima, K. Shively, J.E., and Udenfriend, S.(1982) An 18,200 dalton adrenal protein that contains four (Met)enkephalin sequences. Proc. Natl. Acad. Sci. 79, 3057-3061.
266
6.
7. 8.
9. 10.
11.
12. 13. 14. 15. 16.
R.N., Shively, J.E., Kilpatrick, D.L., Stern, A.S., Lewis, R.V., Kojima, K., and Udenfriend, S. (1982) Adrenal opioid proteins of 8,600 and 12,600 daltons: intermediates in proenkephalin processing. Proc. Natl. Acad. Sci. 79, 2096-2100. and Rossier, J. (1983) Presence in Liston, D.R., Vanderhaegen, J.J. brain of synenkephalin, a proenkephalin-immunoreactive protein which does not contain enkephalin. Nature 302, 62-65. Hook, V.Y.H., Eiden, L.E., and Pruss, R.M. (1985). Selective regulation of carboxypeptidase peptide-hormone-processing enzyme during enkephalin biosynthesis in cultured bovine adrenomedullay chromaffin cells. Biol. Chem. 26D, 5991-5997. J. Laemmli, U.K. (1970) Cleavage of structural proteins during the assemhly of the head of bacteriophage T4. Nature 227, 680-695. Hook, V.Y.H., Mezey, E. Fricker, L.D. Pruss, R.M., Siegel, R., and Brownstein, M.(1985) Immunochemical characterization of carhoxypeptidase B-like peptide hormone processing enzyme. Proc. Natl. Acad. Sci. 82, 4745-49. Birch, N.P., Davies, A.D., and Christie, 0. L. (1986) Identification of a 27-kDa enkephalin-containing protein associated with bovine adrenal FEBS medullary chromaffin granule membranes by immunoblotting. Letters 197, 173-178. Fleminger, G., Ezra, E., Kilpatrick, D.L., and Udenfriend, S. (1983) Processing of enkephalin-containing peptides in isolated bovine adrenal chromaffin granules. Proc. Natl. Acad. Sci. USA 80, 6418-6421. Loh, P.Y., and Tam, W. (1985) Association of newly synthesized proopiomelanocortin with secretory granule membranes in pituitary pars FEBS Letters 184, 40-43. intermedia cells. Noe, B.D. and Moran, M.N. (1984) Association of newly synthesized islet prohormones with intracellular membranes. J.Cell.Biol. 99, 418-424. Chang, T.L. and Loh, Y.P. In vitro processing of proopiocortin by membrane-associated and soluble converting enzyme activities from rat intermediate lobe secretory granules. (1984) Endocrin. 114, 2092-2099. Fletcher, D.J., Duigley, J.P., Bauer, G.E. and Noe, B.D. (1981) Characterization of proinsulin and proglucagon-converting activities in isolated secretory granules. J. Cell. Riol. 9n, 312-322.
Jones,
Received Accepted
23 February 1987 6 March 1987
267
DISTRIBUTION OF ENKEPHALIN-CONTAINING PEPTIDES WITHIN BOVINE CHROMAFFIN GRANULES Vivian Y.H. Hook and Dane Liston* Department of Biochemistry, Uniformed Services University of the Health Sciences, Bethesda, Maryland 20814-5799 (reprint requests to VH) and *Institute for Advanced Biomedical Research, The Oregon Health Sciences University, Portland, Oregon, 97201 ABSTRACT The distribution of large enkephalin-containing peptides (ECP's) between soluble and membrane components of bovine chromaffin granules was examined by immunoblotting with synenkephalin antiserum which recognizes the NH2-terminus of proenkephalin. Immunoblots showed that the 23.3 and 18.2 kilodalton ECP's were present in both soluble and membrane granule compartments but the 12.6 kilodalton ECP was present only in the soluble fraction, These results suggest that the larger ECP's may be preferentially associated with the granule membrane and may be redistributed to the soluble granule compartment upon proteolytic processing. INTRODUCTIUN The enkephalin peptides are initially synthesized as a large precursor (l-3) which must undergo proteolytic processing to form the small biologically active peptides. The bovine adrenal medulla contains the large 23.3, 18.2, 12.6 and 8.6 kilodalton enkephalin-containing peptides (ECP's) (4-6) which are thought to be intermediates in the sequence of proteolytic steps that convert proenkephalin to its smaller products. All of these ECP's contain the amino-terminus of proenkephalin and, thus, can be identified by the synenkephalin antiserum which recognizes the NH2-terminal synenkephalin fragment (4,7). In this report, immunoblots of bovine adrenal medulla chromaffin granule membrane and soluble fractions revealed that the ECP's are differentially distributed between these two granule compartments.
263
MATERIALS
AND METHODS
Chromaffin granules from one bovine Preparation of Chromaffin Granules. adrenal medulla were purified by a modification of the method described previously (8). The medulla was dissected from-a fresh adrenal gland (obtained fresh from Mount Airy, MD) and
) in 1.5 mm thick slab gels (15OV, 5-6 Proteins were then electrophoretically transferred to nitrocelluhours). lose paper in a transblot cell (Hoefer). Immunoblotting was performing as described previously (10). Membranes were blocked with 10% fetal calf serum in phosphate-buffered saline (PBS) pH 7.4 at room temperature and incubated for 2 hours (with rocking) with synenkephalin antiserum (final dilution 1:750 in IO% fetal calf serum/PBS pH 7.4). Antisynenkephalin complexes were visualized with peroxidase-conjugated goat anti-rabbit immunoglobulins (Boehringer Mannheim). RESULTS AND DISCUSSION Immunoblot analysis of chromaffin granule proteins with the synenkephalin antiserum that recognizes the amino-terminal region of proenkephalin (7) revealed that enkephalin-containing peptides (ECP's) are differentially distributed between membrane and soluble components of the chromaffin granule (Figure 1). The synenkephalin antiserum recognized four immunoreactive protein bands in chromaffin granules with apparent molecular weights of 25, 23, 19, and 14 kilodaltons.
264
Characterization of these proteins (4) have shown that they correspond to the 23.3, possible glycosylated 18.2, non-glycosylated 18.2, and 12.6 kilodalton proenkephalin-derived intermediates. The soluble granule component contained the 23.3, 18.2, and 12.6 kioldalton ECP's while only the 23.3 and 18.2 kilodalton species were present in the membrane component. A recent study has shown that an antiersum directed towards residues 95-117 of proenkephalin also detects a 23.3 kilodalton (27 kd, apparent Mr) in the membrane component of chromaffin granules (11). Our studies show that some 18.2 kilodalton species is also associated with the granule membranes. These findings suggest that the larger ECP's are preferentially associated with the granule membrane and that upon proteolytic cleavage they may be transferred to the soluble compartment.
-45K - 31K
- 21K -14K
Figure 1. Enkephalin-Containing Peptides in Membrane and Soluble Components of Chromaffin Granules Fraction 4 from the sucrose gradient was separated into soluble and membrane fractions. Immunoblots with synenkephalin antiserum of purified chromaffin granules (30 IJ~of fraction 4, lane l), soluble granule fraction (from 30 II~fraction 4, lane 2) and membrane granule fraction (from 200 l.11 of fraction 4, lane 3) are shown. Immunoblots of fractions 3 and 5 showed identical results. Immunoreactive bands A, R, C, and D show apparent molecular weights of 26, 23, 19, and 14 kilodaltons.
265
The 8.6 kilodalton proenkephalin derived product (4,11), was detected by the synenkephalin antiersum only when intact granules (in 0.32 M sucrose) were allowed to remain at 4o C for 12 hours before lysis and denaturation by sodium dodecyl sulfate (unpublished observations). Processing of proenkephalin in isolated chromaffin granules has been observed (12). Thus, the presence of the 8.6 kilodalton proenkephalin derived product found in other experiments (4,ll) may depend on the period of time for granule preparation and isolation. Secretory granule membrane localization of prohormones, and not their smaller peptide products has also been found for proopiomelanocortin (POMC) in pituitary pars intermedia (13) and for proglucagon, prosomatostatin, and proinsulin in anglerfish islet cells (14). This membrane association has been suggested to represent a general mechanism for proper intracellular sorting and targeting of prohormones to their secretory granule destination (13,14). Within the granule, the membrane association may also be important for correct orientation between prohormone substrate and processing enzyme to enhance and promote proteolytic activity. This would be important if the processing enzyme also had a particular distribution within the granule. Indeed, several processing enzyme activities (15, 16) have been found to reside with membrane as well as soluble components of secretory granules. Association of large peptide precursors with the granule membrane implies that they are not released to the extracellular environment upon exocytosis. Presumably, only the soluble components are released and membrane-bound proteins become assimilated with the plasma membrane; This may be a mechanism to insure that only the biologically active peptide products and not their inactive precursors are released. Localization of precursors to the membrane may be important for proper processing and release of neuroendocrine peptides. REFERENCES 1.
2.
3.
4.
5.
Comb. M., Seeburg, P.H., Adelman, J., Eiden, I_. and Herbert E. (1982) Primary structure of the human Met- and Leu-enkephalin precursor and its mRNA. Nature 295, 663-666. Gubler, U., Seeburg, P., Hoffman, R.J., Gage, L.P., and Udenfriend S. (1982) Molecular cloning establishes proenkephalin as a precursor of enkephalin-containing peptides. Nature 295, 206-208. Noda, M., Furutani, Y., Takahashi,Toyosato, M., Hirose, T., Inayama, S., Nakanishi, S., and Numa S. (1982) Cloning and sequence Nature 295, 202-206. analysis of cDNA for bovine preproenkephalin. Patey, G., Ciston, D., and Rossier, J. (1984) Characterization of new enkephalin-containing peptides in the adrenal medulla by immunoblotting. FERS Letters 172, 303-308. Kilpatrick, D.L., Jones, R.N., Lewis, R.V., Stern, A.S. Kojima, K. Shively, J.E., and Udenfriend, S.(1982) An 18,200 dalton adrenal protein that contains four (Met)enkephalin sequences. Proc. Natl. Acad. Sci. 79, 3057-3061.
266
6.
7. 8.
9. 10.
11.
12. 13. 14. 15. 16.
R.N., Shively, J.E., Kilpatrick, D.L., Stern, A.S., Lewis, R.V., Kojima, K., and Udenfriend, S. (1982) Adrenal opioid proteins of 8,600 and 12,600 daltons: intermediates in proenkephalin processing. Proc. Natl. Acad. Sci. 79, 2096-2100. and Rossier, J. (1983) Presence in Liston, D.R., Vanderhaegen, J.J. brain of synenkephalin, a proenkephalin-immunoreactive protein which does not contain enkephalin. Nature 302, 62-65. Hook, V.Y.H., Eiden, L.E., and Pruss, R.M. (1985). Selective regulation of carboxypeptidase peptide-hormone-processing enzyme during enkephalin biosynthesis in cultured bovine adrenomedullay chromaffin cells. Biol. Chem. 26D, 5991-5997. J. Laemmli, U.K. (1970) Cleavage of structural proteins during the assemhly of the head of bacteriophage T4. Nature 227, 680-695. Hook, V.Y.H., Mezey, E. Fricker, L.D. Pruss, R.M., Siegel, R., and Brownstein, M.(1985) Immunochemical characterization of carhoxypeptidase B-like peptide hormone processing enzyme. Proc. Natl. Acad. Sci. 82, 4745-49. Birch, N.P., Davies, A.D., and Christie, 0. L. (1986) Identification of a 27-kDa enkephalin-containing protein associated with bovine adrenal FEBS medullary chromaffin granule membranes by immunoblotting. Letters 197, 173-178. Fleminger, G., Ezra, E., Kilpatrick, D.L., and Udenfriend, S. (1983) Processing of enkephalin-containing peptides in isolated bovine adrenal chromaffin granules. Proc. Natl. Acad. Sci. USA 80, 6418-6421. Loh, P.Y., and Tam, W. (1985) Association of newly synthesized proopiomelanocortin with secretory granule membranes in pituitary pars FEBS Letters 184, 40-43. intermedia cells. Noe, B.D. and Moran, M.N. (1984) Association of newly synthesized islet prohormones with intracellular membranes. J.Cell.Biol. 99, 418-424. Chang, T.L. and Loh, Y.P. In vitro processing of proopiocortin by membrane-associated and soluble converting enzyme activities from rat intermediate lobe secretory granules. (1984) Endocrin. 114, 2092-2099. Fletcher, D.J., Duigley, J.P., Bauer, G.E. and Noe, B.D. (1981) Characterization of proinsulin and proglucagon-converting activities in isolated secretory granules. J. Cell. Riol. 9n, 312-322.
Jones,
Received Accepted
23 February 1987 6 March 1987
267