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Glycoproteins of the chromaffin-granule matrix: Use of lectin blotting to distinguish several separate classes
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Neuroscience Vol. 16,No. 2, pp. 477487, 1985
Printed in Great
Pergamon Press Ltd 0 1985IBRO
Britain
GLYCOPROTEINS OF THE CHROMAFFIN-GRANULE MATRIX: USE OF LECTIN BLOTTING TO DISTINGUISH SEVERAL SEPARATE CLASSES D. K. APPS, J. H. PHILLIPS and F. C. PURVES* Department of Biochemistry, University of Edinburgh Medical School, George Square, Edinburgh EH8 9XD, Scotland
Abstract--The soluble proteins released by hypotonic lysis of highly purified bovine adrenal chromaffin granules were analysed by one- and two-dimensional electrophoresis, followed by transfer to nitrocellulose and decoration with lectins or specific antibodies. The effects of neuraminidase treatment, and of chemical deglycosylation by trifluoromethanesulphonic acid, were investigated. It was shown that lectins could be used to distinguish the two major series of chromogranins from each other, from dopamine /?-hydroxylase and from several minor, unidentified glycoprotein components of the lysate. Antibody decoration revealed a complex series of peptides containing enkephalin sequences, some of which changed their electrophoretic mobility on treatment with trifluoromethanesulphonic acid.
The catecholamine-storage vesicles of the adrenal medulla, known as chromaffin granules, contain, as well as catecholamines, nucleotides, ascorbate and divalent cations, several types of soluble protein, some in very high concentrations. The most abundant of these, known as chromogranins A, are a family of acidic glycoproteins derived proteolytically from a common precursor of approximate mol. wt 75,000.5*‘3,24A second series of chromogranins is derived from a different precursor, of approximate mol. wt 130,000; this series has been termed chromogranins B.5,24 Minor protein components of the chromaffin-granule matrix include a soluble form of dopamine /?-hydroxylase (EC 1.14.17. l), the structure of which and relationship to the membranebound form of the enzyme are still controversial,‘6l22 the opioid peptide precursors, prodynorphin’ and proenkephalin,*’ together with a series of enkephalincontaining peptides derived from them, glycoprotein III (called glycoprotein R in Ref. 8), a protein of unknown function which also occurs in a membranebound form’ and two proteases’ which appear to be involved in proenkephalin processing and may presumably also process the chromogranin precursors. The majority of these components have been separated by polyacrylamide gel electrophoresis and identified by Coomassie blue staining and several laboratories have prepared antisera to them. However, many glycoproteins stain poorly with
*Present address: Department of Biochemistry, University of Glasgow, Glasgow G12 SQQ, Scotland. A; HRP, horseradish peroxidase (EC. 1.11.1.7);PNA, peanut agglutinin; PSA, Pisum sativum agglutinin; WGA, wheat-germ agglutinin.
Abbreviations: Con A, concanavalin
Coomassie blue, and therefore may be missed if they are minor components. We now report the use of lectins to distinguish several types of chromaffingranule matrix protein, following their separation by two-dimensional electrophoresis and transfer to nitrocellulose sheets. This approach has already been used to identify proteins of the chromaffin-granule membrane* and offers great sensitivity and resolution in the detection of minor components, as well as providing information about the nature of the exposed carbohydrate residues of the glycoproteins. EXPERIMENTAL PROCEDURES Concanavalin A (Con A), horseradish peroxidase (HRP) (type VI), neuraminidase (type X), protein A and 4-chloro-I-naphthol were from Sigma Chemical Co., St. Louis, MO; the horseradish peroxidase conjugates of Pisum (HRP-PSA), peanut agglutinin sativum agglutinin (HRP-PNA) and wheat-germ agglutinin (HRP-WGA) were from Kern-En-Tee, Lemchesvej 11, DK-2900 Hellerup, Denmark. Soluble dopamine /?-hydroxylase was purified by ion-exchange and lectin chromatography,ls followed by preparative electrophoresis in the absence of disulphidereducing reagents. The protein was electroeluted from stained slab gels and used to immunize female New Zealand white rabbits. Rabbit antiserum to chromogranin A was prepared as previously described.” Rabbit antiserum to haptenized [Leulenkephalin, possessing approximately 10% cross-reactivity with [Metlenkephalin, was the generous gift of Dr. Kwen-Jen Chang.” Bovine chromaffin granules were isolated as described elsewhere’ and lysed by resuspension in 10 mM 4-(2hydroxyethyl)-1-piperazine ethane sulphonate (Hepe.s)NaOH, pH 7.0, containing 0.5 mM ethylenediaminetetraacetate and 0.1 mM phenylmethylsulphonyl fluoride (PMSF). After removal of membranes by centrifugation (60 min, gav= 1.6 x 10s) the lysate was dialysed against 5 mM Hepes, 0.2 mM ethylenediaminetetra-acetate, pH 7.0, then lyophilized and redissolved to give a final protein concentration of 8-12mg/ml; this solution was passed 477
D. K. APPS ef al
478
through a Millipore cellulose nitrate filter (pore size 0.45 pm). Treatment with neuraminidase was for 12 h at 25°C. in 0.05 M 2-(N-morpholino)ethane sulphonate-NaOH, pH containing 2.5 mM benzamidine, 0.1 mM 5.5, 0.1 mM ketone, chloromethyl mu-~-tosyl-L-lysine L-1-tosylamide 2-phenyiethyl chloromethyl ketone, 0.1 mM PMSF, I mM ethylenediaminetetra-acetate, 0.2 Units/ml neuraminidase, and 6-8 mg/ml chromaffin-granule matrix protein. Chemical deglycosylation was performed essentially as described by Stewart et ai.23Chromafin-granule lysate was desalted by passage through a column of Bio-Gel P6DG equilibrated with water and the proteins lyophi~iz~. To 8 mg of dry protein was added 1ml anisole and 2 ml trifluoromethanesulphonic acid; after incubation at 0°C for 2 h, the mixture was treated with 10 ml pyridine-water (4: I, v/v). then exhaustively dialysed against water. The protein was collected by lyophilization and was readily soluble in sodium dodecyl suiphate-containing buffers. Two-dimensional electrophoresis was performed essentially as described by O’Farrell.Zo In the first dimension, electrofocussing was in rod gels containing 8 M urea, 2% Nonidet P40 and I% LKB ampholine pH 4-6.5; in the second dimension, slab gels contained an exponentially increasing concentration of acrylamide (S-150/-,w/v). Transfer to cellulose nitrate sheets (Schleicher and Schull) was performed in an Electra-Blot tank. in 0.02 M Na,HPO,, 0.02:& sodium dodecyl sulphate, 207; (v/v) methanol; electrophoresis was for 90min, 0.8 amp. The transfers were washed for I h in 3”; w/v serum albumin in 0.15 M NaCi, 0.02 M Tris-NaCl, pH 7.4, then for 1 h with lectins or antisera. Antisera were diluted I : 100 in Tris-NaCl buffer containing 3% serum albumin and 5% v/v horse serum and binding was detected by the use of ‘251-labelledprotein A.‘) Horseradish peroxidase-conjugate lectins (3 mg/ml) were diluted I:200 (or 1: 100 for HRP-WGA) in Tris-NaC1 buffer; after washing with these, the transfers were washed in Tris-NaCl and developed in Cchloro-I-naphthol (0.5 mg/ml), H,Oz (0.01% w/v) in Tris-NaCl.’ Decoration with Con A and HRP was essentially as described by Clegg,’ although Triton X-100 was omitted from the buffers and 0.05% v/v Tween-20 included in one of the washing steps; ~chloro-I-naphthol was used as the substrate for colour development. RESULTS
Highly purified bovine chromaffin granules were lysed by osmotic shock and their soluble protein components resolved by two-dimensional electrophoresis (Fig. 1). The pattern is dominated by chromogranin A and its derivatives (for identification, see Refs 5, 13 and 24) but reveals a number of minor components. Nitrocellulose blots of similar electrophoretograms were decorated with lectins (Fig. 2) or antisera (Fig. 3) for the identification of major components. We discuss these and some minor components revealed by the lectin decoration, in the following sections. For electrofocussing, we chose ampholytes that would generate a narrow pH gradient (approx. 4.0-6.0); these are more suitable for study of chromaffin-granule matrix proteins than are wide-range ampholytes.7,24 In case lectin binding should be masked by terminal sialic acid residues, some samples were subjected to neuraminidase treatment before electrophoresis. Although not shown here, Coomassie blue staining of such a gel shows that many components, including
the chromogranin A derivatives (Fig. 1), are shifted to the left on the gels by this treatment (i.e. towards higher PI). Examples of lectin binding before and after such treatment are shown in Figs 2 and 4. Both concanavalin A and Pisum satitl~ agglutinin reveal a variety of components as horizontal rows of spots of similar molecular weight, presumably arising from heterogeneous glycosylation. Neuraminidase treatment generally converts these to components of higher pI and reduces the number of spots. In the case of peanut agglutinin (Figs 2 and 4b) there is virtually no binding before neuraminidase treatment. We used chemical deglycosylation to investigate the polypeptide chains of major components, using lectins (Fig. 4) and antisera (Fig. 5) to reveal the products. Treatment with trifluoromethanesulphonic acid breaks O-glycosyl bonds, including those to protein,J but fails to cleave N-glycosyl bonds, thus leaving asparagine-linked N-acetylglucosamine as a residue of N-linked chains. Following deglycosylation, the only lectin binding seen is thus a weak binding of WGA to the polypeptide arising from dopamine #-hydroxylase and to some other components (Fig. 4c); marked shifts in molecular weight of some components are revealed by antisera (Fig. 5). Chromogranin A series
It has been previously shown” that an antiserum raised against purified chromogranin A (mol. wt 75,000) cross-reacts with a series of proteins of similar isoelectric points (4.7-5.4) and mol. wts 70-40,OOO and two smaller polypeptides (mol. wts 29,000 and 20,000). These components are indicated in Fig. 1. Lectin blotting (Fig. 2) shows that chromogranins A are not decorated by Con A (Figs 2a, b), by the HRP conjugate of PSA, which has a similar sugar specificity to Con A (Figs 2c,d) or by the galactosespecific HRP-PNA (Fig. 2e); in these blots, the chromogranins A are just discernible as white, unstained spots against the pale violet background. Following neuraminidase treatment, however, these proteins do bind HRP-PNA (Fig. 20, but not Con A or PSA. These results are consistent with the reports U’ that the carbohydrate chains of chromogranin A are O-linked tri- or tetrasaccharides containing N-acetylgalactosamine, galactose and sialic acid; only after removal of terminal sialie acid by neuraminidase is the exposed galactose able to bind HRP-PNA. Chemical deglycosylation only produced slight changes in the apparent molecular weights (Fig. 5a), but very significant shifts in the isoelectric points of the chromogranins A (not shown), that of the largest component being raised from 4.7-5.0 to X1-5.4. This is presumably the result of removing all sialic acid residues; treatment with neuraminidase produced smaller shifts in the same direction. It was previously concluded8 that removal of a single sialic acid from glycoprotein R (glycoprotein III) increases the pI by
/lOOO
3
Fig. I. Two-dimensional electrophoretogram of chroma~n-grange matrix proteins (I80 pg), stained with Coomassie blue. (A, B) Components binding antisera to chromogranins A and B. respectively (Refs 5, 13 and 24). DBH, dopamine @-hydroxylase; M,, mol. wt.
479
Fig. 2. Lectin blots of chromaffin-granule matrix proteins, detected by horseradish peroxidase-activity staining. (a, b) Concunavalin A; (c, d) Pisum satiuum agglutinin; (e, f) peanut agglutinin. Proteins from untreated lysate (a, c, e) or neuraminidase-treated lysate (b, d, f) were separated in two dimensions as in Fig. I.
480
3
481
Fig. 4. Effects of chemical deglycosylation on lectin binding by chromaffin-granule proteins (one-dimensional electrophoretograms). (a) Pisum satimm agglutinin; (b) peanut agglutinin; (c) wheat-germ agglutinin. Track I, untreated lysate; track 2, neuraminidase-treated lysate; track 3, lysate deglycosylated with trifluoromethanesulphonic acid; track 4, lysate treated with neuraminidase and trifluoromethanesulphonic acid: track 5. untreated chro~~~n-granule membrane proteins; R, glycoprotein III (Ref. 7) or band R (Ref. 8). M,, mol. wt.
Fig. 5. Effects of chemical deglycosylation on antibody binding by chromaffin-granule matrix proteins. Immune blots of one-dimensional electrophoretograms detected by 1251-labelledprotein A and autoradiography. (a) Anti-chromogranin A; (b) anti-dopamine B-hydroxylase; (c) anti-[Leulenkephalin. Track 1, untreated iysate; track 2, deglycosylated with trifluoromethanesulphonic acid. M,, mol. wt.
483
Glycoproteins of the chromaffin-granule
0.08, so the observed shift in p1 of approx. 0.4 is consistent with the estimated sialic acid content of chromogranin A (mol. wt 75,000), which is 6 mole/mole (Refs 6 and 11). Chromogranin B series
The proteins derived from chromogranin B (mol. wt 130,000; p1 5.4-5.8) form a complex series, less acidic than chromogranin A and its derivatives. Unlike chromogranin A, they bind both Con A and HRP-PSA, both specific for mannose or glucose (Figs 2a,c). The higher molecular weight members of this series (mol. wts SO-130,000) also bind HRP-PNA after neuraminidase treatment (Fig. 2f), suggesting that they have both N-linked chains containing mannose and other chains containing galactose and terminal sialic acid. A series of proteins of similar p1 but lower mol. wt fail to bind HRP-PNA and so appear to lack the latter class of carbohydrate chains, which could be either N- or O-linked. It is not clear from our results whether these are members of the chromogranin B family, but they appear to be identical with the proteins found to react with an antiserum to chromogranin B (Fischer-Colbrie and Frischenschlager, 1985).5 They appear to contain sialic acid, as neuraminidase treatment increases their electrophoretic mobility (Fig. 4a). Dopamine /I-hydroxylase
matrix
485
After chemical deglycosylation of lysate proteins, HRP-WGA bound to a component of apparent mol. wt 72,000 (Fig. 4c) which is probably dopamine /I-hydroxylase-containing residual asparagine-linked N-acetylglucosamine but little or no other carbohydrate. Some new HRP-WGA-reactive bands also appeared on treatment with trifluoromethane sulphonate; these may be degradation products of other N-glycosylated proteins. Opioid peptides
The antiserum used detects both [Leuj- and [Metlenkephalin-containing sequences, and in blots of one-dimensional electrophoretograms (Fig. 5) recognized 6 major bands (apparent mol. wts 28,500, 21,500, 19,150, 17,600, 14,600 and 13,800) and also reacted with unresolved proteins at the dye-front of the gel. Some of the bands presumably correspond to those detected by Patey et al.*’ using an antiserum to synenkephalin (the N-terminal seventy amino acids of proenkephalin). In two dimensions, however, the pattern is far more complex (Fig. 3b). The polypeptides are not sufficiently abundant to be detected by Coomassie blue staining (Fig. 1); the cleavage products appear to fall into two groups, with pI about 5.5 and 6.0, although some heterogeneity is seen. The adrenal medulla contains both proenkephalin and dynorphin, derivates of these polypeptides being released from adrenalineand noradrenalinecontaining vesicles, respective1y.3 Although immunohistochemical studies suggested that [Leulenkephalins were only present in adrenaline-containing cellsI it seems likely that in immune blots the antiserum used would recognize the [Leulenkephalin sequence in dynorphin and its derivatives, as well as enkephalin sequences in proenkephalin-derived proteins. The largest polypeptides detected have apparent mol. wts close to that of uncleaved proenkephalin, which has been deduced to be 27,300 from the sequence of the cDNA.‘* Patey et al.,” however, were unable to detect this species in bovine adrenal medulla. Kilpatrick et al.‘* found no neutral or amino sugars in enkephalin-containing peptides from the adrenal medulla. While we were unable to assign binding of any lectin to opioid peptides, treatment with trifluoromethanesulphonic acid reduced the mobility of some bands (Fig. 5). The electrophoretic mobility of small glycopeptides may not correlate with their molecular weight and it is possible that the observed changes reflect some type of chemical modification other than deglycosylation.
This enzyme is the major Con A- and the glycoprotein of HRP-PSA-binding chromaffin-granule matrix, as would be expected from its high mannose content.6.‘6 In immune blots (Fig. 3a) and lectin blots (Figs 2ad) it appears as a series of three bands of apparent mol. wt 72-78,000 and pI 5.8-6.0; this pattern is indistinguishable from that of the membrane-bound form of the enzyme.’ It fails to bind HRP-PNA, even after neuraminidase treatment, despite the terminal galactose-linked sialic acid found in N-linked biantennary complexes of dopamine /?-hydroxy1ase;‘6 furthermore, neuraminidase treatment produces no detectable shift in its isoelectric point (see Fig. 2). Possibly the carbohydrate chains of dopamine /I-hydroxylase are rather resistant to neuraminidase. Chemical deglycosylation abolished binding of con A and HRP-PSA to dopamine fi-hydroxylase, but the deglycosylated enzyme still bound antiserum (Fig. 5) and appeared as a sharp doublet of apparent mol. wt 72,000. These bands of approximately equal intensity are extremely close together and are hard to resolve in the photograph. They may correspond to two different polypeptide subunits. The identity of the minor bands of slightly lower molecular weight in Other glycoproteins Fig. 5(b) is unknown. The amount of ‘251-labe11ed Lectin blotting reveals some minor components of protein A bound by the immune complex was much unknown identity: reduced by deglycosylation, suggesting that a sub(a) A poorly focused, acidic component of high stantial fraction of the polyclonal antibodies was molecular weight (approx. 100,000; pI 4.4-4.7) binds directed against glycosyl chains.
D. K.
486
PNA-HRP after neuraminidase treatment (Fig. 2f) and is just visible in Fig. 1. This could be a proteoglycan.” (b) Con A binds to about a dozen unidentified proteins, each of which focusses as a series of discrete spots of the same molecular weight (Fig. 2a). Some cover pI ranges of about 0.3 pH units, but two, of mol. wts 40,000 and 37,000. cover at least 1.2 pH units. Four of these minor components are also decorated by PSA. Neuraminidase treatment of the granule lysate produces marked shifts in the patterns; in particular four or five Con A-binding components are shifted from pI of about 5.4 to as high as p1 6.0 (Fig. Zb). However, none of the glycoproteins run as a single spot after this treatment, The identity of these proteins is unknown; their lectin-binding properties distinguish them from the chromogranins A and their isoelectric points are higher than those of chromogranins B or glycoprotein III.’ These minor glycoproteins may of course be contaminants, but this seems unlikely as chromaffin granules were highly purified before lysis and soluble glycoproteins account for at least 70:/, of the total protein.” None of these components is detectable in chromaffin-granule membranes.’ (c) HRP-WGA binds to a heterogeneous glycoprotein of apparent mol. wt 3740,000, variously termed glycoprotein III (Ref. 7) or band R (Ref. 8). This protein is present in both granule membranes and lysate (Fig. 4c) although it is a minor component of the latter and undetectable by Coomassie blue staining. Treatment with trifluoromethanesulphonic acid abolishes its ability to bind WGA-HRP and neuraminidase digestion converts it to a sharper band of increased mobility (Fig. 4~). The lectin-binding properties of the major glycoproteins of the chromaffin-granule matrix are summarized in Table 1. All of the chromogranins A exhibit the same properties but, as noted above, there may be some differences between the high- and low-molecular weight members of the chromogranin B series.
It is shown in Fig. 1 that granules contain several proteins of mol. wt less than 20,000 which do not bind lectins and do not cross-react with the antisera that we have used. These may be non-antigenic degradation products of chromogranins A or B, but
APPS et al.
the shape and positions of these components suggest that they are still to be identified. Although they may yet be found to be glycoproteins, it is notable that all of the lysate proteins of higher molecular weight that are visible in Fig. 1 appear to be glycoproteins, using the few lectins employed in this study.
DISCUSSION
The complexity of two-dimensional gels makes protein and glycoprotein nomenclature difficult; this is particularly a problem with the chromogranins, the major secreted proteins of chromaffin granules. O’Connor and Frigon19 used one-dimensional electrophoresis to separate the proteolytic derivatives of chromogranin A and designated them chromogranins B, C and D; however, it seems likely that these components are themselves heterogeneous and it is therefore safer at present to designate as chromogranins A those proteins derived from the precursor of mol. wt 75,000 and as chromogranins B those derived from the less acidic precursor of mol. wt 130,000.’ Possibly the biogenically related components should be designated A,, A2 etc. (see Ref. 13) but it seems quite likely that the spots in twodimensiona electrophoretograms are themselves heterogeneous; the shape of the major chromogranin A spot, for example, clearly suggests the superimposition of at least two components. Further refinement of electrophoretic procedures is therefore soon likely to render any more detailed classification obsolete. Lectin binding has revealed several soluble glycoproteins of chromafhn granules that are not seen by Coomassie blue staining, and this emphasises the difficulty of making quantitative estimates of the amounts of different proteins present in the granule matrix. It is clear, however, that the chromogranin A family are by far the major constituents: their biological role is quite unknown. The enkephalincontaining polypeptides are very minor components and granules clearly contain at least a dozen other heterogeneous glycoproteins that may be present in equivalent amounts to them. Because of the method of preparation, there seems to be little possibility that these are contaminants, and so it becomes an urgent necessity to define the biological roles of these proteins which are stored within the matrix of chromaffin
Table 1. Binding of lectins by the major matrix proteins of chromaffin granules, in untreated and neuraminidase-treated lysate Con A Chromogranins A Chromogranins B Dopamine B-hydroxylase Glycoprotein III(R)
+ + +
Con AiNEUR + + +
PNA -
PNAiNEUR + +
PSA + +
PSA/NEUR _ + +
WGA WGAJNEUR + + + + + -+
Molecular weights of the chromogranins A (from Fig. 1) were 75, 72, 68, 57, 55, 49, 29 and 20 x 103;chromogranins B, 130, 115, 106, 99, 89 x 10’. NEUR. neuraminidase-treated.
Glycoproteins of the chromaffin-granule
granules and are presumably
matrix
487
co-secreted with cate-
edge the gift of anti-enkephalin antiserum from Dr. Kwen-
Acknowledgements-This work was supported by a grant from the Medical Research Council, We gratefully acknowl-
Jen Chang, Wellcome Research Laboratories and communication of results prior to publication by Professor H. Winkler. We are indebted to Kern-En-Tee for supplying samples of HRP-conjugated lectins.
cholamines.
REFERENCES
1. Apps D. K. and Schatz G. (1979) An adenosine triphosphatase
2. 3. 4. 5. 6. 7. 8. 9. 10. II. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24.
isolated from chromaffin-granule membranes is closely similar to F,-adenosine triphosphatase of mitochondria. Eur. J. Biochem. 100, 411-419. Clegg J. C. S. (1982) Glycoprotein detection in nitrocellulose transfers of electrophoretically separated protein mixtures using concanavalin A and peroxidase: application to arenavirus and flavivirus proteins. Anaiyl. Biochem. 127,389-394. Dumont M., Day R. and Lemaire S. (1983) Distinct dist~bution of immunoreactive dynorphin and leucine enkephalin in various populations of isolated adrenal chromaffin cells. Life Sci. 32, 287-294. Edge A. S. B., Faltynek C. R., Hof L., Reichert L. E. and Weber P. (1981) Deglycosylation of glycoproteins by trifluoromethanesulphonic acid. Analyr. Biochem. 118, 131-137. Fischer-Colbrie R. and Frischenschlager I. (1985) Immunological characterization of secretory proteins of chromaffin granules: chromogranins A, chromogranins B and enkephahn-containing peptides. J. Neuroihem. In press. Fischer-Colbrie R.. Schachinger M.. Zannerle R. and Winkler H. (1982) Dooamine B-hvdroxvlase and other glycoproteins from the SolubIe~onte~~ and the membranes of adrenal chromakin granules: isolation and carbohydrate analysis. f. Neurochem. 38, 725-732. Fischer-Colbrie R., Zangerle R., Frischenschlager I., Weber A. and Winkler H. (1984) Isotation and immunological characterization of a glycoprotein from adrenal chromaffin granules. J. Neurochem. 42, 1008-1016. Gavine F. S., Pryde J. G., Deane D. L. and Apps D. K. (1984) Glycoproteins of the chromaffin-granule membrane: separation by two-dimensional electrophoresis and identification by lectin binding. J. Neurochem. 43, 1243-1252. Hawkes R. (1982) Identification of concanavalin-A binding proteins after SDS-gel electrophoresis and protein blotting. Art@. Biochem. 123, 143-146. Hook V. Y. M. and Eiden L. E. (1984) Two peptidases that convert ‘*~I-Lys-Arg-[Metlenkephalin and ‘zsI-[Met]enkephalin-Arg6, respectively, to I”~-[Met]enkephalin in bovine adrenal medullary chromaffin granules. FEBS Lett. 172, 212-218. Kiang W-L., Krusius T., Finne J., Margolis R. U. and Margolis R. K. (1982) Glycoproteins and proteoglycans of the chromaffin granule matrix. J. biol. Chem. 257, 16.51-1659. Kilpatrick D. L., Gibson K. D. and Jones B. N. (1983) Is adrenal proenkephalin glycosylated? Archs Biochem. Biophys. 224, 402404. Kilpatrick L., Gavine F., Apps D. and Phillips J. (1983) Biosynthetic relationship between the major matrix proteins of adrenal chromafin granules. FEBS Left. 164, 383-388. Livett B. G., Day R., Elde, R. P. and Howe, P. R. C. (1982) Co-storage of enkephalins and adrenaline in the bovine adrenal medulla. Neuroscience 7, 1323-1332. Ljones T., Skotland T. and Flatmark T. (1976) Purification and characterization of dopamine p-hydroxylase from bovine adrenal medulla. Eur. J. Biochem. 61, 525-533. Margolis R. K., Finne J., Krusius T. and Margolis R. U. (1984) Structural studies on glycoprotein oligosaccharides of chromaffin granule membranes and dopamine B-hydroxylase. Archs Biochem. Biophys. 228, 443-449. Miller R. J., Chang K-J., Cooper B. and Quatrecasas P. (1978) Radioimmuno~ay and characterization of enkephalins in rat tissues. J. biof. Chem. 253, 531-538. Noda M., Furutani Y., Takahashi H., Toyosato M., Hirose T., Inayama S., Nakanishi S. and Numa S. (1982) Cloning and sequence analysis of cDNA for bovine adrenal proenkephalin. Narure 295, 202-206. O’Connor D. T. and Frigon R. P. (1984) Chromogranin A, the major catecholamine storage vesicle soluble protein. J. biol. Chem. 259, 3237-3247. O’Farrell D. M. (1975) High resolution two-dimensional electrophoresis of proteins. J. biol. Chem. 250, 4007-4021. Patey G., Liston D. and Rossier J. (1984) Characterization of new enkephalin-containing peptides in the adrenal medulla by immunoblotting. FEBS Left. 172, 303-308. Saxena A. and Fleming P. J. (1983) Isolation and reconstitution of the membrane-bound form of dopamine /?-hydroxylase. J. biol. Chem. 258, 4147-4152. Stewart J. R., Chaplin M. F. and Kenny A. J. (1984) Deglycosylation by trifluoromethane sulphonic acid of endopeptidase-24.11 purified from pig kidney and intestine. Biochem. J. 221, 919-922. Winkler H., Falkensammer G., Patzak A., Fischer-Colbrie R., Schober M. and Weber A. (1984) Life cycle of the catecholaminergic vesicle: from biogenesis to secretion. In Regulation of Transmitter Function. Proceedings of 5th Meeting European Society of Neuroehem~s~ry, Budapest (eds Vizi E. S. and Magyar K.), pp. 65-73. (Accepted 24 March 1985)
Neuroscience Vol. 16,No. 2, pp. 477487, 1985
Printed in Great
Pergamon Press Ltd 0 1985IBRO
Britain
GLYCOPROTEINS OF THE CHROMAFFIN-GRANULE MATRIX: USE OF LECTIN BLOTTING TO DISTINGUISH SEVERAL SEPARATE CLASSES D. K. APPS, J. H. PHILLIPS and F. C. PURVES* Department of Biochemistry, University of Edinburgh Medical School, George Square, Edinburgh EH8 9XD, Scotland
Abstract--The soluble proteins released by hypotonic lysis of highly purified bovine adrenal chromaffin granules were analysed by one- and two-dimensional electrophoresis, followed by transfer to nitrocellulose and decoration with lectins or specific antibodies. The effects of neuraminidase treatment, and of chemical deglycosylation by trifluoromethanesulphonic acid, were investigated. It was shown that lectins could be used to distinguish the two major series of chromogranins from each other, from dopamine /?-hydroxylase and from several minor, unidentified glycoprotein components of the lysate. Antibody decoration revealed a complex series of peptides containing enkephalin sequences, some of which changed their electrophoretic mobility on treatment with trifluoromethanesulphonic acid.
The catecholamine-storage vesicles of the adrenal medulla, known as chromaffin granules, contain, as well as catecholamines, nucleotides, ascorbate and divalent cations, several types of soluble protein, some in very high concentrations. The most abundant of these, known as chromogranins A, are a family of acidic glycoproteins derived proteolytically from a common precursor of approximate mol. wt 75,000.5*‘3,24A second series of chromogranins is derived from a different precursor, of approximate mol. wt 130,000; this series has been termed chromogranins B.5,24 Minor protein components of the chromaffin-granule matrix include a soluble form of dopamine /?-hydroxylase (EC 1.14.17. l), the structure of which and relationship to the membranebound form of the enzyme are still controversial,‘6l22 the opioid peptide precursors, prodynorphin’ and proenkephalin,*’ together with a series of enkephalincontaining peptides derived from them, glycoprotein III (called glycoprotein R in Ref. 8), a protein of unknown function which also occurs in a membranebound form’ and two proteases’ which appear to be involved in proenkephalin processing and may presumably also process the chromogranin precursors. The majority of these components have been separated by polyacrylamide gel electrophoresis and identified by Coomassie blue staining and several laboratories have prepared antisera to them. However, many glycoproteins stain poorly with
*Present address: Department of Biochemistry, University of Glasgow, Glasgow G12 SQQ, Scotland. A; HRP, horseradish peroxidase (EC. 1.11.1.7);PNA, peanut agglutinin; PSA, Pisum sativum agglutinin; WGA, wheat-germ agglutinin.
Abbreviations: Con A, concanavalin
Coomassie blue, and therefore may be missed if they are minor components. We now report the use of lectins to distinguish several types of chromaffingranule matrix protein, following their separation by two-dimensional electrophoresis and transfer to nitrocellulose sheets. This approach has already been used to identify proteins of the chromaffin-granule membrane* and offers great sensitivity and resolution in the detection of minor components, as well as providing information about the nature of the exposed carbohydrate residues of the glycoproteins. EXPERIMENTAL PROCEDURES Concanavalin A (Con A), horseradish peroxidase (HRP) (type VI), neuraminidase (type X), protein A and 4-chloro-I-naphthol were from Sigma Chemical Co., St. Louis, MO; the horseradish peroxidase conjugates of Pisum (HRP-PSA), peanut agglutinin sativum agglutinin (HRP-PNA) and wheat-germ agglutinin (HRP-WGA) were from Kern-En-Tee, Lemchesvej 11, DK-2900 Hellerup, Denmark. Soluble dopamine /?-hydroxylase was purified by ion-exchange and lectin chromatography,ls followed by preparative electrophoresis in the absence of disulphidereducing reagents. The protein was electroeluted from stained slab gels and used to immunize female New Zealand white rabbits. Rabbit antiserum to chromogranin A was prepared as previously described.” Rabbit antiserum to haptenized [Leulenkephalin, possessing approximately 10% cross-reactivity with [Metlenkephalin, was the generous gift of Dr. Kwen-Jen Chang.” Bovine chromaffin granules were isolated as described elsewhere’ and lysed by resuspension in 10 mM 4-(2hydroxyethyl)-1-piperazine ethane sulphonate (Hepe.s)NaOH, pH 7.0, containing 0.5 mM ethylenediaminetetraacetate and 0.1 mM phenylmethylsulphonyl fluoride (PMSF). After removal of membranes by centrifugation (60 min, gav= 1.6 x 10s) the lysate was dialysed against 5 mM Hepes, 0.2 mM ethylenediaminetetra-acetate, pH 7.0, then lyophilized and redissolved to give a final protein concentration of 8-12mg/ml; this solution was passed 477
D. K. APPS ef al
478
through a Millipore cellulose nitrate filter (pore size 0.45 pm). Treatment with neuraminidase was for 12 h at 25°C. in 0.05 M 2-(N-morpholino)ethane sulphonate-NaOH, pH containing 2.5 mM benzamidine, 0.1 mM 5.5, 0.1 mM ketone, chloromethyl mu-~-tosyl-L-lysine L-1-tosylamide 2-phenyiethyl chloromethyl ketone, 0.1 mM PMSF, I mM ethylenediaminetetra-acetate, 0.2 Units/ml neuraminidase, and 6-8 mg/ml chromaffin-granule matrix protein. Chemical deglycosylation was performed essentially as described by Stewart et ai.23Chromafin-granule lysate was desalted by passage through a column of Bio-Gel P6DG equilibrated with water and the proteins lyophi~iz~. To 8 mg of dry protein was added 1ml anisole and 2 ml trifluoromethanesulphonic acid; after incubation at 0°C for 2 h, the mixture was treated with 10 ml pyridine-water (4: I, v/v). then exhaustively dialysed against water. The protein was collected by lyophilization and was readily soluble in sodium dodecyl suiphate-containing buffers. Two-dimensional electrophoresis was performed essentially as described by O’Farrell.Zo In the first dimension, electrofocussing was in rod gels containing 8 M urea, 2% Nonidet P40 and I% LKB ampholine pH 4-6.5; in the second dimension, slab gels contained an exponentially increasing concentration of acrylamide (S-150/-,w/v). Transfer to cellulose nitrate sheets (Schleicher and Schull) was performed in an Electra-Blot tank. in 0.02 M Na,HPO,, 0.02:& sodium dodecyl sulphate, 207; (v/v) methanol; electrophoresis was for 90min, 0.8 amp. The transfers were washed for I h in 3”; w/v serum albumin in 0.15 M NaCi, 0.02 M Tris-NaCl, pH 7.4, then for 1 h with lectins or antisera. Antisera were diluted I : 100 in Tris-NaCl buffer containing 3% serum albumin and 5% v/v horse serum and binding was detected by the use of ‘251-labelledprotein A.‘) Horseradish peroxidase-conjugate lectins (3 mg/ml) were diluted I:200 (or 1: 100 for HRP-WGA) in Tris-NaC1 buffer; after washing with these, the transfers were washed in Tris-NaCl and developed in Cchloro-I-naphthol (0.5 mg/ml), H,Oz (0.01% w/v) in Tris-NaCl.’ Decoration with Con A and HRP was essentially as described by Clegg,’ although Triton X-100 was omitted from the buffers and 0.05% v/v Tween-20 included in one of the washing steps; ~chloro-I-naphthol was used as the substrate for colour development. RESULTS
Highly purified bovine chromaffin granules were lysed by osmotic shock and their soluble protein components resolved by two-dimensional electrophoresis (Fig. 1). The pattern is dominated by chromogranin A and its derivatives (for identification, see Refs 5, 13 and 24) but reveals a number of minor components. Nitrocellulose blots of similar electrophoretograms were decorated with lectins (Fig. 2) or antisera (Fig. 3) for the identification of major components. We discuss these and some minor components revealed by the lectin decoration, in the following sections. For electrofocussing, we chose ampholytes that would generate a narrow pH gradient (approx. 4.0-6.0); these are more suitable for study of chromaffin-granule matrix proteins than are wide-range ampholytes.7,24 In case lectin binding should be masked by terminal sialic acid residues, some samples were subjected to neuraminidase treatment before electrophoresis. Although not shown here, Coomassie blue staining of such a gel shows that many components, including
the chromogranin A derivatives (Fig. 1), are shifted to the left on the gels by this treatment (i.e. towards higher PI). Examples of lectin binding before and after such treatment are shown in Figs 2 and 4. Both concanavalin A and Pisum satitl~ agglutinin reveal a variety of components as horizontal rows of spots of similar molecular weight, presumably arising from heterogeneous glycosylation. Neuraminidase treatment generally converts these to components of higher pI and reduces the number of spots. In the case of peanut agglutinin (Figs 2 and 4b) there is virtually no binding before neuraminidase treatment. We used chemical deglycosylation to investigate the polypeptide chains of major components, using lectins (Fig. 4) and antisera (Fig. 5) to reveal the products. Treatment with trifluoromethanesulphonic acid breaks O-glycosyl bonds, including those to protein,J but fails to cleave N-glycosyl bonds, thus leaving asparagine-linked N-acetylglucosamine as a residue of N-linked chains. Following deglycosylation, the only lectin binding seen is thus a weak binding of WGA to the polypeptide arising from dopamine #-hydroxylase and to some other components (Fig. 4c); marked shifts in molecular weight of some components are revealed by antisera (Fig. 5). Chromogranin A series
It has been previously shown” that an antiserum raised against purified chromogranin A (mol. wt 75,000) cross-reacts with a series of proteins of similar isoelectric points (4.7-5.4) and mol. wts 70-40,OOO and two smaller polypeptides (mol. wts 29,000 and 20,000). These components are indicated in Fig. 1. Lectin blotting (Fig. 2) shows that chromogranins A are not decorated by Con A (Figs 2a, b), by the HRP conjugate of PSA, which has a similar sugar specificity to Con A (Figs 2c,d) or by the galactosespecific HRP-PNA (Fig. 2e); in these blots, the chromogranins A are just discernible as white, unstained spots against the pale violet background. Following neuraminidase treatment, however, these proteins do bind HRP-PNA (Fig. 20, but not Con A or PSA. These results are consistent with the reports U’ that the carbohydrate chains of chromogranin A are O-linked tri- or tetrasaccharides containing N-acetylgalactosamine, galactose and sialic acid; only after removal of terminal sialie acid by neuraminidase is the exposed galactose able to bind HRP-PNA. Chemical deglycosylation only produced slight changes in the apparent molecular weights (Fig. 5a), but very significant shifts in the isoelectric points of the chromogranins A (not shown), that of the largest component being raised from 4.7-5.0 to X1-5.4. This is presumably the result of removing all sialic acid residues; treatment with neuraminidase produced smaller shifts in the same direction. It was previously concluded8 that removal of a single sialic acid from glycoprotein R (glycoprotein III) increases the pI by
/lOOO
3
Fig. I. Two-dimensional electrophoretogram of chroma~n-grange matrix proteins (I80 pg), stained with Coomassie blue. (A, B) Components binding antisera to chromogranins A and B. respectively (Refs 5, 13 and 24). DBH, dopamine @-hydroxylase; M,, mol. wt.
479
Fig. 2. Lectin blots of chromaffin-granule matrix proteins, detected by horseradish peroxidase-activity staining. (a, b) Concunavalin A; (c, d) Pisum satiuum agglutinin; (e, f) peanut agglutinin. Proteins from untreated lysate (a, c, e) or neuraminidase-treated lysate (b, d, f) were separated in two dimensions as in Fig. I.
480
3
481
Fig. 4. Effects of chemical deglycosylation on lectin binding by chromaffin-granule proteins (one-dimensional electrophoretograms). (a) Pisum satimm agglutinin; (b) peanut agglutinin; (c) wheat-germ agglutinin. Track I, untreated lysate; track 2, neuraminidase-treated lysate; track 3, lysate deglycosylated with trifluoromethanesulphonic acid; track 4, lysate treated with neuraminidase and trifluoromethanesulphonic acid: track 5. untreated chro~~~n-granule membrane proteins; R, glycoprotein III (Ref. 7) or band R (Ref. 8). M,, mol. wt.
Fig. 5. Effects of chemical deglycosylation on antibody binding by chromaffin-granule matrix proteins. Immune blots of one-dimensional electrophoretograms detected by 1251-labelledprotein A and autoradiography. (a) Anti-chromogranin A; (b) anti-dopamine B-hydroxylase; (c) anti-[Leulenkephalin. Track 1, untreated iysate; track 2, deglycosylated with trifluoromethanesulphonic acid. M,, mol. wt.
483
Glycoproteins of the chromaffin-granule
0.08, so the observed shift in p1 of approx. 0.4 is consistent with the estimated sialic acid content of chromogranin A (mol. wt 75,000), which is 6 mole/mole (Refs 6 and 11). Chromogranin B series
The proteins derived from chromogranin B (mol. wt 130,000; p1 5.4-5.8) form a complex series, less acidic than chromogranin A and its derivatives. Unlike chromogranin A, they bind both Con A and HRP-PSA, both specific for mannose or glucose (Figs 2a,c). The higher molecular weight members of this series (mol. wts SO-130,000) also bind HRP-PNA after neuraminidase treatment (Fig. 2f), suggesting that they have both N-linked chains containing mannose and other chains containing galactose and terminal sialic acid. A series of proteins of similar p1 but lower mol. wt fail to bind HRP-PNA and so appear to lack the latter class of carbohydrate chains, which could be either N- or O-linked. It is not clear from our results whether these are members of the chromogranin B family, but they appear to be identical with the proteins found to react with an antiserum to chromogranin B (Fischer-Colbrie and Frischenschlager, 1985).5 They appear to contain sialic acid, as neuraminidase treatment increases their electrophoretic mobility (Fig. 4a). Dopamine /I-hydroxylase
matrix
485
After chemical deglycosylation of lysate proteins, HRP-WGA bound to a component of apparent mol. wt 72,000 (Fig. 4c) which is probably dopamine /I-hydroxylase-containing residual asparagine-linked N-acetylglucosamine but little or no other carbohydrate. Some new HRP-WGA-reactive bands also appeared on treatment with trifluoromethane sulphonate; these may be degradation products of other N-glycosylated proteins. Opioid peptides
The antiserum used detects both [Leuj- and [Metlenkephalin-containing sequences, and in blots of one-dimensional electrophoretograms (Fig. 5) recognized 6 major bands (apparent mol. wts 28,500, 21,500, 19,150, 17,600, 14,600 and 13,800) and also reacted with unresolved proteins at the dye-front of the gel. Some of the bands presumably correspond to those detected by Patey et al.*’ using an antiserum to synenkephalin (the N-terminal seventy amino acids of proenkephalin). In two dimensions, however, the pattern is far more complex (Fig. 3b). The polypeptides are not sufficiently abundant to be detected by Coomassie blue staining (Fig. 1); the cleavage products appear to fall into two groups, with pI about 5.5 and 6.0, although some heterogeneity is seen. The adrenal medulla contains both proenkephalin and dynorphin, derivates of these polypeptides being released from adrenalineand noradrenalinecontaining vesicles, respective1y.3 Although immunohistochemical studies suggested that [Leulenkephalins were only present in adrenaline-containing cellsI it seems likely that in immune blots the antiserum used would recognize the [Leulenkephalin sequence in dynorphin and its derivatives, as well as enkephalin sequences in proenkephalin-derived proteins. The largest polypeptides detected have apparent mol. wts close to that of uncleaved proenkephalin, which has been deduced to be 27,300 from the sequence of the cDNA.‘* Patey et al.,” however, were unable to detect this species in bovine adrenal medulla. Kilpatrick et al.‘* found no neutral or amino sugars in enkephalin-containing peptides from the adrenal medulla. While we were unable to assign binding of any lectin to opioid peptides, treatment with trifluoromethanesulphonic acid reduced the mobility of some bands (Fig. 5). The electrophoretic mobility of small glycopeptides may not correlate with their molecular weight and it is possible that the observed changes reflect some type of chemical modification other than deglycosylation.
This enzyme is the major Con A- and the glycoprotein of HRP-PSA-binding chromaffin-granule matrix, as would be expected from its high mannose content.6.‘6 In immune blots (Fig. 3a) and lectin blots (Figs 2ad) it appears as a series of three bands of apparent mol. wt 72-78,000 and pI 5.8-6.0; this pattern is indistinguishable from that of the membrane-bound form of the enzyme.’ It fails to bind HRP-PNA, even after neuraminidase treatment, despite the terminal galactose-linked sialic acid found in N-linked biantennary complexes of dopamine /?-hydroxy1ase;‘6 furthermore, neuraminidase treatment produces no detectable shift in its isoelectric point (see Fig. 2). Possibly the carbohydrate chains of dopamine /I-hydroxylase are rather resistant to neuraminidase. Chemical deglycosylation abolished binding of con A and HRP-PSA to dopamine fi-hydroxylase, but the deglycosylated enzyme still bound antiserum (Fig. 5) and appeared as a sharp doublet of apparent mol. wt 72,000. These bands of approximately equal intensity are extremely close together and are hard to resolve in the photograph. They may correspond to two different polypeptide subunits. The identity of the minor bands of slightly lower molecular weight in Other glycoproteins Fig. 5(b) is unknown. The amount of ‘251-labe11ed Lectin blotting reveals some minor components of protein A bound by the immune complex was much unknown identity: reduced by deglycosylation, suggesting that a sub(a) A poorly focused, acidic component of high stantial fraction of the polyclonal antibodies was molecular weight (approx. 100,000; pI 4.4-4.7) binds directed against glycosyl chains.
D. K.
486
PNA-HRP after neuraminidase treatment (Fig. 2f) and is just visible in Fig. 1. This could be a proteoglycan.” (b) Con A binds to about a dozen unidentified proteins, each of which focusses as a series of discrete spots of the same molecular weight (Fig. 2a). Some cover pI ranges of about 0.3 pH units, but two, of mol. wts 40,000 and 37,000. cover at least 1.2 pH units. Four of these minor components are also decorated by PSA. Neuraminidase treatment of the granule lysate produces marked shifts in the patterns; in particular four or five Con A-binding components are shifted from pI of about 5.4 to as high as p1 6.0 (Fig. Zb). However, none of the glycoproteins run as a single spot after this treatment, The identity of these proteins is unknown; their lectin-binding properties distinguish them from the chromogranins A and their isoelectric points are higher than those of chromogranins B or glycoprotein III.’ These minor glycoproteins may of course be contaminants, but this seems unlikely as chromaffin granules were highly purified before lysis and soluble glycoproteins account for at least 70:/, of the total protein.” None of these components is detectable in chromaffin-granule membranes.’ (c) HRP-WGA binds to a heterogeneous glycoprotein of apparent mol. wt 3740,000, variously termed glycoprotein III (Ref. 7) or band R (Ref. 8). This protein is present in both granule membranes and lysate (Fig. 4c) although it is a minor component of the latter and undetectable by Coomassie blue staining. Treatment with trifluoromethanesulphonic acid abolishes its ability to bind WGA-HRP and neuraminidase digestion converts it to a sharper band of increased mobility (Fig. 4~). The lectin-binding properties of the major glycoproteins of the chromaffin-granule matrix are summarized in Table 1. All of the chromogranins A exhibit the same properties but, as noted above, there may be some differences between the high- and low-molecular weight members of the chromogranin B series.
It is shown in Fig. 1 that granules contain several proteins of mol. wt less than 20,000 which do not bind lectins and do not cross-react with the antisera that we have used. These may be non-antigenic degradation products of chromogranins A or B, but
APPS et al.
the shape and positions of these components suggest that they are still to be identified. Although they may yet be found to be glycoproteins, it is notable that all of the lysate proteins of higher molecular weight that are visible in Fig. 1 appear to be glycoproteins, using the few lectins employed in this study.
DISCUSSION
The complexity of two-dimensional gels makes protein and glycoprotein nomenclature difficult; this is particularly a problem with the chromogranins, the major secreted proteins of chromaffin granules. O’Connor and Frigon19 used one-dimensional electrophoresis to separate the proteolytic derivatives of chromogranin A and designated them chromogranins B, C and D; however, it seems likely that these components are themselves heterogeneous and it is therefore safer at present to designate as chromogranins A those proteins derived from the precursor of mol. wt 75,000 and as chromogranins B those derived from the less acidic precursor of mol. wt 130,000.’ Possibly the biogenically related components should be designated A,, A2 etc. (see Ref. 13) but it seems quite likely that the spots in twodimensiona electrophoretograms are themselves heterogeneous; the shape of the major chromogranin A spot, for example, clearly suggests the superimposition of at least two components. Further refinement of electrophoretic procedures is therefore soon likely to render any more detailed classification obsolete. Lectin binding has revealed several soluble glycoproteins of chromafhn granules that are not seen by Coomassie blue staining, and this emphasises the difficulty of making quantitative estimates of the amounts of different proteins present in the granule matrix. It is clear, however, that the chromogranin A family are by far the major constituents: their biological role is quite unknown. The enkephalincontaining polypeptides are very minor components and granules clearly contain at least a dozen other heterogeneous glycoproteins that may be present in equivalent amounts to them. Because of the method of preparation, there seems to be little possibility that these are contaminants, and so it becomes an urgent necessity to define the biological roles of these proteins which are stored within the matrix of chromaffin
Table 1. Binding of lectins by the major matrix proteins of chromaffin granules, in untreated and neuraminidase-treated lysate Con A Chromogranins A Chromogranins B Dopamine B-hydroxylase Glycoprotein III(R)
+ + +
Con AiNEUR + + +
PNA -
PNAiNEUR + +
PSA + +
PSA/NEUR _ + +
WGA WGAJNEUR + + + + + -+
Molecular weights of the chromogranins A (from Fig. 1) were 75, 72, 68, 57, 55, 49, 29 and 20 x 103;chromogranins B, 130, 115, 106, 99, 89 x 10’. NEUR. neuraminidase-treated.
Glycoproteins of the chromaffin-granule
granules and are presumably
matrix
487
co-secreted with cate-
edge the gift of anti-enkephalin antiserum from Dr. Kwen-
Acknowledgements-This work was supported by a grant from the Medical Research Council, We gratefully acknowl-
Jen Chang, Wellcome Research Laboratories and communication of results prior to publication by Professor H. Winkler. We are indebted to Kern-En-Tee for supplying samples of HRP-conjugated lectins.
cholamines.
REFERENCES
1. Apps D. K. and Schatz G. (1979) An adenosine triphosphatase
2. 3. 4. 5. 6. 7. 8. 9. 10. II. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24.
isolated from chromaffin-granule membranes is closely similar to F,-adenosine triphosphatase of mitochondria. Eur. J. Biochem. 100, 411-419. Clegg J. C. S. (1982) Glycoprotein detection in nitrocellulose transfers of electrophoretically separated protein mixtures using concanavalin A and peroxidase: application to arenavirus and flavivirus proteins. Anaiyl. Biochem. 127,389-394. Dumont M., Day R. and Lemaire S. (1983) Distinct dist~bution of immunoreactive dynorphin and leucine enkephalin in various populations of isolated adrenal chromaffin cells. Life Sci. 32, 287-294. Edge A. S. B., Faltynek C. R., Hof L., Reichert L. E. and Weber P. (1981) Deglycosylation of glycoproteins by trifluoromethanesulphonic acid. Analyr. Biochem. 118, 131-137. Fischer-Colbrie R. and Frischenschlager I. (1985) Immunological characterization of secretory proteins of chromaffin granules: chromogranins A, chromogranins B and enkephahn-containing peptides. J. Neuroihem. In press. Fischer-Colbrie R.. Schachinger M.. Zannerle R. and Winkler H. (1982) Dooamine B-hvdroxvlase and other glycoproteins from the SolubIe~onte~~ and the membranes of adrenal chromakin granules: isolation and carbohydrate analysis. f. Neurochem. 38, 725-732. Fischer-Colbrie R., Zangerle R., Frischenschlager I., Weber A. and Winkler H. (1984) Isotation and immunological characterization of a glycoprotein from adrenal chromaffin granules. J. Neurochem. 42, 1008-1016. Gavine F. S., Pryde J. G., Deane D. L. and Apps D. K. (1984) Glycoproteins of the chromaffin-granule membrane: separation by two-dimensional electrophoresis and identification by lectin binding. J. Neurochem. 43, 1243-1252. Hawkes R. (1982) Identification of concanavalin-A binding proteins after SDS-gel electrophoresis and protein blotting. Art@. Biochem. 123, 143-146. Hook V. Y. M. and Eiden L. E. (1984) Two peptidases that convert ‘*~I-Lys-Arg-[Metlenkephalin and ‘zsI-[Met]enkephalin-Arg6, respectively, to I”~-[Met]enkephalin in bovine adrenal medullary chromaffin granules. FEBS Lett. 172, 212-218. Kiang W-L., Krusius T., Finne J., Margolis R. U. and Margolis R. K. (1982) Glycoproteins and proteoglycans of the chromaffin granule matrix. J. biol. Chem. 257, 16.51-1659. Kilpatrick D. L., Gibson K. D. and Jones B. N. (1983) Is adrenal proenkephalin glycosylated? Archs Biochem. Biophys. 224, 402404. Kilpatrick L., Gavine F., Apps D. and Phillips J. (1983) Biosynthetic relationship between the major matrix proteins of adrenal chromafin granules. FEBS Left. 164, 383-388. Livett B. G., Day R., Elde, R. P. and Howe, P. R. C. (1982) Co-storage of enkephalins and adrenaline in the bovine adrenal medulla. Neuroscience 7, 1323-1332. Ljones T., Skotland T. and Flatmark T. (1976) Purification and characterization of dopamine p-hydroxylase from bovine adrenal medulla. Eur. J. Biochem. 61, 525-533. Margolis R. K., Finne J., Krusius T. and Margolis R. U. (1984) Structural studies on glycoprotein oligosaccharides of chromaffin granule membranes and dopamine B-hydroxylase. Archs Biochem. Biophys. 228, 443-449. Miller R. J., Chang K-J., Cooper B. and Quatrecasas P. (1978) Radioimmuno~ay and characterization of enkephalins in rat tissues. J. biof. Chem. 253, 531-538. Noda M., Furutani Y., Takahashi H., Toyosato M., Hirose T., Inayama S., Nakanishi S. and Numa S. (1982) Cloning and sequence analysis of cDNA for bovine adrenal proenkephalin. Narure 295, 202-206. O’Connor D. T. and Frigon R. P. (1984) Chromogranin A, the major catecholamine storage vesicle soluble protein. J. biol. Chem. 259, 3237-3247. O’Farrell D. M. (1975) High resolution two-dimensional electrophoresis of proteins. J. biol. Chem. 250, 4007-4021. Patey G., Liston D. and Rossier J. (1984) Characterization of new enkephalin-containing peptides in the adrenal medulla by immunoblotting. FEBS Left. 172, 303-308. Saxena A. and Fleming P. J. (1983) Isolation and reconstitution of the membrane-bound form of dopamine /?-hydroxylase. J. biol. Chem. 258, 4147-4152. Stewart J. R., Chaplin M. F. and Kenny A. J. (1984) Deglycosylation by trifluoromethane sulphonic acid of endopeptidase-24.11 purified from pig kidney and intestine. Biochem. J. 221, 919-922. Winkler H., Falkensammer G., Patzak A., Fischer-Colbrie R., Schober M. and Weber A. (1984) Life cycle of the catecholaminergic vesicle: from biogenesis to secretion. In Regulation of Transmitter Function. Proceedings of 5th Meeting European Society of Neuroehem~s~ry, Budapest (eds Vizi E. S. and Magyar K.), pp. 65-73. (Accepted 24 March 1985)