Chromogranin A, the Major Catecholamine Storage Vesicle Soluble Protein MULTIPLE SIZE FORMS, SUBCELLULAR STORAGE, AND REGIONAL DISTRIBUTION IN CHROMAFFIN AND NERVOUS TISSUE ELUCIDATED BY RADIOIMMUNOASSAY*

Chromogranin A (CgA), the major catecholamine storage vesicle (CSV) soluble protein, may index exocytotic sympathoadrenal secretion. To explore CgA in adrenergic tissues, we developed a radioimmunoassay for bovine CgA. Within adrenal medulla CSV, several minor chromogranins had similar amino acid compositions and peptide maps to that of CgA and also showed parallel, partial cross-reactivity in the CgA radioimmunoassay. CgA immunoreactivity represented 7 f 1% of total adrenal medulla cell protein and was localized to adrenal CSV, representing 46 f 2% of CSV soluble protein. In brain, there was 1000-fold less CgA than in ad- renal medulla, with a widespead regional distribution (maximal in neocortex) and an unusual subcellular dis- tribution (maximal in cytosol), both of which differ from reported catecholamine distribution. Brain chro- mogranin immunoreactivity also had a lower Stokes radius than adrenal CgA. Sympathetic nerve and serum had 6,000-fold and 30,000-fold less CgA than that in adrenal medulla. The results suggest a “family” of adrenal medulla chromogranins, similar structurally and immunologi- cally. Adrenal medulla and brain ehromogranin differ in concentration, subcellular localization, and molecu- pestle (Arthur H. Thomas) and filtered through cheesecloth. The homogenate was centrifuged at 1000 X g for 10 min (1 X 10' x g x min) to sediment nuclei and debris and then at 25,000 X g for 20 min (5 X lo5 X g X min) to sediment a crude granule fraction from the supernatant, containing cytosol and microsomes. The crude granule fraction was resuspended in 0.3 M sucrose, layered onto step gradients of 1.6 M sucrose, and centrifuged at 20,000 X g for 5 h (6 X lo6 X g X min) to yield a pink chromaffin granule pellet. Electron microscopy of the granule pellet showed a highly purified chromaffin granule preparation (3). The chromaffin granules were lysed by resuspension in 0.001 M sodium phosphate, pH 6.5, frozen and thawed, and centrifuged at 100,000 X g for 60 min (6 X lo6 X g X min) to separate soluble vesicle lysate from vesicle membranes. The membranes were resuspended in the same buffer, frozen and thawed, and recentrifuged at 100,000 X g for 60 min to wash the membranes free of remaining soluble vesicle lysate. Tissue fractions were frozen at -70 "C prior to assay. Tissue fractions were assayed for protein, chromogranin A, dopa-mine-P-hydroxylase, and catecholamines.

Chromogranin A (CgA), the major catecholamine storage vesicle (CSV) soluble protein, may index exocytotic sympathoadrenal secretion. To explore CgA in adrenergic tissues, we developed a radioimmunoassay for bovine CgA. Within adrenal medulla CSV, several minor chromogranins had similar amino acid compositions and peptide maps to that of CgA and also showed parallel, partial cross-reactivity in the CgA radioimmunoassay. CgA immunoreactivity represented 7 f 1% of total adrenal medulla cell protein and was localized to adrenal CSV, representing 46 f 2% of CSV soluble protein.
In brain, there was 1000-fold less CgA than in adrenal medulla, with a widespead regional distribution (maximal in neocortex) and an unusual subcellular distribution (maximal in cytosol), both of which differ from reported catecholamine distribution. Brain chromogranin immunoreactivity also had a lower Stokes radius than adrenal CgA. Sympathetic nerve and serum had 6,000-fold and 30,000-fold less CgA than that in adrenal medulla.
The results suggest a "family" of adrenal medulla chromogranins, similar structurally and immunologically. Adrenal medulla and brain ehromogranin differ in concentration, subcellular localization, and molecular size. Finally, CgA in serum may provide a useful tool for sympathoadrenal studies in intact organisms.
To explore the utility of chromogranin A as a sympathoad-* This work was supported by the Veterans Administration, the National Institutes of Health (HL-25,457), and the American Heart Association. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "aduertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. -" ----~ " " " " " " ____ renal index, we developed a sensitive and specific RIA' for bovine chromogranin A, initially using the assay to examine the structural and immunologic similarities among several electrophoretically separable chromogranins and to assess chromogranin A in chromaffin and nervous tissue.
Our results suggest that there are multiple immunologically and structurally related adrenal medullary chromogranins, that marked differences exist between adrenal medullary and brain chromogranins, and that radioimmunoassay may be a useful probe of chromogranin in serum as well as in isolated tissues.

Preparation of Chromogranins
Chromogranins were prepared from bovine adrenal medullary chromaffin granules (13). Chromaffin granules were isolated from the adrenal medulla by centrifugation on sucrose density gradients (3,14) and then were lysed in 0.001 M sodium phosphate, pH 6.5, and centrifuged at 100,000 x g for 1 h to separate granule membranes from soluble lysate. After extensive dialysis of the soluble lysate against 0.01 M sodium phosphate, pH 6.5, to remove catecholamines, the lysate was then chromatographed on concanavalin A-Sepharose to remove dopamine-@-hydroxylase (15) and then subjected to preparative polyacrylamide gel electrophoresis in 8 M urea to yield pure chromogranin A (13). The quality of the chromogranin A preparation was verified by re-electrophoresis in SDS gels (16), yielding one band (13), as well as by gel filtration in 6 M guanidine HCI and isoelectric focusing (13). Purified bovine chromogranin A was characterized (13) as a 67,000-dalton, acidic (isoelectric point, 4.68-4.81), monomeric protein.
For some experiments, the lysate, after dopamine-@-hydroxylase removal as described, was preparatively electrophoresed on native polyacrylamide gels (17,18) and not only the major chromogranin

Chromogranin A in Adrenergic Tissues
Human chromogranin A was also prepared from the chromaffin vesicle lysate of a pheochromocytoma, exactly as described above and earlier (13), to be used as an immunogen.
Preparation of Antisera Antisera to chromogranins A (antigen purified from 8 M urea gels, with purity documented by re-electrophoresis on SDS gels) and B (antigen purified on native gels, with purity documented by reelectrophoresis on SDS gels) were prepared in male New Zealand white rabbits by a modification of the schedule of Miras-Portugal and Santos-Ruiz (19). One mg of antigen, emulsified in complete Freund's adjuvant, was injected in multiple intradermal sites, on three occasions a t 2-week intervals. The antisera were harvested 1 month after the last injection, via the central ear artery. The animals received a 500-pg booster injection at 6-month intervals, with antiserum harvested again 1 month later. Antisera were aliquotted and stored a t -70 "C.
Radioiodination of Chromogranin A The purified bovine chromogranin A was radioiodinated by the solid phase, immobilized lactoperoxidase-glucose oxidase method (20) (Enzymobeads, Bio-Rad Laboratories), as previously outlined by us (21). After radioiodination, the labeled chromogranin A was separated from iodide and aggregate on an Ultrogel ACA-22 gel filtration column (25 X 1.5 cm), equilibrated with 0.15 M NaCI, 0.01 M sodium phosphate, p H 7.4, 0.1% (w/v) ovalbumin, 0.1% (w/v) sodium azide, and eluted a t 2 ml/h, collecting 1-ml fractions. This gel filtration resin provided optimal separation of radioiodinated chromogranin A (lZ5Ichromogranin A) from both the void volume ( Vo, determined by blue dextran) and the total internal volume (V,, determined by NaIZ5I) of the column. 1251-Chromogranin A was detected by counting 10 p1 from each fraction for 1 min in a Searle Model 1185 y counter (Searle Analytic, Inc., Des Plaines, IL).

Antibody Titrations
To determine the immunoprecipitability of the radioiodinated antigen and to determine the titer of the first antibody Eor RIA, immunoprecipitation of 1251-chromogranin A was carried out with serial dilutions of first antibody (rabbit anti-chromogranin A). The incubation mixture contained 5000 cpm of 1251-chromogranin A in 100 pI of Buffer PBO-10 (0.15 M NaCI, 0.01 M sodium phosphate, pH 7.4, 1% (w/v) ovalbumin, 0.1% (w/v) sodium azide); 100 pl of 0.1 M EDTA, pH 7.4; 100 pl of various dilutions of first antibody in PBO-10; and 500 pl of PBO-10. After 24 h a t 4 "C, the second antibody (in slight excess) and carrier antiserum were added: 100 pl of a 1:s (v/v) dilution of sheep anti-rabbit y-globulin (SA, P4 titer) in PBO-10 and 100 pl of 2% (v/v) normal rabbit serum diluted in PBO-10. After another 24 h a t 4 "C, bound and free antigen were separated by centrifugation at 5000 X g for 20 min a t 4 "C. The supernatant was aspirated and the antigen-antibody pellet was washed with 500 @I of PBO-10 and recentrifuged a t 5000 X g for 20 min a t 4 "C. After aspiration of the supernatant, the final antigen-antibody pellet was counted in the tube for 1 min in the y counter.
Titration results were graphed as per cent of maximum binding (at low titer of first antibody) uersus the loglo of the first antibody dilution.
A time course analysis of incorporation of '251-chromogranin A into the final antigen-antibody pellet was conducted both for the first antigen-antibody reaction (over 1-3 days, a t various dilutions of first antibody, followed by second antigen-antibody reaction for 24 h) and for the second antigen-antibody reaction (over 24 h, a t the previously noted final dilution of second antibody used in the assay, after a first antigen-antibody reaction of 24 h at working titer of first antibody).

Radioimmunoassay
The working titer of first antibody was selected to immunoprecipitate 30-4096 of 5000 cpm of 1251-bovine chromogranin A, under these conditions. The RIA incubation mixture contained 100 p1 of 5000 cpm of '251-bovine chromogranin A in PBO-10; 100 p1 of first antibody at working dilution in PBO-10 (antibody 137-6, rabbit anti-bovine chromogranin A, 1:lOOO (v/v)); 100 pl of 0.1 M EDTA, pH 7.4; and 500 pl for unlabeled chromogranin A calibration standards (1-2000 ng) or unknown sample, diluted in PBO-10. After 24 h at 4 "C, the second antibody (in slight excess) and carrier antiserum were added: 100 pl of a 1:s (v/v) dilution of sheep anti-rabbit y globulin (SA, P4 titer) in PBO-10 and 100 pl of 2% (v/v) normal rabbit serum in PBO-10. After another 24 h a t 4 "C, the antigen-antibody pellet was harvested and counted as just described (see "Antibody Titrations").
TO exclude tissue proteolytic activity contributions to apparent tissue immunoreactivity, representative tissue samples were boiled (10 min, sealed, a t 100 "C), and others were treated with multiple protease inhibitors (0.01 M EDTA, 0.01 M N-ethylmaleimide, 0.001 M phenylmethylsulfonyl fluoride) prior to RIA.
The RIA standard curve was expressed as counts/min bound (minus blank) in a given tube (B), over counts/min bound (minus blank) at the working titer of first antibody without addition of unlabeled chromogranin A (Bo), plotted uersus log,, of unlabeled chromogranin A added. Typical values for Bo were 1200-1500 cpm, while typical values for the blank (obtained by assay incubations using appropriate dilutions of normal rabbit serum instead of specific antibody) were 140-160 cpm. Samples were assayed in duplicate a t dilutions designed to place pellet counts/min in the 20-70% B/Bo working range of the standard assay curve.
Ouchterlony Immunodiffusion Antigenicity and antigenic cross-reactivity among chromogranins was also examined by Ouchterlony double immunodiffusion (22), performed overnight at 4 "C, in a humid chamber with prepunched agar plates (Hyland Laboratories) soaked in phosphate-buffered saline, containing a central antibody well and peripheral antigen wells. After 16 h of incubation, the plates were soaked overnight in phosphate-buffered saline, then stained for 1 h with 1% (w/v) Amido black in 7% (v/v) acetic acid, and destained with 7% (v/v) acetic acid. After photography, plates were soaked in 10% (v/v) glycerol and airdried for storage.

Characterization of Chromogranins
Amino Acid Composition-The electrophoretically purified proteins, bovine chromogranins A, B, and C, were freed of any contaminating peptides by addition of SDS to a final concentration of 1% (w/v), heating to 100 "C for 5 min, and exhaustive dialysis against 0.05% (w/v) SDS in 0.01 M sodium phosphate, pH 6.5, for 24 h. One hundred pg of protein (in triplicate) was lyophilized and resuspended in a vacuum tube in 300 pl of 6 N HCI with a crystal of phenol (to prevent destruction of tyrosine) and 5 pl of a solution of 12.5 nmol of norleucine (as internal standard). The tubes were sealed under vacuum and each protein was hydrolyzed for 24,48, and 72 h a t 115 "C. The time course served for determination of serine and threonine, by extrapolation back to time zero. After hydrolysis, the tubes were cooled, centrifuged, and broken open, and the HCI was evaporated at 60 "C under N P . The amino acid hydrolysates were then resuspended in 0.15 N lithium carbonate, pH 2.2, and analyzed in a Durrum model D-500 amino acid analyzer (Dionex Corp., Sunnyvale, CA), eluting with a physiological amino acid profile program (23), with data analysis by a Mark I1 software package (Dionex Corp.). Results are expressed as the mean value (2 S.D. or coefficient of variation) of the 3 determinations, except for serine and threonine, where results are extrapolated to time zero to correct for destructive losses during hydrolysis. Results are expressed both as weight per cent (grams of the particular amino acid/100 g of protein) and as residues/mol (nearest whole number with molecular weight taken as the SDS-gel electrophoretic value). The eluate from a blank polyacrylamide preparative gel was also carried through this entire procedure, to correct for amino acid contaminants in the gel buffers or matrix.
Peptide Mapping-One hundred-to two hundred-pg portions (1. parts pyridine, 897 parts HzO, pH 6.5. The buffer was sprayed onto the plate and electrophoresis was performed over a metal cooling plate a t 8-10 "C, for 50 min at 800 V (cathode on left), whereupon the plate was dried, sprayed with 0.025% (w/v) fluorescamine in acetone, then visualized, and photographed under a long wavelength ultraviolet lamp. A control experiment, assessing the peptide map of trypsin alone, in the concentration used in these experiments, incubated without addition of chromogranin substrate, did not result in detectable peptide spots.

Preparation of Other Antigens for Cross-reactiuity Studies in the RIA
Bovine adrenal dopamine 0-hydroxylase was purified by concanavalin A-Sepharose chromatography on bovine adrenal medulla chromaffin vesicle lysates, as previously described (15).

Bovine Adrenal Medulla Subcellular Fractionations
To localize immunoreactive chromogranin A within the cell, adrenal medulla subcellular fractions were obtained by a modification (3) of the sucrose density step gradient method of Smith and Winkler (14). Fractionation was performed in quadruplicate. The glands were obtained within 20 min of death at the slaughterhouse and transported immediately to the laboratory at 0 "C. All steps were conducted in the cold. The medullae were dissected out, weighed, minced finely, homogenized at 20% (w/v) in 0.3 M sucrose in a Potter-Elvejehm tissue grinder with glass mortar and Teflon pestle (Arthur H. Thomas) and filtered through cheesecloth. The homogenate was centrifuged at 1000 X g for 10 min ( 1 X 10' x g x min) to sediment nuclei and debris and then a t 25,000 X g for 20 min (5 X lo5 X g X min) to sediment a crude granule fraction from the supernatant, containing cytosol and microsomes. The crude granule fraction was resuspended in 0.3 M sucrose, layered onto step gradients of 1.6 M sucrose, and centrifuged a t 20,000 X g for 5 h (6 X lo6 X g X min) to yield a pink chromaffin granule pellet. Electron microscopy of the granule pellet showed a highly purified chromaffin granule preparation (3). The chromaffin granules were lysed by resuspension in 0.001 M sodium phosphate, pH 6.5, frozen and thawed, and centrifuged at 100,000 X g for 60 min (6 X lo6 X g X min) to separate soluble vesicle lysate from vesicle membranes. The membranes were resuspended in the same buffer, frozen and thawed, and recentrifuged a t 100,000 X g for 60 min to wash the membranes free of remaining soluble vesicle lysate. Tissue fractions were frozen a t -70 "C prior to assay.
Tissue fractions were assayed for protein, chromogranin A, dopamine-P-hydroxylase, and catecholamines.

Chromogranin in the Nervous System
Bovine brains were obtained within 30 min of death and transported to the laboratory in ice-cold 0.3 M sucrose. All steps were performed in the cold. For regional distribution, dissections were performed in quintuplicate. The tissue was minced, homogenized in 0.3 M sucrose (at 1:5 ratio of tissue:buffer) in a Potter-Elvejebm homogenizer (glass mortar, Teflon pestle), frozen and thawed, and centrifuged a t 1000 X G for 10 min (lo4 X g X min) to sediment debris, whereupon the supernatant was frozen a t -70 "C prior to assay.
For subcellular distribution, a crude synaptosomal preparation (26) was obtained in quintuplicate from frontal cortex, since this was a brain region relatively high in chromogranin concentration (see below). The tissue was minced, homogenized in 0.3 M sucrose (at 1:10 ratio of tissue:buffer) in a Potter-Elvejehm homogenizer (glass mortar, Teflon pestle), then centrifuged a t 1000 X g for 10 rnin (lo4 X g X min) to sediment a nuclear fraction, a t 20,000 X g for 20 min (4 x lo5 X g X min) to sediment a crude synaptosomal fraction, and a t 10,000 X g for 10 h (6X lo6 X g X min) to sediment microsomes and membranes, leaving a cell cytosol supernatant. The pellets were resuspended in 0.01 M sodium phosphate, p H 7.4, and then all cell fractions were frozen at -70 "C prior to assay. The identity of synaptosomes in the crude synaptosomal pellet was verified in two ways: ( a ) morphologically, by transmission electron microscopy on a portion of the synaptosomal pellet (see below) and ( b ) biochemically, by spectrophotometric assay of acetylcholinesterase (27), a synaptosomal marker enzyme localized to the synaptosomal membranes (26).
Tissue fractions were assayed for protein, chromogranin A, and acetylcholinesterase.
Sympathetic nerve homogenates were prepared from sympathetic axons dissected from the intrasplenic (distal) portions of splenic neurovascular bundles (28). Homogenizations were performed in a Polytron (Brinkmann Instruments).

Chrornogranin in Serum
Whole blood samples were freshly obtained from 10 cows, then allowed to clot in the cold, then centrifuged a t 1000 X g for 10 min ( l o 4 X g X min), whereupon the serum was frozen a t -70 "C for future assay.

Chromaffin Vesicle Lysate and Brain Homogenate Gel Filtration
One hundred pl of bovine chromaffin vesicle soluble lysate and 100 p1 of brain homogenate (frontal cortex, homogenized in a protease inhibitor buffer of 0.01 M EDTA, 0.001 M phenylmethylsulfonyl fluoride, 0.01 M N-ethylmaleimide, 0.01 M sodium phosphate, pH 6.5) were gel-filtered using the same Ultrogel ACA-22 column, buffer, and conditions used for isolation of '2sI-chromogranin after radioiodination.

Electron Microscopy
Portions (2 X 2 X 2 mm) of selected, freshly prepared subcellular fractions (chromaffin granule pellets, synaptosomal pellets) were fixed, stained, and viewed by transmission electron microscopy as previously described (13).

Assays
Protein was measured by the Coomassie blue dye binding method (29) as recommended for adrenal subcellular fractions (30). Catecholamines were determined separately as epinephrine and norepinephrine, by the fluorimetric method (31). Dopamine &hydroxylase was assayed spectrophotometrically (32) with inclusion of 30 mM Nethylmaleimide to neutralize endogenous inhibitors; results were expressed as international units, where 1 unit represents conversion of 1 pmol of tyramine substrate to octopamine product per min at pH 5.0 and 37 "C. Acetylcholinesterase (a synaptosomal marker enzyme) (26) was assayed spectrophotometrically (27). Results are recorded as units of acetylcholinesterase activity per mg of protein, where 1 unit represents the amount of enzyme that liberates 1 Kmol of acetic acid from acetylcholine in 30 min a t 25 "C and pH 7.8, with a lower detection limit of 1 unit in the assay. Assay color blanks were tissue fractions heated to 60 "C for 10 min prior to assay, to destroy authentic tissue acetylcholinesterase activity (27).

RESULTS
Chromogranin A was purified to SDS electrophoretic homogeneity from bovine adrenal chromaffin vesicle lysates ( Fig. 1, left). At lower protein loads on the gel, chromogranin A gave the appearance of a doublet band, as previously described (3). After solid phase enzymatic radioiodination, the rial and NalZ5I by gel filtration (Fig. 2). The product had a specific activity of 300,000 cpm/Mg of protein.
Immunoprecipitation of lZ5I-bovine chromogranin A (Fig.  3) was carried out with two antisera to bovine chromogranin A and one antiserum to bovine chromogranin B. Each precipitated 70-85% of the total counts/min at excess titers, indicating intact immunoreactivity of the radioiodinated molecule. The 50% immunoprecipitation titers for the antichromogranin A antisera were approximately 1:lOOO. The antichromogranin B antiserum also precipitated '251-~hromogranin A, albeit a t a somewhat lower titer of approximately 1:200. An antiserum to human chromogranin A also precipitated '251-labeled bovine chromogranin A, indicating at least some interspecies cross-reactivity. An antiserum directed against rat chromogranin A (#137d-l) did not immunoprecipitate '251-labeled bovine chromogranin A, at any titer. Timing of the RIA incubation steps was determined by the time course analysis of the association of first antigen (bovine  (26) and destained in 5% (v/v) acetic acid, and regions corresponding to the stained chromogranin bands (CgA through CgD) were sliced out of the remaining 11 gels and eluted as described under "Materials and Methods." chromogranin A) with first antibody (rabbit anti-bovine chromogranin A) at various dilutions (Fig. 4, left) and the association of second antigen (rabbit y-globulin) (Fig. 4, right) with second antibody (sheep anti-rabbit y-globulin). Both reactions were essentially complete a t 24 h; thus an equilibrium RIA was constructed with sequential 24-h incubations a t 4 "C.

Chromogranin A in Adrenergic Tissues
Neither incubation at 25 "C, nor preincubation for 2 h at X "C, measurably accelerated the association of first antigen with first antibody. chromogranin A from the antigen-antibody pellet, yielding the assay standard curve (Fig. 5 ) . The assay had a sensitivity and working range of 10-100 ng/tube. Repeated measurements on the same unlabeled chromogranin A sample yielded an intra-assay coefficient of variation of 4.8% (n = 20) and an interassay coefficient of variation of 13% (n = 8).
Several other chromogranins, here named B, C, and D, were sliced out of preparative polyacrylamide native gels of chromaffin vesicle lysate (Fig. 1, right). Each of these chromogranins was able to displace '"I-chromogranin A from its antibody in parallel with the pure chromogranin A standard (Fig. 5 ) , suggesting that the antibody recognized the same or a similar site on all the chromogranin antigens. The per cent cross-reactivity, on a weight basis, was 40.7% for chromo-  (Table I).
The electrophoretic pattern of multiple chromogranins (Fig.  1) was unlikely to be the result of artifactual proteolysis during vesicle preparation, since the same patterns were noted even when the vesicle preparation was conducted in sucrose buffers containing 0.001 M phenylmethylsulfonyl fluoride. Furthermore, the electrophoretic patterns of multiple chromogranins persisted in several vesicle lysate preparations.
Chromogranins A and B were subjected to re-electrophoresis in SDS gels (Fig. 1, left), yielding apparent molecular weights (upon interpolation of relative mobilities on a plot of molecular weight standards' relative mobility uersus log,, molecular weight, r = -0.999) of 67,000 and 52,000, respectively, neither of which changed with inclusion or exclusion of sulfhydryl reagents in the electrophoresed sample, precluding intersubunit disulfide links in either. At lighter protein loads on the SDS slab gels, chromogranins A and B each had a doublet appearance (Fig. 1, left), as has been previously described for chromogranin A (13).
Amino acid analysis on chromogranins A, B, and C revealed unusual but similar per cent compositions (Table I1 and Fig.   6)"each was rich in glutamic acid (28.76-30.56%) and proline (7.95-8.03%) and poor in cysteine (0-0.20%). The tryptic digest peptide maps of chromogranins A and B ( Fig. 7) revealed considerable structural homology, with most peptides shared in common. Ouchterlony immunodiffusion (Fig. 8) also revealed chromogranin cross-reactivity-the precipitin lines for total bovine chromogranins, bovine chromogranin A, and bovine chromogranin B all fused, suggesting that the antibody (rabbit anti-bovine chromogranin A) recognizes a similar antigenic determinant in all three. This antibody does not form a precipitin line with human chromogranin A or B (Fig. 8).
Other chromaffin granule soluble constituents-catecholamines, enkephalins, and dopamine-p-hydroxylase-did not cross-react in the RIA with chromogranin A, even at 10,000fold mass excess in the assay ( Table I). The interspecies crossreactivity of the assay was minimal ( Table I).
A chromaffin granule preparation was obtained from bovine adrenal medulla and verified by electron microscopy (Fig. 9) and by biochemical markers (Table 111). Chromogranin immunoreactivity was detected in nanoliter quantities of adrenal medullary subcellular fractions. The immunoreactivity paralleled the assay standard curve (Fig. 10) and was not abolished by boiling or treatment with protease inhibitors (0.01 M EDTA, 0.01 M N-ethylmaleimide, and 0.001 M phenylmethylsulfonyl fluoride); thus the immunoreactivity could not be ascribed to proteolytic activity in the adrenal medulla.
Gel filtration of bovine chromaffin vesicle lysate (Fig. 11) yielded a single immunoreactive chromogranin peak that eluted in the same position as 'ZsI;chromogranin A, with an apparent Stokes radius (15) of 80 A for both. A small trailing shoulder on the peak may represent the minor, lower molecular weight chromogranins.
The subcellular distribution of immunoreactive chromogranin A, catecholamines, and dopamine @-hydroxylase in the adrenal medulla is presented in Table 111. All three were largely localized to the catecholamine storage vesicles-chromogranin A and catecholamines were predominantly in the soluble vesicle lysate, while dopamine @-hydroxylase was distributed between soluble vesicle lysate and vesicle membrane. Immunoreactive chromogranin A was almost completely (69%/74% or 93%) released from the vesicles into the soluble lysate during the first i n vitro lysis cycle, with an additional 3%/74% or 4% released during the second lysis cycle. Only 2%/74% or 3% of the vesicle's chromogranin remains on the vesicle membrane after two in vitro lysis cycles.
The tissue fraction chromogranin A specific activity (immunoreactive chromogranin A protein/total protein) was highest, within the chromaffin cell, in the catecholamine storage vesicle soluble lysate (Table HI), where chromogranin immunoreactivity accounted for 46 t 2% of the total soluble  Table I: chromogranin B, 40.7%; chromogranin C , 8.6%; and chrornogranin D, 1.2%.  vesicle protein. In the initial adrenal medullary cell homogenate, chromogranin immunoreactivity accounted for 7 -t 1% of the cell's total protein.
In the nervous system, chromogranin was detected by parallel titration displacement of '251-chromogranin A from antibody (Fig. 12). Immunoreactive chromogranin had a widespread distribution in various brain regions (Table IV), being maximal in neocortex and minimal in cerebellum, medulla oblongata, and spinal cord. Even in the neocortex, however, there was 1000-fold less chromogranin in brain than in adrenal medulla. The pituitary gland also contained substantial quantities of chromogranin immunoreactivity (Table  IV). Neither boiling nor treatment with several protease inhibitors (0.01 M EDTA, 0.001 M phenylmethylsulfonyl fluoride, 0.01 M N-ethylmaleimide) abolished brain tissue chromogranin immunoreactivity, suggesting that brain proteases were not contributing to apparent tissue immunoreactivity.
Subcellular distribution studies included preparation of a crude brain synaptosomal fraction (Table V, Fig. 13). Electron microscopy of the synaptosomal pellet revealed typical synaptosomes with synaptic vesicles (Fig. 13). The synaptosomal fraction also contained acetylcholinesterase activity (4.14 k 0.97 units/mg of protein, range 2.98 to 6.22 units/mg of protein), while the other cell fractions (nuclear, cytosol, microsome, and membrane) did not contain detected acetylcholinesterase (<1 unit/100 pl of cell fraction). Only 18 +-2% of the cell's immunoreactive chromogranin was found in the synaptosomes, at a specific activity of only 10.4 f 1.4 ng of chromogranin/mg of protein. By contrast, the cytosol contained the majority (69 k 6%) of the cell's chromogranin, at a higher specific activity of 123 f 4.5 ng of chromogranin/mg of protein. In fact, chromogranin specific activity in the cell increased with each successive centrifugation (Table V), CUIminating in a maximal specific activity in the very high speed supernatant (cytosol). This is in marked contrast to the results in the adrenal medulla, where per cent chromogranin localization and specific activity were both maximal in the sedimentable vesicle fraction (Table 111). Similar chromogranin subcellular distribution results in brain frontal cortex were noted ( n = 10) when another buffer (0.32 M sucrose, 0.01

TABLE I1
Amino acid compositions of the chramogranins: A, B, and C The values are presented as weight per cent (grams of that amino acid/100 g of protein) and as residues/mol (to the nearest integral number of residues), taking molecular weights from SDS gel relative electrophoretic mobility (chrornogranin A, M, = 67,000; chromogranin B, M, = 52,000). Numbers shown are the mean value for 3 hydrolysates at 24,48, and 72 h, with coefficient of variation ((standard deviation/mean) (loo)), except for serine and threonine, which were extrapolated back to time zero to correct for destructive losses during acid hydrolysis. The amino acid composition of bovine chromogranin A has been reported previously (13).  subjected to chromatography (dimension 1, from bottom to top) followed by electrophoresis (dimension 2, from left to right). The separated peptides were visualized by long wave ultraviolet irradiation after fluorescamine spraying. The peptide map for chromogranin A (left) has been reported previously (13). also found in the hypothalamus (n = 5).
Brain chromogranin also differed from adrenal medullary chromogranin on gel filtration. While adrenal medullary chromogranin immunoreactivity co-eluted with purified '251-chromogranin A (Fig. l l ) , brain (frontal cortex) chromogranin immunoreactivity eluted from the calibrated column consistently later than purified '251-chromogfanin A (Fig. 14), with an apparent Stokes radius (15) of 54 A. The result could not easily be ascribed to proteolytic degradation of chromogranin during homogenization and preparation of the brain tissue sample for gel filtration, since these steps were carried out in a buffer containing several protease inhibitors: 0.01 M EDTA, 0.001 M phenylmethylsulfonyl fluoride, and 0.01 M N-ethylmaleimide. Similar results were obtained (n = 2) when brain homogenates were prepared and gel-filtered in the absence of protease inhibitors.
Chromogranin immunoreactivity was also detected in sympathetic nerve and in serum (Fig. 12, Table IV), although the relative amounts were 6,000-fold and 30,000-fold, respectively, less than that found in the adrenal medulla. Equivalent results were obtained in bovine serum, EDTA plasma, or heparinized plasma. Immunoreactive chromogranin was detectable with this assay in bovine and sheep sera, although not in sera from pig, rat, rabbit, or man.

DISCUSSION
We sought to develop an immunoassay for chromogranin A because it is a major soluble component of catecholamine storage vesicles and may be a useful probe of catecholamine storage and exocytotic release (9)(10)(11)(12). Prior attempts to measure chromogranin A used microcomplement fixation; while useful information emerged, the technique was insensitive and prone to large errors (-50% to +ZOO%) because the results emerge from an analysis of degree of antigen dilution required to fix complement (9-12). Our assay is sensitive (working range, 10-100 ng/tube) and reproducible (intra-and interassay coefficients of variation, 4.8% and 13%). Within the chromaffin vesicle, the minor chromogranins (B, C, D) cross-react to some extent (1.2%-40.7%) ( Fig. 5; Table I), while other vesicle constituents do not cross-react ( Table I). The parallel displacement of "'1chromogranin A from antibody by all four chromogranins suggests that the antibody recognizes a similar antigenic determinant on all four chromogranins. Further evidence for immunologic relatedness is the ability of anti-chromogranin B to precipitate chromogranin A (Fig. 3). In addition, antichromogranin A forms fused precipitin lines with both chromogranin A and chromogranin B during double immunodiffusion (Fig. 8). Antigenic cross-reactivity among the chromogranins has also been reported by Hortnagl et al. (17), who found that anti-chromogranin A also recognized minor chro-mogranins with faster electrophoretic mobility than chromogranin A.

Chromogranin A in Adrenergic Tissues
These immunologically cross-reacting chromogranins also possess considerable structural homology, as judged by similar amino acid compositions ( Table 11, Fig. 6) and similar tryptic digest peptide maps (Fig. 7). Thus, there seems to be a "family" of chromogranins, structurally and immunologically related, with a spectrum of sizes. The similarities of chromogranins A and B are consistent with Smith and Kirshner's report (2) that chromaffin granule proteins SI and S, (analogous to our chromogranins A and B) have similar amino acid compositions and peptide maps.
It should be noted that the biosynthetic relationship of the minor chromogranins to chromogranin A has not been established. The minor, lower molecular weight chromogranins could be either separate gene products from chromogranin A or post-translational (e.g. proteolytic) modifications of chromogranin A, even though they are present when vesicles are prepared in the presence of 0.001 M phenylmethylsulfonyl fluoride to inhibit serine proteases. This issue could perhaps be addressed by in vitro translation of adrenal medullary messenger ribonucleic acid, with immunoprecipitation of chromogranin-like translation products prior to in vivo posttranslational modifications. Also, we have not entirely excluded the possibility that some of the minor chromogranins might have been contaminated with fragments of chromogranin A. In particular, we have not shown that all of the chromogranin B, C , and D preparations can be quantitatively precipitated with antisera to chromogranin A. However, chromogranin B's homogeneous appearance on re-electrophoresis in SDS (Fig. l ) , coupled with its substantial (40.7%) crossreactivity in the RIA, argue against the notion that contaminating chromogranin A fragments completely account for our results. Not all vesicle soluble peptides cross-react in the assay, however; the assay recognizes only 46 f 2% of the soluble vesicle proteins (Table 111) and does not recognize enkephal-

Subcellular distribution of chromogranin A, epinephrine, dopamine-&hydroxylase activity, and total protein in the bovine adrenal medulla
Results are recorded as mean f S.E. for the four subcellular preparations. The subcellular fractions were prepared by differential sucrose gradient centrifugation, as described under "Materials and Methods." The supernatant over the crude chromaffin granule pellet constituted the cytosol plus microsomes. Chromogranin A was quantitated by radioimmunoassay, epinephrine was measured fluorimetrically, and dopamine-&hydroxylase was measured spectrophotometrically. n = 4 for all determinations.  Fig. 2. Eluted fractions were tested for chromogranin A by incubating 100 p1 from each fraction in the RIA. The chromogranin immunoreactive peak eluted in the same position as purified lZ5Ichromogranin A, suggesting a similar effective hydrodynamic (Stokes) radius of 80 A. A small trailing shoulder in the immunoreactive peak may represent the minor, lower molecular weight chromogranins. Vo = void volume; V, = total internal column volume; lZ5Ichromogranin A = elution volume for purified '251-chromogranin A.

12511-labeled bovine chromogranin
A can be immunoprecipitated by anti-human chromogranin A (Fig. 3), albeit at a low titer of approximately 1:lOO. However, unlabeled human chromogranins (pheochromocytoma chromaffin vesicle lysate) do not cross-react in the bovine chromogranin A RIA, even at 10,000-fold mass excess (Table I)   FIG. 13. Electron microscopy of the crude synaptosomal pellet after differential centrifugation of brain (frontal cortex) homogenate. Typical synaptosomal elements are identified, including the presynaptic cell of the synaptosome (Sy), synaptic vesicles (SV), and the postsynaptic density (PSD). The magnification is 10,000 diameters. dulla (Table III), immunoreactive chromogranin A was localized along with catecholamines to the soluble phase of chromaffin vesicles, where it accounted for a major portion (46 & 2%) of the soluble protein. By contrast, dopamine p-hydroxylase, another chromaffin vesicle marker (2,4,5,15) is distributed between the membrane phase and the soluble phase of the vesicles, as has been found by others (4,5). This comparatively complete release of chromogranin A into the soluble phase may be an advantage to the molecule's status Gel filtration of brain (frontal cortex) chromogranin immunoreactivity. The column, elution buffer, and conditions were the same as those in Figs. 2 and 11. Eluted fractions were tested for chromogranin by incubating 200 pl from each fraction in the RIA. The chromogranin immunoreactive peak eluted consistently later than the peak for purified '251-chromogranin A, suggesting an effective hydrodynamic (Stokes) radius of 54 A for brain chromogranin. VO = void volume; V, = total internal volume; '=I-chromogranin A = elution position for purified 1251-chromogranin A.
as an indicator of exocytotic release of protein along with neurotransmitter from the vesicles; that is, unlike dopamine @-hydroxylase, a major fraction of chromogranin does not remain behind, bound to the vesicle membrane, during discharge of vesicle contents to the extracellular space.
Chromogranin was present in the nervous system, although it apparently differed from adrenal medullary chromogranin in several fashions. First, there was far less chromogranin immunoreactivity in nervous tissue than in the adrenal medulla (1000-fold less in frontal cortex; 6000-fold less in sympathetic nerve). Second, the regional distribution of immunoreactive chromogranin in brain (maximal in neocortex, minimal in cerebellum, medulla oblongata, and spinal cord) is quite different from the regional distribution of norepinephrine (37), suggesting that chromogranin in brain may have a function independent of catecholamines. This dissociation is reinforced by the subcellular finding that chromogranin immunoreactivity is most prominent in the brain cell cytosol (Table V), rather than the synaptosomes, the usual storage site for neurotransmitters, including catecholamines (26). Finally, the molecular size of immunoreactive chromogranin, estimated by g ! l filtration (Figs. 11 and 14), yas smaller in brain (at 54 A) than in adrenal medulla (at 80 A). All of these observations suggest that chromogranin in brain may have a very different role from adrenal chromogranin, although no function has yet been postulated for brain chromogranin.
Chromogranin A in Also of note, substantialamounts of chromogranin were detected in the pituitary gland and the pineal gland ( Table  4). In the pituitary gland, there was 30-fold less chromogranin than that in the adrenal medulla, but 30-fold more than that in any other brain region. This further suggests a distribution of chromogranin independent of catecholamines and opens the possibility that chromogranin may be associated with polypeptide hormone producing tissues other than the adrenal medulla alone, as may also be the case in the parathyroid gland (38).
Easily detectable chromogranin in serum (Fig. 12, Table  IV) provides a means of studying chromogranin's release and significance as a potential sympathoadrenal index molecule in intact animals via this accessible tissue.
In summary, we have used a chromogranin RIA, coupled with electrophoretic, amino acid, and peptide studies, to elucidate several structurally and immunologically related chromogranins. Application of the RIA to the adrenal medulla revealed a soluble phase, vesicular localization of chromogranin along with catecholamines. Analysis of brain, however, revealed an unusual chromogranin distribution (regional and subcellular) and molecular size. Measurement of chromogranin in serum may provide a tool for assessing chromogranin release in vivo and hence for investigation of exocytotic sympathoadrenal catecholamine release.

D T O'Connor and R P Frigon
and nervous tissue elucidated by radioimmunoassay.
Multiple size forms, subcellular storage, and regional distribution in chromaffin Chromogranin A, the major catecholamine storage vesicle soluble protein.