THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1988 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 263, No. 17, , Issue of June 15, pp. 8371-8379,1988 Printed in U.S.A.

(Received for publication, January 15, 1988)

Betty A. Eipper, Victor May, and Karen M. Braas From the Department of Neuroscience, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205

Recent investigations have shown that the heart atrium is an endocrine tissue. In the present studies, high levels of peptidylglycine a-amidating monooxy- genase (PAM), which catalyzes the formation of bioac- tive a-amidated peptides from their glycine-extended precursors, have been found in particulate fractions from bovine and rat heart atrium; only low levels of PAM activity were present in soluble fractions. Cor- responding fractions from the ventricles contained 20- fold less activity. Immunocytochemical studies dem- onstrated that PAM was localized primarily to atrial cardiocytes, with a distribution resembling that of atriopeptin. Following differential centrifugation of rat atrial homogenates, most of the PAM activity was associated with crude granule fractions, with lesser amounts of activity associated with crude microsomal fractions. Upon further subcellular fractionation, PAM activity in the rat atrium was found primarily with immunoactive atriopeptin in fractions enriched in secretory granules. Following sodium dodecyl sul- fate-polyacrylamide gel electrophoresis, antisera to purified bovine pituitary PAM identified a 113,000- dalton protein in bovine atrial microsomes and secre- tory granules; the protein predicted from the sequence of the cDNA encoding bovine pituitary PAM is of sim- ilar size (Eipper, B. A., Park, L. p., Dickerson, I. M., Keutmann, H. T., Thiele, E. A., Rodriguez, H., Scho- field, P. R., and Mains, R. E. (1987) Mol. Endocrinol. 1, 777-790). Northern blot analysis using cDNA probes encoding bovine pituitary PAM demonstrated higher levels of PAM mRNA in heart atrium than in anterior pituitary. Rat heart contains PAM mRNA species of 3.6 and 3.8 kilobases, the smaller mRNA species corresponding in size to the PAM mRNA ex- pressed in rat anterior pituitary.

A pituitary enzyme responsible for the post-translational generation of a-amidated peptides from precursors containing a carboxyl-terminal glycine residue has recently been purified and cloned (1-4). Bovine pituitary peptidylglycine a-amidat- ing monooxygenase (PAM)’ is dependent on copper, ascor- bate, and molecular oxygen (3,4). Two forms of PAM differing in apparent molecular weight (PAM-A, 54,000; PAM-B,

* This work was supported by National Institute on Drug Abuse Grants DA-00266 and DA-00098 and by National Institutes of Health Grant DK-32949. 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.

The abbreviations used are: PAM, peptidylglycine a-amidating monooxygenase; ANP, atrial natriuretic peptide, atriopeptin; TES, N-tris[hydroxymethyl]methyl-2-aminoethanesulfonic acid; MOPS, 3-(N-morpholino)propanesulfonic acid; SDS, sodium dodecyl sulfate; kb, kilobase(s).

38,000) were purified from the bovine neurointermediate pi- tuitary (3). Although PAM was purified from the soluble fraction of the bovine neurointermediate pituitary, analysis of the cDNA encoding PAM revealed the presence of a hydro- phobic putative membrane spanning domain near the car- boxyl terminus of the initial transcript (1). Subsequent analy- sis of rat anterior and neurointermediate pituitary revealed a tissue-specific distribution of PAM activity among soluble and membrane-associated forms (5 ) . Analysis of the cDNA encoding PAM also indicated the presence of an amino- terminal signal peptide and 10 pairs of basic amino acids that may undergo endoproteolytic cleavage to generate the soluble forms of PAM (1).

In our earlier studies on the distribution of PAM activity in various rat tissues, little soluble PAM activity was observed in the heart (6). In the course of subsequent studies on soluble and membrane-associated PAM activity and the distribution of PAM mRNA in different rat tissues, the heart was found to contain extremely high levels of PAM.

It has recently been appreciated that the heart serves as an endocrine organ (7-9). Secretory granules resembling those in polypeptide hormone producing cells were described in cardiocytes of the mammalian heart atrium over 30 years ago and subsequently shown to contain the precursor to atrial natriuretic peptide (ANP) or atriopeptin, a potent diuretic and natriuretic factor (9-18). Generation of the major storage form of rodent atriopeptin requires only the removal of the signal peptide from the amino-terminal end of prepro-ANP and the removal of 2 arginine residues from the carboxyl- terminal end of pro-ANP (12, 15, 19, 20). Signal peptidase is commonly found in all tissues (21), and high levels of a secretory granule-associated carboxypeptidase with the nec- essary specificity, carboxypeptidase E, have been identified in cardiac atrium (22). The localization of the endoproteolytic cleavage enzyme that releases bioactive ANP(1-28) from its prohormone is not yet clear (9, 12, 19, 23).

PAM is thought to be uniquely associated with neuroen- docrine tissues that actively produce a-amidated bioactive peptides. Although nearly one-half of the bioactive peptides currently identified terminate with a carboxyl-terminal a- amide, neither ANP(1-28) nor the major amino-terminal fragment of pro-ANP is a-amidated. The high levels of PAM in the heart, however, have led us to begin studies aimed at examining the functional role of the enzyme in cardiac tissue. The studies described here were conducted to identify soluble and particulate forms of PAM in the heart and to define their cellular and subcellular localization using immunocytochem- ical and subcellular fractionation techniques.

MATERIALS AND METHODS

RNA Isolation and Northern Blot Analysis-Total RNA was iso- lated by the guanidine thiocyanate/CsCl procedure (24) or the acid guanidine thiocyanate/phenol/chloroform procedure (25). RNA was

8371

8372 Peptide Amidation Activity in Heart TABLE I

PAM activity in the heart Tissues were homogenized in 20 mM NaTES, pH 7.4, containing

10 mM mannitol and separated into soluble and particulate fractions as described (5). Particulate fractions were washed three times and solubilized by incubation with buffer containing 1% Triton X-100 for 30 min at 4 "C. Levels of PAM activity reported were determined in the presence of optimal levels of CuSO.. Data represent the average of duplicate determinations from two separate preparations; ranges given reflect the difference between the two samples.

PAM activity

Soluble Particulate ' Partic'- Tissue

late pmolf#! proteinfh

Rat atrium 0.20 f 0.08 2.04 f 0.68 70 Ventricle <0.003 0.04 & 0.01

Bovine right atrium 0.02 f 0.01 0.98 f 0.14 90 Left atrium 0.02 f 0.01 0.68 & 0.02 90 Ventricle c0.002 0.03 f 0.02

fractionated on 1% agarose gels containing 2.2 M formaldehyde, 20 mM MOPS, 5 mM sodium acetate, 1 mM EDTA, pH 7.0, and trans- ferred to Nytran (Schleicher & Schuell) by capillary action; additional 5-pg aliquots were fractionated and visualized with acridine orange to monitor the quality of the RNA. Filters were hybridized with nick- translated probes as described (1); prehybridization and hybridization were carried out at 42 "C in 50% deionized formamide, 5 X SSC (20 X SSC is 3.0 M NaCI, 0.3 M sodium citrate, pH 7.0), 4 X Denhardt's solution (50 X Denhardt's solution is 1% Ficoll-400, 1% polyvinyl- pyrrolidone, 1% bovine serum albumin), 0.1 mg/ml sonicated carrier herring sperm DNA, 20 mM sodium phosphate, pH 6.5, 0.1% SDS. Three EcoRI fragments of bovine PAM cDNA (1) were used as probes (1 X lo6 cpm/ml): 0.8 kb (bpl-781), 0.7 kb (bp782-1503). and 2.2 kb (bp1504-3724). DNA fragments (100 ng) recovered from agarose gels were radiolabeled by nick translation (Du Pont-New England Nu- clear) with 100 pCi of [a-'*P]dCTP (3000 Ci/mmol; Amersham Corp.) to a specific activity of 0.5-1 X lo9 cpmlpg DNA. For molecular weight determination rRNA was visualized with acridine orange, and an RNA ladder (Bethesda Research Laboratories) fractionated in an adjacent lane was transferred to nitrocellulose and visualized by hybridization to nick-translated wild type X DNA. Autoradiograms were densitized using the LOATS RAS-1000 Image Analysis Pro- gram.

Subcellular Fractionation-The subcellular fractionation scheme adopted was modified from that of deBold and Bencosme (10) and deBold (11). Tissue was collected into 0.25 M sucrose, 20 mM NaTES, pH 7.0, blotted, weighed, and minced. The minced tissue was trans- ferred to 10 volumes of 0.25 M sucrose, 0.6 M KCI, 10 m M NaTES, pH 7.0, containing 0.3 mg/ml phenylmethylsulfonyl fluoride, 2 pg/ml leupeptin, 16 pg/ml benzamidine and dispersed using a Polytron (PT10 probe, setting 6 for 12 s), followed by 5 strokes with a motor- driven Potter-Elvehjem homogenizer. The homogenate was centri- fuged at 2,000 X gmaX for 10 min to yield a pellet containing nuclei, unbroken cells, and connective tissue (crude nuclear pellet). Centri- fugation of the supernatant at the same speed for another 10 min yielded a crude mitochondrial pellet. Centrifugation of the superna- tant a t 32,000 X gmax for 15 min yielded a crude granular pellet. A crude microsomal pellet was obtained by centrifugation of the super- natant at 145,000 X g,. for 60 min with the remaining supernatant constituting the soluble fraction. The crude granular and microsomal pellets were resuspended by homogenization in 0.25 M sucrose, 0.6 M KC1.10 mM NaTES, pH 7.0. Samples containing up to approximately 6 mg of protein were layered onto gradients consisting of 0.5 ml of 2.0 M sucrose, 1.0 ml of 1.6 M sucrose, 1.0 ml of 1.17 M sucrose, 1.0 ml of 0.93 M sucrose, and 0.5 ml of 0.44 M sucrose, and the gradients were centrifuged in a Beckman SW 55-Ti rotor a t 50,000 rpm (304,000 X gmX) for 90 min; all sucrose solutions contained 0.6 M KCI, 10 mM NaTES, pH 7.0. Samples containing approximately 15 mg of protein were centrifuged in a Beckman SW 41 rotor a t 38,000 rpm (247,000 X g-) for 120 min; the volumes of the sucrose solutions were doubled. Fractions were collected from the top of the gradient and particulate fractions were washed by centrifugation following dilution with 0.25 M sucrose, 0.6 M KCl, 10 mM NaTES, pH 7.0, and resuspended by homogenization in the same buffer.

Enzyme Assays-PAM assays were performed as described previ-

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FIG. 1. Northern blot. A, atria ( A ) and ventricles ( V ) from 4 adult male rats were minced, and total RNA was extracted. Aliquots

Peptide Amidation Activity in Heart 8373 ously using 0.5 pM D-Tyr-Val-Gly, '2SI-labeled D-Tyr-Val-Gly, 0.5 mM ascorbate, and 10 p~ CuSO, in 110 mM NaTES, pH 8.5 (4); samples were assayed in duplicate a t each of several dilutions to establish assay linearity, and duplicates differed from the mean by less than 10%. Prior to assay, samples were solubilized by the addition of Triton X-100 to a final concentration of 1% (5). The reaction veloc- ities are initial velocities using a concentration of substrate a t least 10-fold below the K , of the enzyme for the substrate; less than 10% of the substrate was converted to product.

Immunoassays for ANP were performed to localize atrial secretory granules (7, 17, 26). Rabbit antiserum to ANP(99-126) (RAS8798, Peninsula Laboratories, Inc., Belmont, CA) was used essentially as described by Glembotski et al. (27). Synthetic human ANP(99-126) (Peninsula Laboratories, Inc.) was used as the assay standard (mid- point, 22 fmol/tube) and trace; the peptide was iodinated by the chloramine-T method and purified by gel filtration.

Assays for galactosyltransferase (28) localized Golgi complex mem- branes. Assays for fumarase (29) identified mitochondria. Assays for lactate dehydrogenase (30) localized cytosol. Protein concentrations were determined with the BCA protein assay reagent (Pierce Chem- ical Co.) using bovine serum albumin as standard.

Antisera to PAM-Four rabbit antisera were used in these studies. Two antisera, Ab35 and Ab36, were generated by injection of a mixture of purified bovine PAM-A and PAM-B (1). For some exper- iments these antisera were purified by ammonium sulfate precipita- tion followed by affinity purification using CH-Sepharose 4B to which a mixture of purified bovine PAM-A plus PAM-B had been linked (1). Two antisera, Ab45 and Ab46, were generated by injection of partially purified j3-galactosidaselPAM fusion protein produced by Escherichia coli CAG456 cells infected with phage XPAM-1 (1). To remove antibodies to @-galactosidase from the fusion protein antisera, ammonium sulfate-precipitated antibodies from 5 ml of serum were incubated for 4 h a t 4 'C with 1 ml of CH-Sepharose 4B resin to which 2.4 mg of E. coli j3-galactosidase (Sigma (2-5635) had been linked; the resin was removed, and the remaining solution was sub- jected to affinity purification on a PAM resin.

Immunocytochemistry-Bovine heart atria and ventricles, obtained approximately 30 min post-mortem, were cut into 1-mm slices, im- mersion-fixed in 4% paraformaldehyde for 2 h, washed overnight in 0.15 M sodium phosphate buffer, pH 7.4, containing 0.32 M sucrose, dehydrated, and embedded in paraffin. These conditions were optimal for preserving the antigenicity of PAM in similar studies on bovine pituitary tissue (31). Tissue sections (8 pm) were deparaffinized, rehydrated, and immunocytochemically stained using the following modification of the avidin-biotin-peroxidase complex technique (32- 34): 1) incubate with 0.10 M sodium phosphate, pH 7.4, 0.9% NaCl containing 0.1% Triton X-100 for 10 min; 2) block with 0.1% gelatin in 0.1 M sodium phosphate buffer, pH 7.4, for 15 min; 3) incubate with primary rabbit antiserum (1:5,000 to 1:10,000) for 48 h at 4 "C; 4) wash with 0.10 M sodium phosphate buffer, pH 7.4; 5) block; 6) incubate with biotinylated goat anti-rabbit IgG (Vector Laboratories, Burlingame, CA) (1:400) for 60 min; 7) wash; 8) block; 9) incubate with avidin-biotin-peroxidase complex (1:200) for 60 min; 10) wash; 11) incubate with peroxidase substrates diaminobenzidine (0.3 mg/ ml) and H202 (0.03%) in 0.05 M Tris-HCI, pH 7.6, for 10-30 min. Antisera to purified bovine pituitary PAM (Ab35) and to j3-galacto- sidase/PAM fusion protein (Ab46) were diluted in sterile 0.10 M sodium phosphate, pH 7.4, containing 0.1% gelatin for use in the immunocytochemical stain. As a control, tissue sections were also

of total RNA (20 pg) were fractionated on agarose-formaldehyde gels and transferred to Nytran. Following hybridization with the 0.7-kb cDNA probe, the blot was stripped and reprobed with the 0.8-kb cDNA probe followed by the 2.2-kb cDNA probe (data not shown). To allow visualization of the two species of mRNA in both atrium and ventricle, shorter exposures (20 h for 0.8-kb probe; 64 h for 0.7- kb probe) are shown in the first two panels and longer exposures (4 days for 0.8-kb probe, 8 days for 0.7-kb probe) in the third and fourth panels. Similar results were obtained in 4 separate experiments. B, total RNA was extracted from the anterior pituitaries (Ant), atria and a corresponding amount of ventricular tissue from 2 adult male rats, and aliquots containing 20 pg of total RNA were fractionated as above; the blot was hybridized with the 0.7-kb cDNA probe, and the autoradiogram shown was exposed for 42 h at -70 'C; the blot was stripped and reprobed with the 0.8-kb cDNA probe (data not shown) followed by the 2.2-kb cDNA probe (24 h at -70 "C). The positions of the molecular weight markers are indicated at the left; the large arrowhead marks the position of sample application.

FIG. 2. Immunocytochemical localization of PAM in the bo- vine heart. Paraffin tissue sections of bovine atrial (A) and ventric- ular ( B ) tissue were immunocytochemically stained with a 1:5000 dilution of antiserum to j3-galactosidase/PAM fusion protein (Ab461 as described under "Materials and Methods." The prominent punc- tate perinuclear staining in the atrial cardiocytes (A) was absent in the ventricular cells ( B ) . Staining in the left and right atria was similar (data not shown). Scale bur, 10 pm.

stained using normal rabbit (preimmune) serum to replace the pri- mary antiserum or omitting primary antibody, biotinylated goat anti- rabbit IgG, or avidin-biotin-peroxidase complex. Staining with a working dilution of Ab35 was previously shown to be absorbed upon preincubation with 1 pg/ml purified PAM enzyme (31).

Western Blot Analysis-Samples were fractionated on slab gels containing 10% acrylamide and 0.25% N,N'-methylenebisacrylamide using the buffer system of Laemmli (35). Proteins were electropho- retically transferred to nitrocellulose (Bio-Rad) in 25 mM Tris, 192 mM glycine, pH 8.3, containing 20% methanol (36). Proteins were visualized on the nitrocellulose with Ponceau S (Sigma); molecular weights were estimated by comparison with standard proteins (Rain- bow markers; Amersham Corp.). Nitrocellulose strips were immuno- stained using reagents from Bio-Rad as follows: 1) block for 1 h at room temperature with 500 mM NaC1, 20 mM Tris-HCI, pH 7.5 (TBS), containing 0.05% Tween 20,10 mg/ml bovine serum albumin; 2) wash for 2 X 5 min with TBS containing Tween 20; 3) incubate overnight at 4 "C with primary rabbit antiserum diluted in blocking solution; 4) wash as in step 2; 5) incubate for 1 h at room temperature with alkaline phosphatase conjugated to affinity purified goat anti- rabbit immunoglobulin (1:3000 in blocking solution); 6) wash as in step 2; 7) wash 1 X 5 min with TBS; 8) incubate a t room temperature for 1-30 min with alkaline phosphatase substrates 5-bromo-4-chloro-

8374 Peptide Amidation Activity in Heart

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Atrium

Ventricle

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and a similar weight of ventricular tissue were homogenized and subjected to differential centrifugation. Particulate fractions were resuspended by homogenization in 0.25 M sucrose, 0.6 M KCl, 10 mM NaTES, pH 7.0. Aliquots were assayed for PAM activity, fumarase activity, galactosyltransferase activity, ANP immunoactivity, and protein. Note the 25-fold expanded scale used for the PAM activity in the ventricular samples. Fumarase specific activity was highest in the crude nuclear and mitochondrial fractions; ANP immunoactivity was highest in the crude granular fraction; galactosyltransferase activity was highest in the crude granular and microsomal fractions. Similar distributions were observed in 3 independent experiments.

3-indolyl phosphate p-toluidine salt (0.3 mg/ml) and p-nitro blue tetrazolium chloride (0.15 mg/ml) in 0.1 M NaHC03, 1 mM MgC12, pH 9.8; 9) stop reaction by washing in water.

RESULTS

Identification of PAM in Rat and Bovine Heart-With the demonstration of a tissue-specific distribution of pituitary PAM activity between soluble and membrane-associated forms (5) and the availability of cDNA probes for PAM (l) , a reexamination of the levels of PAM in various tissues in the adult male rat was undertaken. High levels of PAM activity and PAM mRNA were shown to be present in the heart. Homogenates of atrium and ventricle were separated into crude soluble and particulate fractions; the particulate frac- tions were washed, solubilized by incubation with detergent, and soluble and particulate fractions were assayed for PAM activity (Table I). High levels of PAM activity were observed in the particulate fraction from rat atrium, but not in corre- sponding fractions from rat ventricle. Approximately 70% of the total PAM activity in the rat atrium was membrane associated, and the specific activity of PAM in the particulate fraction was 10-fold greater than in the soluble fraction. The specific activity of PAM in the particulate fraction from rat atrium exceeded the specific activity of PAM in corresponding fractions from rat anterior or neurointermediate pituitary by 4- and ZO-fold, respectively (5).

A similar enrichment of PAM activity in particulate frac- tions was observed in bovine atrium (Table I). Both right and

left atria contained high levels of membrane-associated PAM activity, with a slightly higher specific activity in the right atrium. Approximately 90% of the total PAM activity in both atria was membrane associated. The specific activity of PAM in the particulate fraction from bovine atrium was 30-fold greater than in the soluble fraction. The specific activity of PAM in particulate fractions from the ventricles was at least 20-fold lower than in the corresponding atrial fractions.

The presence of PAM in heart tissue was also demonstrated using probes derived from the cDNA encoding bovine neu- rointermediate pituitary PAM (1). Total RNA was extracted from rat heart atrium and ventricle, fractionated on denatur- ing agarose gels, and transferred to Nytran. Multiple probes spanning the entire bovine PAM cDNA were used to visualize PAM mRNA. Two size forms of PAM mRNA were visualized with all three cDNA probes (Fig. 1A); the amount of the 3.8 & 0.1-kb form was consistently slightly greater (1.3-fold) than the amount of the 3.6 & 0.1-kb form. High levels of PAM mRNA were detected in both the left and right atria (data not shown). The amount of PAM mRNA in the ventricles was much less than the amount of PAM mRNA in the atrium, but the same two size forms were present. Consistent with the levels of PAM activity measured, rat atrium contained approximately 6-fold higher levels of PAM mRNA than the rat anterior pituitary (Fig. 1B); PAM mRNA found in the rat anterior pituitary corresponded in size to the smaller form observed in the atrium. Bovine atrial tissue also contained

Peptide Amidation Activity in Heart 8375

SUCROSE G R A D I E N T

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R A T A T R I A L M I C R O S O M E S FIG. 4. Further fractionation of rat atrial homogenates. The crude granular and microsomal fractions

from the differential centrifugation of rat atria described in Fig. 3 were resuspended and fractionated on discontinuous sucrose gradients as shown. All fractions were assayed for PAM activity, protein, and galactosyl- transferase activity (Gal Tr); fractions from the crude granule sample were also assayed for fumarase activity and ANP immunoactivity (ZR-ANP). Data plotted are percent of total recovered activity in each fraction. For the crude granule fraction, the washed fractions from the gradient contained 70% of the protein, 68% of the PAM activity, 144% of the ANP immunoactivity, 89% of the galactosyltransferase activity, and 81% of the fumarase activity applied to the gradient. For the crude microsomal fraction, the washed fractions from the gradient contained 52% of the protein, 84% of the PAM activity, and 75% of the galactosyltransferase activity applied to the gradient. Similar results were obtained in 2 separate experiments.

high levels of PAM mRNA (3.8 k 0.1 kb), with very little PAM mRNA present in ventricular tissue (data not shown).

Immunocytochemical Localization of PAM in Bovine Heart-Immunocytochemical studies were performed using antisera to purified bovine pituitary PAM and B-galactosid- ase/PAM fusion protein produced by bacteria infected with phage XPAM-1 (Fig. 2). A dark punctate staining pattern consistent with the characteristics of secretory granules was observed in atrial cardiocytes stained with antisera to the fusion protein (Fig. 2A) . The staining was predominantly perinuclear, although there was cell to cell variability in staining intensity and fraction of cell volume stained. Staining was absent over nuclei. Less staining was observed in the more distal portions of the cell. The staining pattern produced by the antiserum to purified bovine pituitary PAM was similar to that shown, although the staining was more diffuse. The

staining pattern for PAM resembled that reported for ANP (14, 17, 26). In contrast to atrial tissue, bovine ventricular cardiocytes were nearly devoid of stain except for a few isolated cells that occassionally stained for PAM (Fig. 2B) . Staining was absent when the primary antibody, secondary antibody, or avidin-biotin-peroxidase complex was omitted from the staining protocol or when normal rabbit serum replaced primary antibody (data not shown). The absence of staining in the ventricle confirmed the tissue specificity of the immunocytochemical reaction. These results were con- sistent with the low levels of PAM activity and PAM mRNA in ventricular tissue.

Subcellular Fractionation of Atrial and Ventricular Homog- enates-A modification of the fractionation scheme of deBold and Bencosme (10) was used to separate homogenates of rat atrium and ventricle into crude mitochondrial, granular, mi-

8376 Peptide Amidation Activity in Heart

Rat Ventricle: Granules vs. Microsomes

Granule8

Microuome8

Soluble 0.44/0.93 0.93/1.2 1.2/1.6 1.6/2.0 FIG. 5. Further fractionation of rat ventricular homogenates. Rat ventricular tissue was processed as

described for atrial tissue. The crude granular and microsomal fractions prepared by differential centrifugation were resuspended and fractionated on discontinuous sucrose gradients. Fractions were assayed for PAM activity, protein, fumarase activity, and galactosyltransferase activity; 68% of the fumarase activity from the crude granule fraction was recovered at the interface of the 0.93 and 1.2 M sucrose; for both samples, approximately 80% of the galactosyltransferase activity was recovered at the interface of the 0.44 and 0.93 M sucrose solutions. A similar distribution of PAM activity was observed in 2 independent experiments.

crosomal, and soluble fractions (Fig. 3). Marker enzyme as- says verified the identification of the major constituents en- riched in each fraction. In the atrium, 64 f 5% of the PAM activity from the post-nuclear supernatant was contained in the crude secretory granule fraction and 18 f 2% in the crude microsomal fraction. In the ventricle, the specific activity was many-fold lower than in the atrium and the subcellular dis- tribution differed significantly, with 18 f 5% of the PAM activity from the post-nuclear supernatant contained in the crude secretory granule fraction and 42 f 2% in the crude microsomal fraction.

The crude granular and microsomal fractions from the rat atrium were further fractionated on discontinuous sucrose gradients (Fig. 4). The PAM activity from the crude granular fraction was co-localized with ANP immunoactivity at the interface of the 1.2 and 1.6 M sucrose solutions (Fig. 4, upper); the specific activity of PAM in this fraction was 6.9 pmol/pg protein/h, more than 3-fold enriched over the crude granular fraction. The parallel distribution of PAM activity and im- munoactive ANP is consistent with the suggestion that PAM is localized in secretory granules along with ANP. The mito- chondrial marker enzyme, fumarase, and total protein were primarily localized at the interface of the 0.93 and 1.2 M sucrose solutions. The PAM activity in the crude microsomal

fraction was co-localized with the Golgi complex marker en- zyme, galactosyltransferase, at the interface of the 0.44 and 0.93 M sucrose solutions (Fig. 4, lower); the specific activity of PAM in this fraction was 1.4 pmollpg protein/h, not as high as in the atrial granule fraction.

Similar studies were carried out to determine the subcellular localization of the low levels of PAM activity in the rat ventricle. The specific activity of PAM in the crude microso- mal fraction from the ventricle exceeded the specific activity of PAM in the crude granule fraction (Fig. 3). Fumarase and galactosyltransferase activity fractionated in a similar fashion in atrial and ventricular homogenates. In both the crude granule and crude microsomal fractions from the ventricle, much of the PAM activity fractionated at the interface of the 0.44 and 0.93 M sucrose solutions, along with the Golgi marker enzyme (Fig. 5); the crude granule fraction from ventricles did not contain PAM activity fractionating with the density expected of secretory granules. Thus there are both quanti- tative and qualitative differences in the properties of the PAM activity in the atrium and the ventricles.

Similar studies were carried out to localize PAM in bovine heart. Upon subcellular fractionation of bovine atrium, ap- proximately equivalent amounts of PAM activity were found in the crude granular and microsomal fractions (data not

Peptide Amidation Activity in Heart 8377

shown). The relative lack of concentration of PAM activity in the crude granule fraction from bovine atrium is consistent with the relative paucity of secretory granules in bovine versus rat atrium (7). PAM activity in the crude subcellular fractions from bovine ventricle was barely detectable, with the specific activity at least 40-fold lower than in the corresponding atrial fractions (data not shown).

To compare the subcellular localization of PAM activity in bovine and rat heart, crude granular and microsomal fractions from the bovine atrium were further fractionated on discon- tinuous sucrose gradients (Fig. 6). As observed for rat atrium, most of the PAM activity in the crude granular fraction from bovine atrium was found along with immunoactive ANP at the interface of the 1.2 and 1.6 M sucrose solutions. The mitchondrial marker enzyme was concentrated at the inter- face of the 0.93 and 1.2 M sucrose solutions while the Golgi complex marker enzyme was concentrated at the interface of the 0.44 and 0.93 M sucrose solutions. Substantial amounts of the PAM activity in the crude microsomal fraction from the bovine atrium were found along with the Golgi marker enzyme at the interface of the 0.44 and 0.93 M sucrose solutions, although more PAM activity was found at the interface of the 0.93 and 1.2 M sucrose solutions (Fig. 6, lower). Thus the subcellular distribution of PAM activity is quite similar in rat and bovine heart atrium.

Further Characterization of Atrial PAM-When fractions containing bovine or rat atrial granules or microsomes were

subjected to freezing and thawing and separated into soluble and particulate fractions by centrifugation, over 90% of the PAM activity was recovered in the particulate fraction. The basic properties of the enzyme activity associated with heart membranes are similar to those of soluble PAM purified from bovine neurointermediate pituitary (3, 4) and membrane- associated PAM from rat pituitary (5). Enzyme solubilized from rat atrial membranes was stimulated over 10-fold by the addition of micromolar concentrations of CuS04 and over 100-fold by the addition of 1 mM ascorbate. The enzyme exhibited an alkaline pH optimum and a K,,, of approximately 5 pM for D-Tyr-Val-Gly. Approximately 80% of the enzyme activity in atrial granule membranes was solubilized upon incubation with 1% Triton X-100 at 4 "C for 1 h; approxi- mately one-third of the solubilized enzyme bound to columns of wheat germ agglutinin or concanavalin A and was eluted with the appropriate carbohydrate.

To determine the apparent molecular weight of the PAM protein present in the granular and microsomal fractions, samples were fractionated by SDS-polyacrylamide gel electro- phoresis; crude granular and microsomal fractions as well as discontinuous sucrose gradient-purified granular and micro- somal fractions were analyzed. The proteins were transferred to nitrocellulose, and the PAM protein present was visualized using antisera to purified bovine neurointermediate pituitary PAM or to the 8-galactosidaselPAM fusion protein (Fig. 7). Equal amounts of atrial and ventricular protein from corre-

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B . O V I N E A T R I A L M I C R O S O M E S FIG. 6. Further fractionation of bovine atrial homogenates. Approximately 3 g of bovine atrial tissue was

minced, homogenized, and subjected to differential centrifugation as described in Fig. 3. The crude granular and microsomal pellets were resuspended and fractionated on discontinuous sucrose gradients as described in Fig. 4. AI1 fractions were assayed for PAM activity, protein, and galactosyltransferase (Gal Tr) activity; fractions from the crude granule gradient were also assayed for fumarase activity and ANP immunoactivity (ZR-ANP). A similar distribution of PAM activity was observed in 2 independent experiments.

8378 Peptide Amidation Activity in Heart

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FIG. 7. Western blot analysis of bovine heart. Samples from bovine atrium and ventricle were fractionated by SDS-polyacrylamide gel electrophoresis and visualized with antisera to purified PAM ( A ) or 6-galactosidaselPAM fusion protein ( B ) . A, for each group of samples, equal amounts of atrial and ventricular protein were ana- lyzed; the amount of PAM activity in each sample is given in paren- thesis. Crude manules from ventricle (0.3 Dmol/h: lane 1) and atrium

sponding fractions were analyzed; the ventricular samples contained low levels of PAM activity and served as a control for the specificity of the immunochemical procedure.

In both microsomal and granular fractions from the bovine atrium, affinity purified antisera to purified bovine neuroin- termediate pituitary PAM visualized a protein with an appar- ent molecular weight of 113,000 f 4,000 (Fig. 7 A ) . As expected due to the low levels of PAM activity present, a corresponding band was not found when equivalent amounts of protein from ventricular fractions were analyzed. When antisera to the B- galactosidase/PAM fusion protein were used to analyze sam- ples of bovine atrial granules, a protein of the same apparent molecular weight was observed (Fig. 7 B ) . Based on the se- quence of the PAM cDNA analyzed, removal of the pre- and pro-segments from the intact PAM precursor would yield a protein with a molecular weight of 104,926 (1); use of either of the two potential sites for addition of N-linked oligosac- charides could increase the molecular weight to the range observed in the Western blots. Purified bovine neurointer- mediate pituitary PAM-B analyzed at the same time yielded a single band with an apparent molecular weight of 37,000. The greater staining intensity observed with purified bovine neurointermediate pituitary PAM-B could reflect the exist- ence of tissue-specific forms of PAM, the accessibility of the antigenic determinants, or different intrinsic activities for the soluble and particulate forms.

The 113,000-dalton species was the only one whose presence correlated with the amount of PAM activity in the sample applied to the gel. When samples from bovine atrial granule or microsomal preparations differing almost 10-fold in specific activity were analyzed, the staining intensity of the 113,000- dalton band corresponded to the total amount of PAM activity applied to the gel (Fig. 7A, lanes 5-7 and 8-10). Thus, both microsomal and granular fractions from bovine atrium con- tain a membrane-associated form of PAM that is similar in size to the protein predicted by analysis of the cDNA.

DISCUSSION

The heart atrium was found to contain unexpectedly high levels of PAM activity, mRNA, and protein. Immunocyto- chemical studies localized PAM to atrial cardiocytes. Al- though the PAM activity in atrium is primarily particulate, its enzymatic properties are similar to those of purified soluble bovine neurointermediate pituitary PAM (3, 4). However, atrial PAM has an apparent molecular weight of 113,000 on Western blots, while soluble bovine neurointermediate pitui-

(0.4 pmol/h; lune 3) and atrium (15 pmol/h; lane 4), 23 pg of protein; sucrose gradient-purified granules (1.2/1.6 M sucrose interface) from ventricle (0.05 pmol/h; lane 5 ) and atrium (7.5 pmol/h; lane 6 ) , 11 pg of protein; separate preparation of purified atrial granules (15 pmol/ h; lane 7), 2.6 pg of protein; sucrose gradient-purified microsomal fraction (0.44/0.93 M sucrose interface) from ventricle (0.3 pmol/h; lane 8) and atrium (7.5 pmol/h; lane 9), 17 pg of protein; separate preparation of purified atrial microsomes (15 pmol/h; lane lo), 1 pg of protein; purified bovine neurointermediate pituitary PAM-B (15 pmol/h; lane I I ) , approximately 0.015 pg of PAM protein plus 4 pg of carrier bovine serum albumin. Affinity purified Ab36 was used at a dilution of 1:lOO. B, in a separate analysis, the crude atrial granule fraction shown in lune 2 of A was again visualized with Ab36. Lanes 2-4 were visualized with affinity purified Ab46; lanes 2 and 3, sucrose gradient-purified atrial granule and microsomal fractions (7.5 pmol/ h), 11 and 17 pg of protein, respectively; lane 4, purified bovine neurointermediate pituitary PAM-B as in lane 11 of A. The band at 180 kDal in lanes 2 and 3 coincides with a major protein component in the sample and is observed in the corresponding ventricular sam- ples, which lack significant amounts of PAM activity. Large arrow- heads mark the interface of the stacking and running gels; locations

(15 pmol/h; la& 2), 20 pg of protein; crude microsomes from ventricle of standard proteins are indicated. . . , ,

Peptide Amidation Activity in Heart 8379

tary PAM has an apparent molecular weight of 37,000 (3, 4). These results suggest that atrial PAM, which is similar in size to the protein predicted by the cloned PAM cDNA (l), is likely to be anchored to the membrane through its hydropho- bic putative membrane spanning domain (PAM(864-887)). It is not clear from the studies carried out here whether the putative pro-peptide (PAM(21-30)) has been cleaved from the precursor. That both PAM and ANP are stored in atrial secretory granules in a high molecular weight relatively un- processed form suggests that similar endoproteases may be involved in the cleavage of both the enzyme and the peptide precursor.

The cellular and subcellular distributions of PAM in the heart resemble those of ANP. Levels of PAM are much higher in the atrium than in the ventricle and immunocytochemically stained atrial cardiocytes exhibit punctate perinuclear stain- ing for PAM. In the atrium, PAM is primarily associated with secretory granules, although significant amounts also frac- tionate with a Golgi complex marker enzyme. In the rat ventricle, the small amount of PAM activity present is pri- marily microsomal, consistent with the relative lack of secre- tory granules in the ventricle (7,37-39). Rat atrial cardiocytes contain more secretory granules than bovine atrial cardiocytes (7, 37), and a greater fraction of the PAM activity in rat atrium is associated with secretory granules while the PAM activity in bovine atrium is more associated with microsomes.

Two forms of PAM mRNA (3.6 and 3.8 kb) were consist- ently observed in Northern blots of rat atrial RNA, with the lower molecular weight form corresponding to the PAM mRNA in rat anterior pituitary. The difference between the two mRNA forms has not yet been investigated, but cDNAs differing by the presence of a 54-base pair segment were identified in a bovine intermediate pituitary library (1). Rat atrium contains approximately 6-fold higher levels of PAM activity and mRNA than rat anterior pituitary.

The presence of very high levels of PAM in heart atrium was unanticipated; Sakata et al. (40) found levels of soluble PAM activity in rat atrium that were 40% as high as in the pituitary. Heart atrium contains high levels of other peptide processing enzymes including a carboxypeptidase B-like en- zyme (carboxypeptidase E or enkephalin convertase) (22) and a dipeptidyl carboxyhydrolase distinct from angiotensin con- verting enzyme (41). Since atrial natriuretic peptide, the primary secretory product of these cells, is not an a-amidated peptide, the role of PAM in heart atrium is not known. The presence of such elevated levels of PAM in the atrial granules, however, suggests that other peptide precursors may serve as substrates in the production of bioactive peptides or that the membrane-associated PAM stored in atrial secretory granules may play a role when exposed on the cell surface.

Acknowledgments-We thank Cris Green, Ed Cullen, Elizabeth Thiele, and Dick Mains for helping to perform and write about the experiments and Dave Lynch for a preprint of his work.

REFERENCES 1. Eipper, B. A., Park, L. P., Dickerson, I. M., Keutmann, H. T., Thiele, E.

A,, Rodriguez, H., Schofield, P. R., and Mains, R. E. (1987) Mol. Endo- crinol. 1,777-790

2. Park, L. P., Thiele, E. A., Dickerson, I. M., Mains, R. E., and Eipper, B. A.

eds) pp. 133-140, Norhaven Bogtrykkeri, Copenhagen (1987) in Highlights on Endocrinology (Christiansen, C., and Riis, B. J.,

3. Murthv. A. S. N.. Mains. R. E.. and EiDDer. B. A. (1986) J. Biol. Chem. 261;'1815-1822

4. Murthy, A. S. N., Keutmann, H. T., and Eipper, B. A. (1987) Mol. Endo-

5. Mains, R. E., May, V., Cullen, E. I., and Eipper, B. A. (1988) in Molecular crinol. 1 , 290-299

Biology of Brain and Endocrine Peptidergic Systems (MeKerns, K. W., and Chretien, M., eds) Plenum Publishing Corp. New York, in press

6. Eipper, B. A., Myers, A. C., and Mains, R. E. (1985) Endocrinology 1 1 6 , 2497-2504

7. deBold, A. J. (1985) Science 230,767-770 8. MacGregor, G. A., and Sagnella, G. A. (1987) Eur. Heart J. 8 , Suppl. B,

.. I . ,

I l l - l l f i 9. Needleman, P., and Greenwald, J. E. (1986) New Engl. J. Med. 314,828-

"* "-

10. deBold, A. J., and Bencosme, S. A. (1973) Cardwuusc. Res. 7,351-363 11. deBold, A. J. (1982) Can. J. Physiol. Phurmol . 60,324-330 12. Michener, M. L., Giene, J. K., Seetharam, R., Fok, K. F., Olins, P. O., Mai,

13. Schwartz, D., Kataube, N. C., and Needleman, P. (1986) Fed. Proc. 4 6 , M. S., and Needleman, P. (1987) Mol. PhurmoL 30,552-557

14. Zisfein, J. B., Matsueda, G. R., Fallon, J. T., Bloch, K. D., Seidman, C. E., 2361-2365

Seidman, J. G., Homcy, C. J., and Graham, R. M. (1986) J. Mol. Cell. Cardlol. 18,917-929

15. Kangawa, K., Tawaragi, Y., Oikawa, S., Mizuno, A., Sakuragawa, Y., Nakazato, H., Fukuda, A,, Minamino, N., and Matsuo, H. (1984) Nature 312,152-155

16. Miyata, A., Kangawa, K., Toshimori, T., Hatoh, T., and Matauo, H. (1985)

17. Rinne, A., Vuolteenaho, O., Jarvinen, M., Dorn, A., and Arjamaa, 0. (1986) Biochem. Biophys. Res. Commun. 129,248-255

18. Thibault, G., Lazure, C., Schiffrin, E. L., Gutkowska, J., Chartier, L., Acta Histochem. 80,19-28

Garcia, R., Seidah, N. G., Chretien, M., Genest, J., and Cantin, M. (1985) Biochem. Bio hys Res Commun. 130,981-986

19. Gibson, T. R., lhielhs, P. P., and Glembotski, C. C. (1987) Endocrinology 120,764-772

20. Oikawa, S., Imai, M., Inuzuka, C., Tawaragi, Y., Nakazato, H., and Matauo, H. (1985) Biochem. Biophys. Res. Commun. 132,892-899

21. Walter, P., and Lingappa, V. R. (1986) Annu. Rea Cell. Biol. 2,499-516 22. Lynch, D. R., Venable, J. C., and Snyder, S. H. (1988) Endocrinology 122 ,

23. Bloch, K. D., Scott, J. A,, Zisfein, J. B., Fallon, J. T., Margolies, M. N., in press

Seidman, C. E., Matsueda, G. R., Homcy, C. J., Graham, R. M., and Seidman, J. G. (1985) Science 230,116%1171

24. Chirgwin, J. M., Przybyla, A. E., MacDonald, R. J., and Rutter, W. J. (1979) Biochemistry 18,5294-5299

25. Chomczynski, P., and Sacchi, N. (1987) AmL Biochem. 162 ,156159 26. Vuolteenaho. 0.. Ariamaa. 0.. Jarvinen. M.. and Rinne. A. (1985) Acta

834

Histochem; 77,199-203' '

Kallen, R. G., and Gibson, T. R. (1987) Endocrinology 121,843-852

17.143-153

. . , . ~,

27. Glembotski, C. C., Oronzi, M. E., Li, X., Shields, P. P., Johnston, J. F.,

28. Rome, L. H., Garvin, A. J., Allietta, M. M., and Neufeld, E. F. (1979) Cell

Histochem; 77,199-203' '

Kallen. R. G.. and Gibson. T. R. (1987) Endocrinohm 121.843-852

. . , . ~,

27. Glembotski, C. C., Oronzi, M. E., Li, X., Shields, P. P., Johnston, J. F.,

I ' d . 148-1x3 979) Cell

29. Kanakk, L.,~and Hill, R. L. (1964) J. Biol. Chem. 239,4202-4206 30. Schwartz, M. K., and Bodansky, 0. (1966) Methods Enzyml. 9,294-302 31. May, V., Braas, K. M., and Eipper, B. A. (1987) Neurosci. Abstr. 13,1277 32. Braas, K. M., Newby, A. C., Wilson, V. S., and Snyder, S. H. (1986) J.

33. Hsu, S.-M., Raine, L., and Fanger, H. (1981) J. Histochem. Cytochem. 2 9 ,

34. Childs, G., and Unabia, G. (1982) J. Histochem. Cytochem. 30,713-716 35. Laemmli, U. K. (1970) Nature 227,680-685 36. Towbin, H., Staehelin, T., and Gordon, J. (1979) Proc. Natl. Acd. Sei.

37. Jamieson, J. D., and Palade, G. E. (1964) J. Cell Biol. 23,151-172 38. Bloch, K. D., Seidman, J. G., Naftilan, J. D., Fallon, J. T., and Seidman,

C. E. (1986) Cell 47,695-702 39. Day, M. L., Schwartz, D., Wlegand, R. C., Stockman, P. T., Brunnert, S.

R., Tolunay, H. E., Currie, M. G., Standaert, D. G., and Needleman, P.

40. Sakata. J.. Mizuno. K.. and Matsuo. H. (1986) Biochem. Bio~hvs. Res. (1987) Hypertension 9,485-491

Neurosci. 6, 1952-1961

577-580

U. S. A. 76,4350-4354

I . ~,~ Cornmu;. 140,230-236

675

~ "- -"-. ~~~~

41. Harris, R. B., and Wilson, I. B. (1984) Arch. Biochem. Biophys. 2 3 3 , 667-