1-Antichymotrypsin-Like Proteins I and II Purified from Bovine Adrenal Medulla Are Enriched in...

a1-Antichymotrypsin-Like Proteins I and II Purified fromBovine Adrenal Medulla Are Enriched in Chromaffin Granules

and Inhibit the Proenkephalin Processing Enzyme“Prohormone Thiol Protease”

Vivian Y. H. Hook, *Nikolaos Tezapsidis, Shin-Rong Hwang, Catherine Sei, Michael Byrne,and Sukkid Yasothornsrikul

Department of Medicine, University of California, San Diego, La Jolla, California; and*Department of Psychiatry andNeurobiology, Mt. Sinai School of Medicine, New York, New York, U.S.A.

Abstract: Proteolytic processing of inactive proenkephalinand proneuropeptides is essential for the production ofbiologically active enkephalins and many neuropeptides.The incomplete processing of proenkephalin in adrenal me-dulla suggests that endogenous protease inhibitors mayinhibit proenkephalin processing enzymes. This study dem-onstrates the isolation and characterization of two isoformsof adrenal medullary a1-antichymotrypsin (ACT), referred toas ACT-like proteins I and II, which are colocalized withenkephalin in chromaffin granules and which inhibit theproenkephalin processing enzyme known as prohormonethiol protease (PTP). Subcellular fractionation demonstratedenrichment of 56- and 60-kDa ACT-like proteins I and II,respectively, to enkephalin-containing chromaffin granules(secretory vesicles). Immunofluorescence cytochemistry ofchromaffin cells indicated a discrete, punctate pattern ofACT immunostaining that resembles that of [Met]enkephalinthat is stored in secretory vesicles. Chromatography of ad-renal medullary extracts through DEAE-Sepharose andchromatofocusing resulted in the separation of ACT-likeproteins I and II that possess different isoelectric points of5.5 and 4.0, respectively. The 56-kDa ACT-like protein I waspurified to apparent homogeneity by Sephacryl S200 chro-matography; the 60-kDa ACT-like protein II was isolated bybutyl-Sepharose, Sephacryl S200, and concanavalin A-Sepharose columns. The proenkephalin processing en-zyme PTP was potently inhibited by ACT-like protein I, witha Ki,app of 35 nM, but ACT-like protein II was less effective.ACT-like proteins I and II had little effect on chymotrypsin.These results demonstrate the biochemical identification oftwo secretory vesicle ACT-like proteins that differentiallyinhibit PTP. The colocalization of the ACT-like proteins andPTP within chromaffin granules indicates that they couldinteract in vivo. Results from this study suggest that theseACT-like proteins may be considered as candidate inhibi-tors of PTP, which could provide a mechanism for limitedproenkephalin processing in adrenal medulla. Key Words:Adrenal medulla—a1-Antichymotrypsin-like proteins I andII—Proenkephalin processing enzyme—“Prohormone thiolprotease”—Chromaffin granules—Secretory vesicles.J. Neurochem. 73, 59–69 (1999).

Peptide neurotransmitters and hormones are synthe-sized as proproteins that require proteolysis to generatethe smaller active neuropeptides. Proneuropeptides arerouted in the cell through the rough endoplasmic reticu-lum and Golgi apparatus to secretory vesicles, whichsynthesize, store, and secrete neuropeptides (Gainer etal., 1985; Hook et al., 1994; Teter and Moore, 1998). Thepresence of precursor and processed peptides within se-cretory vesicles indicates that corresponding proteasesfor proneuropeptide processing are present within theseorganelles.

Secretory vesicles of adrenal medulla, known as chro-maffin granules, have been used as a model system forstudies of proteolytic processing enzymes that generateactive peptide neurotransmitters and hormones (Hook etal., 1994, 1996). Chromaffin granules contain high levelsof enkephalin opioid peptides (Schultzberg et al., 1978;Lundberg et al., 1979; Udenfriend and Kilpatrick, 1983),as well as several other neuropeptides, including neu-ropeptide Y (Carmichael et al., 1990), galanin (Rokaeusand Brownstein, 1986), and somatostatin (Lundberg etal., 1979). Therefore, chromaffin granules serve as anexcellent model neurosecretory vesicle system for iden-tifying endogenous regulators of proneuropeptide pro-cessing enzymes.

Our studies of proteases in chromaffin granules havedemonstrated the cysteine protease “prohormone thiolprotease” (PTP) as a major proenkephalin processingenzyme (Krieger and Hook, 1991; Schiller et al., 1995;

Received February 3, 1999; revised manuscript received February18, 1999; accepted February 19, 1999.

Address correspondence and reprint requests to Dr. V. Y. H. Hook atDepartment of Medicine, University of California, San Diego, 9500Gilman Drive, #0822, La Jolla, CA 92093-0822, U.S.A.

Abbreviations used:ACT, a1-antichymotrypsin; ICE, interleukinconverting enzyme; i.r., immunoreactivity; PBS, phosphate-bufferedsaline; pI, isoelectric point; PTP, prohormone thiol protease; SDS-PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis.

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Journal of NeurochemistryLippincott Williams & Wilkins, Inc., Philadelphia© 1999 International Society for Neurochemistry

Tezapsidis et al., 1995). PTP cleaves proenkephalin atpaired basic residues (Lys-Arg, Arg-Arg, and Lys-Lys),as well as at monobasic residues, to generate enkephalin-containing intermediates and enkephalin peptides(Krieger and Hook, 1991; Krieger et al., 1992; Schiller etal., 1995) that resemble enkephalin-related peptides invivo (Fleminger et al., 1983; Udenfriend and Kilpatrick,1983). Proenkephalin processing in chromaffin granulesis also mediated, to a lesser degree, by the subtilisin-likeprohormone convertase enzymes PC1/3 and PC2 (Az-aryan et al., 1995a) and by an aspartyl protease resem-bling the pituitary proopiomelanocortin converting en-zyme (Azaryan et al., 1995b).

Proenkephalin processing in vivo, however, is limited,because only 10% of [Met]enkephalin in adrenal medullaexists as completely processed pentapeptide and 90% ofenkephalins in this tissue are present as high-molecular-mass intermediates of 10–25 kDa (Fleminger et al.,1983). However, in vitro processing of proenkephalin byPTP proceeds completely, with extensive conversion ofproenkephalin into peptide products (Krieger and Hook,1991; Schiller et al., 1995). These studies suggest thepossibility that endogenous protease inhibitors of PTPmay be present in vivo to limit the extent of proenkepha-lin processing.

Several enkephalin-containing regions in brain, in-cluding the hippocampus, contain the protease inhibitora1-antichymotrypsin (ACT) (Abraham et al., 1988,1990), which is a member of the serpin family of pro-tease inhibitors. ACT possesses an NH2-terminal signalpeptide sequence (Gierasch, 1989) that is typical of pro-teins that are routed to the secretory vesicle pathway. Inaddition, ACT is transported within neurosecretory ves-icles to nerve terminals (Akaaboune et al., 1993). Theseobservations suggest the presence of ACT within secre-tory vesicles that process, store, and secrete enkephalinsor other neuropeptide transmitters.

To assess the hypothesized role of ACT in regulatingchromaffin granule proteases involved in proenkephalinprocessing, this study has purified and characterized twoisoforms of ACT, referred to as ACT-like proteins I andII, that are present in chromaffin granules. Moreover,these isoforms of ACT demonstrate differences in theirability to inhibit the PTP processing enzyme. Thesefindings demonstrate the presence of biochemical iso-forms of ACT-like proteins in bovine adrenal medullathat are proposed as endogenous regulators of proen-kephalin processing enzymes such as PTP.

EXPERIMENTAL PROCEDURES

ACT-like immunoreactivity (i.r.) inchromaffin granules

Chromaffin granules were isolated from fresh bovine adrenalmedulla as previously described (Yasothornsrikul et al., 1998).In brief, adrenal medulla homogenate (in 0.32M sucrose) wassubjected to successive differential centrifugation at 365,12,000, and 120,000g to obtain highly purified chromaffingranules. After the first centrifugation (365g) of the homoge-nate, the resultant supernatant (S1) and pellet (P1) fractions

were collected as chromaffin granule and nuclear fractions,respectively. Centrifugation of the S1 sample (12,000g) re-sulted in a pellet (P2) fraction that represents enriched chro-maffin granules. After the P2 fraction was washed three timesin 0.32M sucrose (12,000g), ultracentrifugation on a discon-tinuous sucrose gradient (0.32 and 1.6M sucrose steps) resultedin purified chromaffin granules obtained as the pellet.

Purified granules (obtained from the discontinuous sucrosegradient) were lysed in isotonic buffer (containing 150 mMNaCl) and separated into soluble and membrane fractions asdescribed previously (Yasothornsrikul et al., 1998). The result-ant membranes were further incubated in high-salt buffer (con-taining 500 mM NaCl) and centrifuged (100,000g for 30 min)to obtain a soluble membrane-associated fraction and remain-ing membranes. Protein content of fractions was determined bythe method of Lowry et al. (1951), and ACT-like immunore-activity was analyzed by western blots using anti-ACT serum(antiserum directed against human liver ACT; Calbiochem) asdescribed previously (Hook et al., 1993).

Partially purified chromaffin granules (P2 fraction) were alsoanalyzed on a multistep sucrose gradient consisting of 2.2–1.2M sucrose (2.2, 2.1, 2.0, 1.9, 1.8, 1.7, 1.6, 1.5, 1.4, and 1.2Msucrose steps, each consisting of 2.5 ml) that was subjected toultracentrifugation (120,000g for 2 h in an SW28 rotor) asdescribed previously (Yasothornsrikul et al., 1998). The chro-maffin granule fractions were identified by the presence of[Met]enkephalin and catecholamines, quantified as describedpreviously (Hook et al., 1985);b-glucuronidase was also as-sayed as a lysosomal enzyme marker, as previously described(Russell, 1981). ACT-like i.r. in gradient fractions was assessedby western blots with antisera against human liver ACT, asdescribed previously (Hook et al., 1993).

Immunofluorescence cytochemistry of ACT-like i.r.in primary cultures of chromaffin cells

Immunofluorescence cytochemistry of ACT-like i.r. and[Met]enkephalin was examined in chromaffin cells. Primarycultures of chromaffin cells were prepared from fresh bovineadrenal medulla by enzymatic dissociation with collagenaseand DNase, as described previously (Hook et al., 1985; Tez-apsidis et al., 1995). Chromaffin cells were plated on two-chamber glass slides (Nunc Lab-Tek slides) that had beencoated with poly-D-lysine (according to the manufacturer’sdirections; Sigma) at a density of 0.53 106 cells per chamberin Dulbecco’s modified Eagle’s medium (high glucose; Gibco-BRL) containing penicillin and streptomycin (100 units/ml and100 mg/ml, respectively), 1 mM sodium pyruvate, 10% heat-inactivated fetal bovine serum (Irvine Scientific), and 1025 Mcytosine arabinofuranoside (Sigma). Cells were maintained in ahumidified air/CO2 incubator (95% air/5% CO2) at 37°C. After5–7 days in culture, cells were fixed in 3.7% formaldehyde [inphosphate-buffered saline (PBS), pH 7.4] for 10 min, washedtwice with PBS (pH 7.4), and stored in 0.5% formaldehyde at4°C. Cells were permeabilized by incubating cells in ice-coldacetone for 2–3 min, and slides were then air-dried. Cells werewashed twice with PBS, washed three times for 5 min eachwith PBS containing 0.5% bovine serum albumin (fraction V;Sigma), and incubated with antisera against human liver ACTor antisera against [Met]enkephalin (final dilutions of 1:500and 1:1,000, respectively) in PBS containing 0.5% bovineserum albumin for 60 min at room temperature. Cells werewashed three times for 5 min each with PBS containing 0.5%bovine serum albumin and incubated with secondary goat anti-rabbit conjugated to a sulfonated-rhodamine derivative knownas Alexa 488 (Molecular Probes; final concentration of second-

J. Neurochem., Vol. 73, No. 1, 1999

60 V. Y. H. HOOK ET AL.

ary antibody, 1mg/ml in PBS containing 0.5% bovine serumalbumin). Cells were again washed three times for 5 min eachin PBS containing 0.5% bovine serum albumin. Coverslipswere mounted with 50% glycerol buffered in PBS, and immu-nofluorescence was visualized in a Nikon Diaphot TE300 mi-croscope using a fluorescein isothiocyanate cube.

Purification of ACT-like proteins from bovineadrenal medulla

Separation of ACT-like proteins I and II from bovine adrenalmedulla used DEAE-Sepharose and chromatofocusing chroma-tography. The ACT-like protein I was then purified by gelfiltration on Sephacryl S200. The ACT-like protein II waspurified by chromatography on butyl-Sepharose, SephacrylS200, and concanavalin A-Sepharose. Specifically, fresh adre-nal medullae from 80 bovine adrenal glands were homogenized(with a Polytron) in 60 mM histidine-HCl, pH 6.5. After cen-trifugation at 10,000 rpm for 30 min at 4°C, the resultantsupernatant was collected and dialyzed (Spectrapor membrane;10–14-kDa cutoff) against 25 mM histidine-HCl, pH 6.5. Thisextract was applied to a DEAE-Sepharose column (353 2.5cm); after washing in equilibration buffer (25 mM histidine-HCl, pH 6.5), the column was subjected to a 0–0.5M NaClgradient in equilibration buffer (300-ml gradient). Fractions (4ml per fraction) were assayed for ACT-like i.r. by westernblots, as described previously (Hook et al., 1993), and bymonitoring relative protein levels by absorbance at 280 nm.ACT-positive fractions were pooled, dialyzed against 25 mMhistidine-HCl, and concentrated by Amicon ultrafiltration. Thissample was chromatographed a second time on the DEAE-Sepharose column with a modified NaCl gradient of 0.1–0.5MNaCl. ACT-positive fractions from the second DEAE-Sepha-rose column were pooled and dialyzed in 25 mM histidine-HCl,pH 6.5.

The second purification step used chromatofocusing on aPolybuffer Exchanger 94 column (253 0.9 cm; Pharmacia),equilibrated with 25 mM histidine-HCl, pH 6.5. The pool ofACT-like i.r. from the DEAE-Sepharose step was applied to thechromatofocusing column, which was eluted with a pH gradi-ent of 6.5–4.0 generated with Polybuffer 96 solution (Pharma-cia). Fractions were monitored by anti-ACT western blots, andthe pH of individual fractions was measured. Two peaks ofACT i.r. eluted at pH 5.5 and 4.0, which were designated asACT-like proteins I and II, respectively.

Purification of the ACT-like protein I to apparent homoge-neity was achieved by gel filtration on a Sephacryl S200column (903 1.5 cm), as described previously (Krieger andHook, 1991). Isolation of the ACT-like protein II was achievedin three additional steps using butyl-Sepharose, SephacrylS200, and concanavalin A-Sepharose columns. For chromatog-raphy on butyl-Sepharose (11.53 2 cm column), the ACT-likeprotein II sample was adjusted to 50% ammonium sulfate,applied to the column equilibrated in 1.95M ammonium sulfate(in 100 mM potassium phosphate), and eluted with a gradient of1.95–0M ammonium sulfate in 100 mM potassium phosphate.ACT-positive fractions were pooled, concentrated by Amiconfiltration (YM10 membrane), and subjected to gel filtration onSephacryl S200 (903 1.5 cm column) with 25 mM histidine-HCl (pH 6.5) buffer. The peak of ACT immunoreactivity wasthen subjected to chromatography on concanavalin A-Sepha-rose (53 1.5 cm column); after loading the sample onto thecolumn and washing with equilibration buffer [100 mM citricacid-NaOH (pH 6.0), 1 mM MgCl2, 1 mM MnCl2, 1 mMCaCl2, and 0.5M NaCl], ACT-like protein II was eluted withmethyl-a-D-mannoside in equilibration buffer, as describedpreviously (Krieger and Hook, 1991). Isolated ACT-like pro-teins I and II were analyzed by sodium dodecyl sulfate–poly-acrylamide gel electrophoresis (SDS-PAGE) of gels stainedwith Coomassie Blue and by western blots with anti-ACTserum, as described previously (Hook et al., 1993). Each puri-fication step was performed at least two or three times.

Effect of ACT-like proteins on PTP andchymotrypsin activities

PTP was purified from chromaffin granules of bovine adre-nal medulla as described previously (Krieger and Hook, 1991),by chromatography on concanavalin A-Sepharose, SephacrylS200, chromatofocusing, and thiol-propyl-Sepharose. PTP ac-tivity was monitored with recombinant35S-enkephalin precur-sor as substrate, generated by in vitro transcription and trans-lation of the rat enkephalin precursor cDNA, as describedpreviously (Krieger and Hook, 1991). PTP activity was moni-tored as the formation of trichloroacetic acid-soluble35S radio-activity, measured in duplicate, with replicates varying by,10% of average values, as described previously (Krieger andHook, 1991). PTP cleavage of the enkephalin precursor wasalso assessed by SDS-PAGE gels and autoradiography.

FIG. 1. Purification of chromaffin granules(CG). CG were isolated by differential centrif-ugation of bovine adrenal medulla homoge-nate; the resultant supernatant (S) and pellet(P) fractions from each centrifugation are il-lustrated. The enriched fraction of chromaffingranules (P2) was subjected to a discontinu-ous sucrose gradient (0.32 and 1.6 M su-crose) to obtain purified chromaffin granules.The P2 fraction (after washing) was also sub-jected to analyses on a multistep sucrosegradient of 2.2–1.2 M sucrose.


61ADRENAL MEDULLARY ACT-LIKE PROTEINS I AND II

Chymotrypsin activity was assayed with Suc-Ala-Ala-Pro-Phe-4-methylcoumaryl-7-amide substrate (1 mM) in 50 mMTris-HCl (pH 8.3), 0.15M NaCl, and 0.1% bovine serumalbumin by incubation at 37°C for 10–60 min, as describedpreviously (Hook et al., 1993). Activity was monitored byproduction of free 7-amino-4-methylcoumarin detected in aPerkin-Elmer fluorometer (model 650-40) at excitation andemission wavelengths of 385 and 465 nm, respectively.

When protease inhibitors were included in assays, inhibitorand enzyme were preincubated (in the absence of substrate) at4°C for 15 min on ice, substrate was added, and assays of each

enzyme were performed as described here. The inhibition con-stant, Ki,app, was calculated by the equation [I]/(12 a) 5Ki,app(1/a) 1 [E], where [I] and [E] represent concentrations ofinhibitor and enzyme, respectively, anda is fractional activity(Van Nostrand et al., 1990).

RESULTS

ACT-like i.r. in chromaffin granulesThe presence of ACT-like i.r. within secretory ves-

icles of bovine adrenal medulla (chromaffin granules)was first investigated. Chromaffin granules were puri-fied by a series of differential centrifugation steps(Fig. 1). Western blots of homogenate, P1 and S1, P2and S2, and purified granules (from the discontinuoussucrose gradient) demonstrated enrichment of 56- and60-kDa ACT-like bands at each step of the subcellularfractionation procedure (Fig. 2). The 56- and 60-kDaACT-like proteins were enriched in S1 compared withP1, in P2 compared with S2, and in purified chromaf-fin granules compared with earlier steps of the frac-tionation procedure. When identical amounts of pro-teins (45mg) from these fractions were analyzed inanti-ACT western blots, relative levels (determined bydensitometry) of the 56- and 60-kDa ACT-like bands

FIG. 2. Enrichment of ACT-like i.r. in chromaffin granules (CG).Relative levels of ACT-like i.r. were assessed in subcellular frac-tions during isolation of CG. ACT i.r. (in duplicate lanes) wasassessed in western blots of samples containing identicalamounts of protein (45 mg) from bovine adrenal medulla homog-enate (H; lanes 1 and 2), P1 (lanes 3 and 4) and S1 (lanes 5 and6) from the first centrifugation (365 g), P2 (lanes 7 and 8) and S2(lanes 9 and 10) from the subsequent centrifugation at 12,000 g,and purified CG obtained from the discontinuous sucrose gra-dient (lanes 11 and 12). Arrows indicate ACT-positive bands of56 and 60 kDa.

FIG. 3. ACT-like i.r. in chromaffin granule fractions on a multistep sucrose gradient. The enriched chromaffin granule fraction (washedP2) was subjected to ultracentrifugation on a multistep gradient of 2.2–1.2 M sucrose, and fractions of the gradient (1 ml per fraction,collected from 2.2 to 1.2 M sucrose) were collected. Fractions were assayed for (a) ACT-like i.r. on western blots, (b) [Met]enkephalin[(Met)enk] and catecholamines (CA) as secretory vesicle markers, and (c) b-glucuronidase (b-Glu) as an enzyme marker for lysosomes.ACT-like i.r. was present in (Met)enk- and CA-containing secretory vesicle fractions.



in S1, P2, and purified chromaffin granules were 0.5,0.6, and 1.0, respectively, thus indicating enrichmentof ACT-like immunoreactivity in chromaffin granule-

containing fractions. The microsomal S2 fraction con-tained a lower level of ACT-like i.r. (relative level of0.2 in S2 compared with 1.0 in purified chromaffingranules), which is consistent with the presence in S2of membranes from rough endoplasmic reticulum andGolgi apparatus where proteins are routed beforepackaging into secretory vesicles.

The localization of ACT to chromaffin granules wasalso analyzed by subjecting partially purified chromaffingranules (washed P2 fraction) to fractionation on a mul-tistep sucrose gradient (2.2–1.2M sucrose), followed byanalyses of gradient fractions for ACT-like i.r. and thesecretory vesicle markers [Met]enkephalin and cat-echolamines.b-Glucuronidase was also assayed as alysosomal marker. Results (Fig. 3) showed that ACT-likei.r., corresponding to 56- and 60-kDa bands on westernblots, was present in chromaffin granule fractions thatcontained [Met]enkephalin and catecholamines (frac-tions 5–12). Moreover, ACT-like i.r. was not enriched infractions containing the lysosomal markerb-glucuroni-dase. These results show that ACT-like i.r. is present inchromaffin granule fractions that contain [Met]enkepha-lin and catecholamines.

FIG. 4. ACT-like i.r. in soluble (SOL.) and membrane (MEM.)components of chromaffin granules. Purified chromaffin gran-ules (5-ml aliquot) were lysed in isotonic 150 mM NaCl salt bufferand separated into SOL. (CGs150; 5 ml) and MEM. (CGm150;resuspended in 5-ml buffer) fractions, as described in Experi-mental Procedures. Membranes were further incubated in highsalt 500 mM NaCl buffer and centrifuged to obtain a solublefraction (CGs500; 5 ml) representing membrane-associated(MEM. ASSOC.) proteins and a pellet fraction representing highsalt-extracted membranes (CGm500; 5 ml) (MEM./NaCl). Thelocalization of ACT-like i.r. was assessed in western blots (20-mlaliquots of granule fractions) of CGs150, CGm150, CGs500, andCGm500 fractions (lanes 1–4, respectively) of chromaffin gran-ules.

FIG. 5. Immunofluorescence cytochemistry of ACT-like i.r. and [Met]enkephalin (ME) in chromaffin cells. Immunofluorescence cyto-chemistry of ACT (with anti-human liver ACT sera) and ME of chromaffin cells in primary culture were performed as described inExperimental Procedures. ACT-like i.r. (a) was observed with a 1003 objective, and ME immunofluorescence (b) was also observed witha 1003 objective. Controls showed no immunostaining when the primary antibody was omitted from the procedures (data not shown).Bar 5 10 mm.



Within chromaffin granules, the 56- and 60-kDa bandsof ACT-like i.r. were present in both the soluble andmembrane components (Fig. 4). Membrane-bound ACTi.r. was not removed with high-salt extraction of mem-branes, indicating that ACT was within the membranesof chromaffin granules. Thus, these ACT-like proteinsmay function in both soluble and membrane componentsof chromaffin granules.

Immunofluorescence cytochemistry of ACT-like i.r.and [Met]enkephalin in chromaffin cells

The presence of ACT-like proteins within secretoryvesicles predicts that these proteins should possess adiscrete subcellular pattern of distribution that can beassessed by immunocytochemistry. In primary culturesof chromaffin cells, ACT-like i.r. appeared as a discretepattern of localization that was similar to that of [Met]-

enkephalin that is stored in secretory vesicles (Fig. 5).The similar discrete pattern of ACT-like i.r. and [Met]-enkephalin immunostaining in chromaffin cells and theirabsence in nuclei are consistent with their localization tochromaffin granules.

Purification and characterization of two isoforms ofACT-like proteins with distinct isoelectric point (pI)values from adrenal medulla

Purification of the 60- and 56-kDa ACT-like proteinswas performed to assess possible inhibition of PTP, aproenkephalin processing enzyme. The soluble extractobtained from adrenal medulla homogenate was sub-jected to ion exchange chromatography on DEAE-Sepharose. The first DEAE-Sepharose column waseluted with a 0–0.5M NaCl gradient, and the secondDEAE column was eluted with a modified gradient of0.1–0.5M NaCl (Fig. 6a). ACT i.r. was eluted as a singlepeak at;0.3 M NaCl. This ACT-like peak was thensubjected to chromatofocusing, with elution by a pH

FIG. 6. ACT-like proteins I and II are resolved by DEAE-Sepha-rose and chromatofocusing. a: DEAE-Sepharose. A soluble ex-tract of bovine adrenal medulla homogenate was subjected tochromatography on DEAE-Sepharose that was eluted with agradient of 0.1–0.5 M NaCl. ACT-like i.r. in column fractions wasmonitored by western blots with anti-ACT serum. Densitometryof ACT i.r. (56- and 60-kDa bands on westerns) is indicated (F).In addition, the NaCl gradient ({) and relative absorbance at 280nm in each fraction (h) were monitored. b: Chromatofocusing.The peak of ACT-like i.r. from the DEAE-Sepharose step wasfractionated on a chromatofocusing column, with elution of pro-teins by a pH gradient. ACT-positive fractions were determinedby anti-ACT western blots and subjected to densitometry (F).The pH (E) and relative absorbance at 280 nm (h) were alsomonitored.

FIG. 7. Purification of ACT-like protein I by Sephacryl S200chromatography. a: Sephacryl S200. The ACT-like i.r. eluting atpH 5.5 from the chromatofocusing column was subjected to gelfiltration on a Sephacryl S200 column. Relative ACT-i.r. wasmonitored (F), as well as absorbance at 280 nm in columnfractions (E). Arrows indicate elution positions of standard pro-teins, 68-kDa bovine serum albumin and 30-kDa carbonic anhy-drase, as molecular mass markers. Arrows also indicate the voidvolumn (Vo) and total volume (Vt ). b: Purified ACT-like protein Ion SDS-PAGE. Purification of ACT-like protein I to apparenthomogeneity was indicated by a single 56-kDa (1 mg) banddetected on SDS-PAGE stained with Coomassie Blue. c: Puri-fied ACT-like protein I on anti-ACT western blot. Purified ACT-like protein I (1 mg) was subjected to western blot analysis withantisera against human liver ACT. The arrow indicates the 56-kDa purified protein that is recognized by anti-ACT.



gradient of pH 6.5–4.0 (Fig. 6b). The chromatofocusingstep resolved two peaks of ACT-like i.r. that eluted at pH5.5 and pH 4.0, respectively. Elution at these pH valuesindicated that these two ACT-like proteins, I and II,possess pI values of 5.5 and 4.0, respectively.

ACT-like protein I was isolated by gel filtration on aSephacryl S200 column (Fig. 7a). A single peak ofACT-like i.r. eluted with an apparent molecular mass of55 kDa. SDS-PAGE analysis of the peak of ACT i.r.indicated a single 56-kDa band detected by CoomassieBlue protein staining (Fig. 7b). This 56-kDa band wasrecognized by anti-ACT antibody (anti-human liver ACTserum) in western blots (Fig. 7c). These results indicatepurification to apparent homogeneity of the 56-kDaACT-like protein I from adrenal medulla.

The ACT-like protein II was isolated in three steps bychromatography on butyl-Sepharose, Sephacryl S200,and concanavalin A-Sepharose columns (Fig. 8). Thepeak of ACT-like i.r. eluting at pH 4.0 from the chro-matofocusing column was chromatographed on a butyl-Sepharose column eluted with a gradient of 1.95–0Mammonium sulfate (Fig. 8a). Subsequent gel filtration onSephacryl S200 yielded a peak of ACT i.r. eluting withan apparent molecular mass of;75 kDa (Fig. 8b). Bind-ing to concanavalin A-Sepharose and elution with meth-yl-a-D-mannoside (Fig. 8c) indicated the glycoproteinnature of ACT-like protein II. SDS-PAGE analysis indi-cated isolation to apparent homogeneity of the 60-kDaACT-like protein II (Fig. 8d) that was recognized by theanti-ACT sera in western blots (Fig. 8e). These resultsdemonstrate isolation of the 60-kDa ACT-like protein IIfrom adrenal medulla.

The isolation procedure yielded 850 and 660mg ofACT-like proteins I and II, respectively (Table 1). Itis notable that ACT-like proteins I and II were recog-nized by the anti-ACT sera (anti-human liver ACT)with different sensitivities compared with humanliver ACT. The anti-ACT sera recognized micro-gram amounts of ACT-like proteins I and II, whereasthe antibody readily detected nanogram levels of hu-man liver ACT (Fig. 9). Furthermore, lower levelsof the 56-kDa ACT-like protein I (1mg on westernblots) were detected by the antibody compared withthe 60-kDa ACT-like protein II (15mg on westernblots). These differences in antibody reactivity wereindicated by specific ACT-like i.r. (Table 1) of 6and 0.4mg of ACT i.r./mg of protein for ACT-likeproteins I and II, respectively. The values for spe-cific ACT (in micrograms of ACT i.r. per milligramof protein) of purified ACT-like proteins I and II

FIG. 8. Isolation of ACT-like protein II by butyl-Sepharose,Sephacryl S200, and concanavalin A-Sepharose. a: Butyl-Sepharose. The ACT-positive fractions, eluting at pH 4.0, fromthe chromatofocusing step were subjected to butyl-Sepharosechromatography, with elution by an ammonium sulfate gradientfrom 1.95 to 0 M ammonium sulfate. ACT-positive fractions weremonitored by densitometry of anti-ACT western blots (F). Theammonium sulfate gradient (E) and absorbance at 280 nm (h) ofcolumn fractions were also monitored. b: Sephacryl S200. Thepeak of ACT i.r. from the butyl-Sepharose step was subjected togel filtration on Sephacryl S200. ACT-positive fractions (F) andabsorbance at 280 nm of column fractions (E) were monitored.c: Concanavalin A-Sepharose. ACT i.r. from the Sephacryl S200step was subjected to lectin affinity chromatography on con-canavalin A-Sepharose. Elution was conducted with methyl-a-D-mannoside, indicated by the arrow. ACT-positive fractions (F)and relative protein levels indicated by absorbance at 280 nm (E)

were monitored. d: ACT-like protein II on SDS-PAGE. Isolation ofthe ACT-like protein II to apparent homogeneity was indicatedby a single 60-kDa band (0.5 mg) detected on SDS-PAGEstained with Coomassie Blue. e: ACT-like protein II on anti-ACTwestern blot. Purified ACT-like protein II (1 mg) was subjected towestern blot analysis with antisera against human liver ACT. Thearrow indicates the 60-kDa purified protein that is recognized byanti-ACT.



indicate;1,000-fold differences in antibody reactiv-ity to human liver ACT compared with the purifiedadrenal medullary ACT-like proteins. These datasuggest that ACT-like proteins I and II may repre-sent isoforms of ACT that are not identical to humanliver ACT.

ACT-like proteins I and II inhibit PTP,a proenkephalin processing enzyme

PTP represents the major enkephalin precursorcleaving activity in chromaffin granules (Hook et al.,1994). PTP activity, assayed by measuring the con-version of recombinant35S-enkephalin precursor intotrichloroacetic acid-soluble radioactivity, which rep-resents small peptide products, was inhibited by nano-

molar levels of ACT-like protein I (Fig. 10a), with aKi,app of 35 nM. ACT-like protein II was less effectivebecause micromolar levels were necessary to reducePTP activity (Fig. 10b). As a control, PTP activity wasnot affected by an unrelated protein, bovine serumalbumin (Hook et al., 1993). These results demonstratethat ACT-like proteins I and II differed in their effec-tiveness at inhibiting PTP.

PTP cleavage of35S-enkephalin precursor, detected byautoradiography of SDS-PAGE gels, was inhibited byACT-like proteins I and II. ACT-like protein I (at 110–240 nM) completely inhibited PTP cleavage of the en-kephalin precursor (Fig. 10c). Higher concentrations ofACT-like protein II (3.4 and 6.0mM), however, wererequired to inhibit PTP cleavage of the enkephalin pre-cursor (Fig. 10d). Both the ACT-like proteins (at micro-molar concentrations), however, had minimal effects onchymotrypsin activity (Table 2). These results indicatethat ACT-like proteins I and II demonstrate differencesin their ability to inhibit the PTP processing enzyme andthat they do not inhibit chymotrypsin. Moreover, thecolocalization of ACT-like proteins I and II with PTP inchromaffin granules indicates that inhibition of PTP andproenkephalin processing by these ACT-like proteinsmay occur in vivo.

DISCUSSION

Specific proteolytic processing of proenkephalin andproneuropeptides is essential for the production of bio-logically active enkephalins and many other neuropep-

TABLE 1. Purification of ACT-like proteins I and II to apparent homogeneity from chromaffin granules

Purification stepVolume

(ml)

Proteinconcentration

(mg/ml)

Totalprotein(mg)

ACT i.r.(mg of i.r./ml)

Total ACTi.r. (mg i.r.)

Specific ACT i.r.(mg of i.r./mg of

protein)Fold

purification Yield

A. Separation of ACT I and IIHomogenate 400 37.7 15,080

ACT I 0.45 182 0.012 100ACT II 0.30 122 0.008 100

Soluble extract 380 28.7 10,906ACT I 0.71 270 0.024 2 148ACT II 0.54 204 0.018 2 167

DEAE-Sepharose 54 8 432ACT I 0.32 18 0.041 3.4 10ACT II 0.72 39 0.090 11 32

ChromatofocusingACT I 14 3.3 47 0.38 5.3 0.12 10 3ACT II 16 13.2 211 1.35 21.6 0.10 12 18

B. ACT I isolationSephacryl S200 13 0.065 0.85 0.40 5.2 6.1 508 3

C. ACT II isolationButyl-Sepharose 38 0.23 8.7 0.07 2.7 0.3 37 2.2Sephacryl S200 13 0.22 2.9 0.04 0.5 0.2 25 0.4Concanavalin A-Sepharose 1.0 0.66 0.66 0.25 0.25 0.4 50 0.2

Chromatography steps of part A separated the 56- and 60-kDa forms of ACT-like proteins. Part B represents purification of the 56-kDa ACT-likeprotein I. Part C represents isolation of the 60-kDa ACT-like protein II. Purification of 56- and 60-kDa bands of ACT-like i.r. was assessed by westernblots with anti-human liver ACT sera. It must be realized that western blots (with densitometry) are not quantitative; therefore, relative levels ofACT-like i.r. are only estimates of ACT-like i.r. In addition, it should be noted that levels of ACT-like i.r. of the purified ACT-like proteins were lessthan the actual amount of purified protein; measurements of specific ACT i.r. showed that ACT-like proteins differ in reactivity to anti-ACT comparedwith human liver ACT.

FIG. 9. ACT antibody detects different levels of ACT-like pro-teins I and II, compared with human liver ACT in western blots.Western blots with anti-human liver ACT sera required micro-gram levels of purified ACT-like proteins I (1 mg) and II (15 mg)in lanes 1 and 2, respectively. However, human liver ACT (20ng; lane 3) was detected at nanogram levels in the westernblot.



tides. Control of biological proteolytic systems ofteninvolves endogenous protease inhibitors as regulators ofproteolytic processing pathways. The limited extent of

proenkephalin processing in adrenal medulla (Flemingeret al., 1983) suggests the presence of endogenous pro-tease inhibitors that may inhibit proenkephalin process-ing protease(s).

This study demonstrates that two serpin-like proteins,ACT-like proteins I and II, are present in enkephalin-containing secretory vesicles (chromaffin granules) andthat these ACT-like isoforms possess different potenciesto inhibit PTP, a proenkephalin processing enzyme. Sub-cellular fractionation of adrenal medulla tissue demon-strated enrichment and localization of 56- and 60-kDaACT-like proteins to chromaffin granules. The parallelpattern of discrete ACT and [Met]enkephalin immuno-staining in chromaffin cells is also consistent with thepresence of ACT-like i.r. in secretory vesicles. Chroma-tography of adrenal medullary extracts through DEAE-

FIG. 10. ACT-like protein I and II differentially inhibit PTP, a proenkephalin processing enzyme. a: ACT-like protein I inhibition of PTPprocessing of 35S-enkephalin precursor to trichloroacetic acid-soluble radioactivity. Inhibition of PTP by ACT-like proteins I (■) wasassessed by measuring the conversion of 35S-enkephalin precursor to trichloroacetic acid-soluble radioactivity. ACT-like protein Iinhibited PTP with a calculated Ki,app of 35 nM. b: ACT-like protein II inhibition of PTP processing of 35S-enkephalin precursor totrichloroacetic acid-soluble radioactivity. Inhibition of PTP by ACT-like proteins II (■) was assessed by measuring the conversion of35S-enkephalin precursor to trichloroacetic acid-soluble radioactivity. ACT-like protein II inhibited PTP with a calculated Ki,app of ;0.5mM. c: ACT-like protein I inhibition of PTP cleavage of 35S-enkephalin precursor. PTP cleavage of the 35S-enkephalin precursor wasassessed by SDS-PAGE gels and autoradiography. 35S-Enkephalin precursor (36 kDa) was incubated without PTP (lane 1), with PTP(lanes 2 and 3), and with PTP in the presence of ACT-like protein I at 240, 160, 110, and 73 nM (lanes 4–7, respectively). Densitometryof the 35S-enkephalin precursor band (indicated by the arrow) indicated that PTP cleavage of the enkephalin precursor was inhibited by;94, 83, 75, and 21% at 240, 160, 110, and 73 nM ACT-like protein I, respectively. d: ACT-like protein II inhibition of PTP cleavage of35S-enkephalin precursor. PTP assays were assessed by SDS-PAGE gels and autoradiography. 35S-Enkephalin precursor wasincubated without PTP (lane 1), with PTP (lanes 2 and 3), and with PTP in the presence of ACT-like protein II at 6.0, 3.4, 1.7, and 1.1mM (lanes 4–7, respectively). Densitometry of the 35S-enkephalin precursor band (indicated by the arrow) indicated that PTP cleavageof its substrate was inhibited by ;55 and 27% by 6.0 and 3.4 mM ACT-like protein II, respectively.

TABLE 2. ACT-like proteins I and II andchymotrypsin activity

Inhibitor Chymotrypsin activity

None 100%ACT-like protein I 92%ACT-like protein II 92%

The effects of ACT-like proteins I and II on chymotrypsin activity weretested at 3.0 and 1.7mM final concentrations, respectively, as described inExperimental Procedures. Chymotrypsin activity was assayed with thesubstrate Suc-Ala-Ala-Pro-Phe-7-methylcoumaryl-7-amide.



Sepharose and chromatofocusing resulted in the separa-tion of the 56- and 60-kDa bands, representing ACT-likeproteins I and II, which possess different pI values of 5.5and 4.0, respectively. Each ACT-like protease was puri-fied to apparent homogeneity by a series of chromato-graphic steps. The proenkephalin processing enzymeknown as PTP, purified from chromaffin granules, wasdifferentially inhibited by the two isoforms of ACT.ACT-like protein I was a potent inhibitor of PTP, with aKi,appof 35 nM. In contrast, ACT-like protein II was lesseffective, requiring micromolar levels for inhibition.These levels of ACT-like proteins that inhibit PTP areconsistent with estimated in vivo micromolar levels ofACT-like proteins within chromaffin granules, indicatingthat inhibition of PTP could occur in vivo. These resultsdemonstrate the presence of two putative ACT-like ser-pins that possess differences in their abilities to inhibitPTP. The colocalization of ACT-like proteins I and IIwith PTP in chromaffin granules indicates that theycould interact in vivo to limit the extent of proenkephalinprocessing.

The anti-ACT serum (directed against human liverACT) detected different levels of ACT-like proteins Iand II compared with human liver ACT. Microgramamounts of the bovine ACT-like proteins were detectedby the antibody, whereas the same anti-ACT serum de-tected nanogram levels of the human liver ACT. Thesefindings suggest that chromaffin granule ACT-like pro-teins I and II may represent isoforms of ACT that maynot be identical to human liver ACT.

The isolation of two biochemical isoforms of ACT-like proteins from adrenal medulla is consistent with theknowledge that multiple bovine ACT genes exist(Hwang et al., 1994). Two cloned isoforms of bovineliver ACT cDNAs have been identified (Hwang et al.,1994), consistent with multiple ACT genes. In addition,a neuroendocrine-specific isoform of ACT expressed inbovine adrenal medulla and pituitary has been identifiedas a partial adrenal medulla ACT cDNA (Hwang et al.,1995). It will be of interest in future studies to obtainprimary sequence characterization of ACT-like proteins Iand II through peptide microsequencing and molecularcloning. Future determination of the structural identitiesof ACT-like proteins I and II will provide knowledge ofhow they compare with previously identified isoforms ofbovine neuroendocrine ACTs (Hwang et al., 1994,1995). In fact, preliminary peptide sequencing of ACT-like protein II indicates homology to ACT-like serpins(authors’ unpublished data).

The proposed inhibition of PTP by endogenous ACT-like inhibitors within chromaffin granules provides anexplanation for the limited extent of proenkephalin pro-cessing in adrenal medulla in vivo. It will be of interest,in future studies, to investigate regulation of ACT-likeproteins during altered production of enkephalin peptides(Liston et al., 1984). The potential role of ACT-likeproteins to regulate the extent of proenkephalin process-ing could provide a novel control mechanism for pro-duction of enkephalins and neuropeptides.

ACT and members of the serpin protease inhibitorfamily typically inhibit serine proteases (Travis andSalvesen, 1983). However, inhibition of the cysteineprotease PTP by ACT-like proteins I and II suggestscross-class inhibition in which the target protease differsin protease class compared with the serpin. Other exam-ples of cross-class inhibition are the cowpox virus CrmAserpin, which inhibits the cysteine protease interleukinconverting enzyme (ICE) (Ray et al., 1992), as well asCrmA inhibition of the ICE-related caspases involved inapoptosis (Takahashi et al., 1996; Zhou et al., 1997).

In summary, this study provides evidence for the lo-calization of ACT-like proteins I and II in adrenal med-ullary secretory vesicles and their different abilities toinhibit a proenkephalin processing enzyme, PTP. Potentinhibition of PTP by ACT-like protein I and lesser inhi-bition by ACT-like protein II suggest that the limitedextent of proenkephalin processing in adrenal medulla invivo may be explained by the presence of endogenousprotease inhibitors. These findings provide evidence for aproposed role of endogenous ACT-like protease inhibi-tors in proteolytic processing mechanisms that generatebioactive enkephalins and neuropeptides.

Acknowledgment: This work was supported by grants fromthe National Institute of Neurological Disorders and Stroke andthe National Institute on Drug Abuse, National Institutes ofHealth. The technical assistance of Tonya Purviance is appre-ciated.

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