Bovine Adrenal Chromaffin Granules Research Articles

Is the vesicular nucleotide transporter a molecular target of eicosapentaenoic acid?

Vesicular nucleotide transporter (VNUT), an active transporter for nucleotides in secretory vesicles, is responsible for the vesicular storage of ATP and plays an essential role in purinergic chemical transmission. Inhibition of VNUT decreases the concentration of ATP in the luminal space of secretory vesicles, followed by decreased vesicular ATP release, resulting in the blockade of purinergic chemical transmission. Very recently, Miyaji and colleagues reported that eicosapentaenoic acid (EPA) is a potent VNUT inhibitor and effective in treating neuropathic and inflammatory pain and insulin resistance through inhibition of vesicular storage and release of ATP. However, our validation study indicated that, in bovine adrenal chromaffin granule membrane vesicles, EPA inhibited the formation of an electrochemical gradient of protons across the membrane with the concentration of 50% inhibition (IC50) being 1.0μM without affecting concanamycin B-sensitive ATPase activity. Essentially, similar results were obtained with proteoliposomes containing purified vacuolar H+-ATPase. Consistent with these observations, EPA inhibited the ATP-dependent uptakes of ATP and dopamine by chromaffin granule membrane vesicles, with ID50 being 1.2 and 1.0μM, respectively. Furthermore, EPA inhibited ATP-dependent uptake of L-glutamate by mouse brain synaptic vesicles with ID50 being 0.35μM. These results indicate that EPA at sub-μM acts as a proton conductor and increases proton permeability across the membrane, regardless of the presence or absence of VNUT, thereby inhibiting non-specifically the vesicular storage of neurotransmitters. Thus, EPA may affect a broader range of chemical transmission than proposed.

Perturbation of Dopamine Metabolism by 3-Amino-2-(4′-halophenyl)propenes Leads to Increased Oxidative Stress and Apoptotic SH-SY5Y Cell Death

We have recently characterized a series of 3-amino-2-phenyl-propene (APP) derivatives as reversible inhibitors for the bovine adrenal chromaffin granule vesicular monoamine transporter (VMAT) that have been previously characterized as potent irreversible dopamine-beta-monooxygenase (DbetaM) and monoamine oxidase (MAO) inhibitors. Halogen substitution on the 4'-position of the aromatic ring gradually increases VMAT inhibition potency from 4'-F to 4'-I, parallel to the hydrophobicity of the halogen. We show that these derivatives are taken up into both neuronal and non-neuronal cells, and into resealed chromaffin granule ghosts efficiently through passive diffusion. Uptake rates increased according to the hydrophobicity of the 4'-substituent. More importantly, these derivatives are highly toxic to human neuroblastoma SH-SY5Y but not toxic to M-1, Hep G2, or human embryonic kidney 293 non-neuronal cells at similar concentrations. They drastically perturb dopamine (DA) uptake and metabolism in SH-SY5Y cells under sublethal conditions and are able to deplete both vesicular and cytosolic catecholamines in a manner similar to that of amphetamines. In addition, 4'-IAPP treatment significantly increases intracellular reactive oxygen species (ROS) and decreases glutathione (GSH) levels in SH-SY5Y cells, and cell death is significantly attenuated by the common antioxidants alpha-tocopherol, N-acetyl-l-cysteine and GSH, but not by the nonspecific caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone. DNA fragmentation analysis further supports that cell death is probably due to a caspase-independent ROS-mediated apoptotic pathway. Based on these and other findings, we propose that drastic perturbation of DA metabolism in SH-SY5Y cells by 4'-halo APP derivatives causes increased oxidative stress, leading to apoptotic cell death.

Purification and characterization of bovine adrenal cytochrome b561 expressed in insect and yeast cell systems

Bovine adrenal chromaffin granule cytochrome (cyt) b 561 is a transmembrane hemoprotein that plays a key role in transporting reducing equivalents from ascorbate to dopamine-β-hydroxylase for catecholamine synthesis. We have developed procedures for expression and purification of functional bovine adrenal cyt b 561 in insect and yeast cell systems. The bovine cyt b 561 coding sequence, with or without a hexahistidine-tag sequence at the C-terminus, was cloned into the pVL1392 transfer vector under the control of the polyhedrin promoter to generate recombinant baculovirus for protein expression in Sf9 insect cells (∼0.5 mg detergent-solubilized cyt b 561/L culture). For the yeast system, the cyt b 561 cDNA was modified with a hexahistidine-tag sequence at the C-terminus, and inserted into the pPICZB vector under the control of the alcohol oxidase promoter. The recombinant plasmid was transformed into Pichia pastoris GS115 competent cells to give methanol-inducible cyt b 561 expression (∼0.7 mg detergent-solubilized cyt b 561/L culture). Recombinant His-tagged cyt b 561 expressed in Sf9 or Pichia cells was readily solubilized from membrane fractions with dodecyl maltoside and purified to electrophoretic homogeneity by one-step chromatography on Ni–NTA affinity resin. The purified recombinant cytochrome from both systems had a heme to protein ratio close to two and was fully functional, as judged by comparison with the spectroscopic and kinetic parameters of the endogenous cytochrome from chromaffin granules. A novel procedure for isolation of chromaffin granule membranes was developed to utilize frozen adrenal glands instead of fresh tissue.

Characterization and location of post-translational modifications on chromogranin B from bovine adrenal medullary chromaffin granules.

Bovine chromoganin B (CGB)/secretogranin I, an acidic protein with a sequence of 626 residues and an isoelectric point of 5.2 is a major member of the chromogranin/secretogranin (CG/Sg) family. The difference between the theoretical molecular mass (76 kDa) and the value estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis results from post-translational modifications (glycosylation, phosphorylation and sulfation) and from the abundance of acidic residues (D 4.6%, and E 16.5%). Although the sequence of CGB is known, the structural analyses of the post-translational modifications have so far not been carried out. In the present study, using a combination of proteomic techniques including two-dimensional gel electrophoresis, Western blot, high-performance liquid chromatography purification, enzymatic digestion, sequencing, carbohydrate analysis, matrix-assisted laser desorption/ionization-time of flight and liquid chromatography mass spectrometry analysis, we have located 18 post-translational modifications on bovine CGB, isolated from adrenal medulla chromaffin granules. Furthermore, we have identified at the molecular level the presence of a mutation M/V on position 577 of natural CGB. All together these data reflect the complex structure of this protein marker of the neuroendocrine system.

Ubiquitination of soluble and membrane-bound tyrosine hydroxylase and degradation of the soluble form.

Tyrosine hydroxylase (TH) demonstrates by two-dimensional electrophoresis a microheterogeneity both as a soluble recombinant human TH (hTH1) and as a membrane-bound bovine TH (bTHmem). Part of the heterogeneity is likely due to deamidation of labile asparagine residues. Wild-type (wt)-hTH1 was found to be a substrate for the ubiquitin (Ub) conjugating enzyme system in a reconstituted in vitro system. When wt-hTH1 was expressed in a coupled transcription-translation TnT(R)-T7 reticulolysate system 35S-labelled polypeptides of the expected molecular mass of native enzyme as well as both higher and lower molecular mass forms were observed. The amount of high-molecular-mass forms increased by time and was enhanced in the presence of Ub and clasto-lactacystin beta-lactone. In pulse-chase experiments the amount of full-length hTH1 decreased by first-order kinetics with a half-time of 7.4 h and 2.1 h in the absence and presence of an ATP-regenerating system, respectively. The ATP-dependent degradation was inhibited by clasto-lactacystin beta-lactone. Our findings support the conclusion that hTH1 is ubiquitinated and at least partially degraded by the proteasomes in the reticulocyte lysate system. Finally, it is shown that the integral TH of the bovine adrenal chromaffin granule membrane (bTHmem) is ubiquitinated, most likely monoubiquitinated. Additional Ub-conjugates of this membrane, detected by Western blot analysis, have not yet been identified.

Asymmetrical membrane fluidity of bovine adrenal chromaffin cells and granules and effect of trichosporin-B-VIa

We examined membrane fluidity of bovine adrenal chromaffin cells and chromaffin granules using cationic trimethylammonium derivative of diphenylhexatriene (TMA-DPH) as a fluorescence probe. After adding TMA-DPH to the suspension of chromaffin cells and that of granules, it first bound to the outer layer of the plasma membrane of the cells and that of the granule membrane, then gradually penetrated the inner layer of each membrane and distributed to both leaflets of the respective membranes. Accompanying increases in the ratio of incorporated probe on the cytoplasmic side of the chromaffin cell membrane, its fluorescence anisotropy gradually decreased. However, in chromaffin granules, the fluorescence anisotropy gradually increased with increases in the ratio of incorporated probe. These findings suggest that the inner layer of the plasma membrane and outer layer of the granular membrane are more fluid than the corresponding side of each membrane, which is suitable for the fusion between both membranes. We also examined the effect of trichosporin-B-VIa, a fungal ion channel forming α-aminoisobutyric acid-containing peptide, on the fluidity of chromaffin cells using TMA-DPH. The peptide decreased the fluorescence anisotropy and increased the fluorescence intensity in the concentration range that induced Ca 2+ dependent catecholamine secretion, suggesting that a change in lipid dynamics of the lipid bilayer of the plasma membrane was induced by this peptide.

Interaction of sulfhydryl reagents with K+ transport in adrenal chromaffin granules.

In the present study the functional role of SH groups in the Ca(2+)-independent K(+)-selective channel activity in the membrane of bovine adrenal gland chromaffin granules has been studied. Ionic channel activity has been estimated using 86Rb+, a K+ analogue, flux measurements. The inhibition of chromaffin granules K+ channel by SH modifying agents, such as N-ethylmaleimide, mersalyl and phenylarsenoxide, is described.

Isolation and identification of intact chromogranin A and two N-terminal processing products, vasostatin I and II, from bovine adrenal medulla chromaffin granules by chromatographic and mass spectrometric methods

Chromogranin A (CGA) is the most abundant protein of the bovine adrenal medulla and plays an important role as precursor protein of several peptides that act as modulators for endocrine cell secretory activity. Furthermore, it is presumed to play a role in the targeting of peptide hormones and neurotransmitters to granules of the regulated pathway. However, its complete primary structure and proteolytic processing have not yet been identified. This study describes a rapid and efficient procedure for the high yield isolation of bovine CGA and its N-terminal processing products, vasostatin I and II. Using the lysate from bovine adrenal medulla chromaffin granules, the soluble proteins were purified by three consecutive HPLC steps, thereby avoiding the use of buffer solutions. The protein fractions were isolated and characterized by SDS-PAGE and Western blot analysis as well as by mass spectrometry. In the latter analysis, the efficiency of matrix-assisted laser desorption ionization-mass spectrometry (MALDI-MS) was demonstrated, enabling the unequivocal and sensitive characterization of proteins from crude mixtures. Sufficient amounts of pure protein were obtained by the present procedure to form the basis for detailed structural studies by spectroscopic methods and X-ray crystallography.

Effects of K+ channel inhibitors on potassium transport in bovine adrenal chromaffin granules.

The Ca(2+)-independent K+ selective channel in the membrane of adrenal gland chromaffin granules has earlier been identified. In the present report we describe new properties of this potassium channel using 86Rb+, a K+ analogue, flux measurements. The studies are performed in membrane vesicles prepared from chromaffin granules. The electrogenic 86Rb+ transport is inhibited by quinacrine, barium, zinc and magnesium. The effects of other potassium channel blockers on 86Rb+ transport into chromaffin granules are also reported.

Membrane and soluble proteins of adrenal chromaffin granules

Bovine adrenal chromaffin granules are useful ‘model’ neurosecretory vesicles, particularly for biochemical studies. The granule matrix contains three major secretory proteins (chromogranin A and secretogranins I and II) together with peptides derived from them, and smaller amounts of neuropeptides (enkephalins and neuropeptide Y). Several different endo- and exo-proteinases are also present in both soluble and membrane-bound forms. The major membrane proteins are those involved in catecholamine biosynthesis (dopamine β-monooxygenase and cytochrome b561), active transport of granule components (vacuolar-type proton-translocating ATPase, and carriers for monoamines, nucleotides and small ions) and exocytosis (synaptotagmin, synaptobrevin and other proteins). In addition, the functions of a number of major granule membrane proteins remain unknown.

Antibacterial Activity of Glycosylated and Phosphorylated Chromogranin A-derived Peptide 173-194 from Bovine Adrenal Medullary Chromaffin Granules

Recently, we have isolated from bovine chromaffin granules and identified two natural peptides possessing antibacterial activity: secretolytin (chromogranin B 614-626) and enkelytin (proenkephalin-A 209-237). Here, we characterize a large natural fragment, corresponding to chromogranin A 79-431, that inhibits growth of both Gram-positive and Gram-negative bacteria. The aim of the present work was to determine the shortest active peptide located in the 79-431 chromogranin A region. Three peptides, which shared the same 173-194 chromogranin A sequence (YPGPQAKEDSEGPSQGPASREK) but differed in post-translational modifications, including O-glycosylation and tyrosine phosphorylation, were isolated. A detailed study using microsequencing and mass spectrometry allowed us to correlate their antibacterial activity with these post-translational modifications. The chromogranin A precursor fragment (79-431) and the active glycosylated and phosphorylated peptides were, respectively, named prochromacin and chromacin (P, G, and PG for phosphorylated, glycosylated, and phosphorylated-glycosylated form).

Chromaffin granule membrane glycoprotein IV is identical with Ac45, a membrane-integral subunit of the granule's H +-ATPase

Glycoprotein IV of bovine adrenal chromaffin granule membranes was purified by membrane fractionation with Triton X-114 and lectin affinity chromatography. An antiserum raised against this protein recognized the same component as one directed against subunit Ac45 of the proton-translocating adenosine triphosphatase in the granule membrane. Amino acid sequencing confirmed that glycoprotein IV and Ac45 are identical proteins, and also showed that they are derived from a larger precursor by removal of a 246-amino acid N-terminal sequence. Enzymatic deglycosylation indicated an apparent polypeptide molecular mass of 29 kDa of the mature Ac45/glycoprotein IV. Blue Native electrophoresis confirmed that this protein is a component of the membrane sector of the V-ATPase.

Interaction of Organotins with a Vacuolar-Type H+-ATPase

Organotin-flavone complexes of 3-hydroxyflavone, 3,5,7-trihydroxyflavone (galangin) and 2′,3,4′,5,7-pentahydroxyflavone (morin) are potent inhibitors of the vacuolar H+-translocating ATPase from bovine adrenal chromaffin granules, with Kivalues around 0.3 μM. The fluorescence of the 3-hydroxyflavone complex is enhanced on binding to the purified, reconstituted V-ATPase, and tributyltin reduces this fluorescence enhancement, though not strictly competitively. Radioiodinated derivatives of galangin and morin were synthesized and their organotin complexes were crosslinked to the ATPase by ultraviolet irradiation. Subunit A (72 kDa) of the V-ATPase was labelled, and tributyltin strongly inhibited this labelling. Subunits M115 and M39 were labelled less strongly, and tributyltin did not affect this labelling. We conclude that there is a specific interaction between organotin compounds and V-ATPase subunit A, in the catalytic sector of the enzyme. This approach did not detect the recently-reported interaction between dibutyltin-3-hydroxyflavone bromide and the 16 kDa ‘proteolipid’ subunit of the V-ATPase.

Purification and Characteristics of the Candidate Prohormone Processing Proteases PC2 and PC1/3 from Bovine Adrenal Medulla Chromaffin Granules

The prohormone-processing proteases PC1/3 and PC2 belong to the family of mammalian subtilisin-related proprotein convertases (PC) possessing homology to the yeast Kex2 protease. The presence of PC1/3 and PC2 in secretory vesicles of bovine adrenal medulla (chromaffin granules) implicates their role in the processing the precursors of enkephalin, neuropeptide Y, somatostatin, and other neuropeptides that are present in chromaffin granules. In this study, PC1/3 and PC2 were purified to apparent homogeneity from the soluble fraction of chromaffin granules by chromatography on concanavalin A-Sepharose, Sephacryl S-200, pepstatin A-agarose, and anti-PC1/3 or anti-PC2 immunoaffinity resins. PC1/3 and PC2 were monitored during purification by measuring proteolytic activities with 35S-enkephalin precursor and Boc-Arg-Val-Arg-Arg-methylcoumarin amide (MCA) substrates and by following PC1/3 and PC2 immunoreactivity with specific anti-PC1/3 and anti-PC2 sera generated in this study. Purified PC1/3 and PC2 on SDS-polyacrylamide gels each show a molecular mass of 66 kDa. PC2 in the soluble fraction of chromaffin granules was present at 5- and 10-fold higher enzyme protein and activity, respectively, compared with that of PC1/3. PC1/3 and PC2 cleaved paired basic and monobasic sites within peptide-MCA substrates, with Boc-Arg-Val-Arg-Arg-MCA and pGlu-Arg-Thr-Lys-Arg-MCA as the most effectively cleaved peptides tested. PC1/3 and PC2 showed pH optima of 6.5 and 7.0, respectively. Kinetic studies indicated apparent Km values for hydrolysis of Boc-Arg-Val-Arg-Arg-MCA as 66 and 40 microM, with Vmax values of 255 and 353 nmol/h/mg for PC1/3 and PC2, respectively. Specificity of the PC enzymes for dibasic sites was confirmed by potent inhibition by the active site-directed peptide inhibitors (D-Tyr)-Glu-Phe-Lys-Arg-CH2Cl and Ac-Arg-Arg-CH2Cl. Inhibition by EGTA and activation by Ca2+ indicated PC1/3 and PC2 as Ca(2+)-dependent proteases. In addition, PC enzymes were activated by dithiothreitol and inhibited by thiol-blocking reagents, p-hydroxymercuribenzoate and mercuric chloride. These results illustrate the properties of endogenous PC1/3 and PC2 as prohormone-processing enzymes.

Quinacrine mustard and lipophilic cations inhibitory to both vacuolar H +-ATPase and F 0F 1-ATP synthase

Various lipophilic cations, such as quinacrine mustard and dequalinium, which are known to inhibit mitochondrial F 1-ATPase, strongly inhibited vacuolar H +-ATPase purified from bovine adrenal chromaffin granules. Quinacrine mustard bound irreversibly to vacuolar H +-ATPase subunit A, and the 115 kDa accessory polypeptide and dithiothreitol had no effect. The binding was competitively inhibited by chlorpromazine and quinacrine, and these compounds specifically reduced the amount of labeling of subunit A. Quinacrine mustard also prevented the binding of [α- 32P]ATP to subunit A but had no effect on the binding of [ 3 H]N- ethylmaleimide to either subunit A or the 115 kDa accessory polypeptide. These results suggest that the binding site of quinacrine mustard in subunit A is not related to the N-ethylmaleimide-binding site(s), which is important for activity.

Monoclonal antibodies reactive with a monoamine transporter preparation purified from bovine adrenal chromaffin granule membranes

There have been no reports of monoclonal antibodies reactive with vesicular monoamine transporters from any source. Western blotting and ELISA data obtained using polyclonal serum from a mouse immunized with a highly purified bovine chromaffin granule monoamine transporter preparation yielded data consistent with the presence of antibodies to the transporter. Hybridomas produced by polyethylene glycol fusion of spleen cells from the mouse with X63/Ag8.653 myeloma cells were screened in an ELISA using partially purified transporter as coating agent. Of the 1142 wells containing colonies, 14 were positive in the initial screen. Hybridomas from wells testing positive were transferred to 24-well plates, grown up, and rescreened. Those still testing positive were subcloned, and the resulting positive wells containing single colonies were grown up and stocked. Of the 6 positive clones that tolerated freeze/thaw (0.5% of the wells tested), 1 was IgGI k , 2 were IgG2a k , and 3 were IgG2b k isotypes. Ascites fluid was generated in pristane-primed BALB/c mice using hybridomas that had been cloned 2–3 times, and antibodies purified on immobilized Protein A. Immunoreactivity with a mixture of these antibodies, or with only one of them, coincided with dihydrotetrabenazine (TBZOH) binding activity in fractions eluted from all columns employed in the transporter purification. Antibody from at least one of the clones was capable of removing [ 3H]TBZOH binding activity from a partially purified preparation of transporter. Monoclonal antibodies exhibiting these properties have not been reported previously.

Presence of Laminin B Chain-Like Protein in Bovine and Rat Adrenal Chromaffin Granules

The presence of a glycoprotein laminin in bovine adrenal chromaffin granules was examined by SDS-PAGE followed by immunoblotting. The two chromaffin granule membrane fractions were obtained by linear sucrose gradient centrifugation followed by freezing and thawing and gel-filtration of the chromaffin granule-rich fraction, respectively. The purity of the granules in these fractions was examined by electron microscopy. These fractions contained laminin B chain-like immunoreactivity as a major immunoreactive component against anti-laminin. Laminin A chain-like immunoreactive protein was undetectable. The soluble fraction of the chromaffin granules contained no immunoreactive peptide. The presence of laminin-like immunoreactivity in the chromaffin granules was confirmed by immunocytochemical study. Laminin B chain-like immunoreactivity was also identified in the rat adrenal chromaffin granule fraction. Laminin A chain was hardly detected, as in the case of bovine adrenals. Structure of laminin in chromaffin granules in bovine and rat adrenals may be different from that of mouse Englebrethe-Holm-Swarm sarcoma laminin. The functional significance of laminin B chain-like protein in the granules is unknown at present.

Diadenosine 5′,5′′′- P1, P4-tetraphosphate (Ap 4A), ATP and catecholamine content in bovine adrenal medulla, chromaffin granules and chromaffin cells

The level of diadenosine 5′,5′′′- P 1- P 4-tetraphosphate (diadenosine tetraphosphate or Ap 4A), catecholamines, ATP and other nucleotides has been investigated in perchloric acid extracts of bovine adrenal medulla, chromaffin granules and cultured chromaffin cells. As a control, the amount of Ap 4A and ATP has also been measured in human blood platelets. The following values (nmol/mg protein) were found in adrenal medulla: Ap 4A, 0.019 ± 0.004; ATP, 109 ± 11; ADP, 23.8 ± 5.8; AMP, 11.3 ± 1.5; p 4A, 0.18 ± 0.08; catecholamines, 460 ± 57. The level of Ap 4A, catecholamines and ATP (nmol/mg protein) found in chromaffin granules and in chromaffin cells were, respectively: (0.15 ± 0.07; 2175 ± 99; 531 ± 66) and (0.22 ± 0.14; 1143 ± 277; 222 ± 53). In all the cases investigated, the ratio catecholamines/ATP and catecholamines/Ap 4A were around 5 and in the order of 10 3, respectively. The amount of Ap 4A found here, in bovine adrenal medulla, chromaffin granules and chromaffin cells, is two orders of magnitude lower than previously reported.

Energy-Dependent Accumulation of Neuron Blockers Causes Selective Inhibition of Neurotransmitter Uptake by Brain Synaptic Vesicles

The effects of neuron blockers on neurotransmitter accumulation in synaptic vesicles were investigated. Upon addition of ATP, brain synaptic vesicles accumulated chlorpromazine, haloperidol, and propranonol against concentration gradients of more than 100-fold. Bioenergetic analysis indicated that the transmembrane pH gradient (ΔpH) established by the vacuolar-type H+-ATPase is a direct driving force for these uptakes. Essentially the same results were obtained with vesicles from bovine adrenal chromaffin granules and proteoliposomes reconstituted with purified vacuolar H +-ATPase, indicating that the energy-dependent accumulation is due to diffusion and does not involve transport carriers specific for the blockers. Incubations of the two organelles with the blockers resulted in dissipation of ΔpH and slight increase of membrane potential (ΔΨ) without affecting ATPase activity. Under the same conditions, uptake of dopamine or γ-aminobutyrate (ΔpH-driven transport) was inhibited by neuron blockers, whereas uptake of glutamate (ΔΨ-driven transport) was slightly stimulated. Thus, neuron blockers inhibited ΔpH-driven uptake of neurotransmitter by dissipating the driving force. These results strongly suggest that synaptic vesicles are one of the target sites of neuron blockers.

Characterization of glycoprotein II from bovine adrenal medullary chromaffin granules. Identification of components representing the secretory vesicle counterparts of the lysosomal-associated membrane glycoproteins (lamp-1 and lamp-2)

Glycoprotein II (GpII) is a heterogenous glycoprotein isolated from the membranes of bovine chromaffin granules in the adrenal medulla. When viewed by two-dimensional electrophoresis this glycoprotein consists of two components, upper (GpIIa) and lower (GpIIb), with a molecular mass of 80,000-100,000 daltons and a pI of 4.2-4.7. NH2-terminal sequence analysis of GpIIa and GpIIb revealed sequence similarity with lysosomal membrane glycoproteins (lamp-1 and lamp-2), which was supported by sequence data of peptides from trypsin and cyanogen bromide digestions. An oligonucleotide probe was used to isolate a cDNA clone encoding the nucleotide sequence of GpIIa. The predicted amino acid sequence of GpIIa shares a 72% identity with the human lamp-1 type protein, which belongs to a highly conserved group of lysosomal-associated membrane glycoproteins (lamp proteins), whose function is still unknown. The COOH-terminal region of GpIIa was identical to the COOH-terminal region of lamp proteins. This COOH-terminal determinant has been demonstrated to be essential for the intracellular targeting of lamp proteins to lysosomes. A synthetic peptide antisera to the COOH-terminal region of GpIIa was used to show that this region is present on purified chromaffin granules and not proteolytically processed. The sequence analysis of GpIIa and immunological data confirm GpII as the secretory granule counterpart of lamp proteins and raise some questions regarding intracellular targeting between lysosomes and secretory granules within the chromaffin cell.