A peptidyl alpha-amidation activity in chromaffin granules of bovine adrenal medulla

Peptidyl alpha-amidation activity in bovine adrenal medulla has been localized in chromaffin granules by density gradient centrifugation. The activity was found to be both soluble and membrane-associated. Both enzymatic activities were stimulated by the addition of Cu2+ and ascorbate. The pH maximum for alpha-amidation in the chromaffin granules in pH 8.0-8.5. By gel filtration, the soluble enzyme activity appeared as a protein of approx. 40 kDa. It is suggested that this enzyme is involved in the carboxyl-terminal amidation of metorphamide, amidorphin and neuropeptide Y.     

Localization of lysophosphatidylcholine in bovine chromaffin granules

One of the unique features of the chromaffin granule membrane is the presence of about 17 mol% lysophosphatidylcholine. Lysophosphatidylcholine isolated from the granules could be degraded by approx. 94% by lysophospholipase. This result is consistent with chemical analyses data showing that about 9% of this lysophospholipid is 1′-alkenyl glycerophosphocholine.The localization of the acylglycerophosphocholine in the chromaffin granule membrane was studied by using pure bovine liver lysophospholipases. In intact granules only about 10% of the total lysophosphatidylcholine was directly available for enzymic hydrolysis. In contrast, when granule membranes (ghosts) were treated with lysophospholipases approx. 60% of the lysophosphatidylcholine was deacylated. These values did not increase after pre-treatment of intact granules or ghosts with trypsin. Added $\text{1-[1-}{}^{14}\text{C]palmitoyl}\text{-sn-}\text{glycero-3-phosphocholine}$ did not mix with the endogenous lysophosphatidylcholine pool(s) and remained completely accessible to added lysophospholipases.     

Localization of lysophosphatidylcholine in bovine chromaffin granules.

One of the unique features of the chromaffin granule membrane is the presence of about 17 mol% lysophosphatidylcholine. Lysophosphatidylcholine isolated from the granules could be degraded by approx. 94% by lysophospholipase. This result is consistent with chemical analyses data showing that about 9% of this lysophospholipid is 1'-alkenyl glycerophosphocholine. The localization of the acylglycerophosphocholine in the chromaffin granule membrane was studied by using pure bovine liver lysophospholipases. In intact granules only about 10% of the total lysophosphatidylcholine was directly available for enzymic hydrolysis. In contrast, when granule membranes (ghosts) were treated with lysophospholipases approx. 60% of the lysophosphatidylcholine was deacylated. These values did not increase after pre-treatment of intact granules or ghosts with trypsin. Added 1-[1-14C]palmitoyl-sn-glycero-3-phosphocholine did not mix with the endogenous lysophosphatidylcholine pool(s) and remained completely accessible to added lysophospholipases.     

Regulation by Ca2+ of membrane elasticity of bovine chromaffin granules

In a range of [Ca2+] similar to cytosolic transient, a drastic reduction from about 20 dyn/cm to almost zero was observed in the membrane elastic modulus of bovine chromaffin granules, isolated in a solution containing 0.3 M sucrose and 5 mM Hepes at pH 7.0, and measured by combination of osmotic swelling and dynamic light-scattering (DLS) methods. This result suggests that the granule membrane becomes extremely flexible as a prelude to exocytosis.     

The adenosine-triphosphatase activity of adrenal chromaffin granules

1. The preparation of a fraction containing highly purified chromaffin granules from the bovine adrenal medulla is described. 2. The fraction contains an adenosine-triphosphatase activity that is stimulated by Mg²⁺ and that cannot be explained by contamination with mitochondria or microsomes. 3. It is suggested that the adenosine-triphosphatase activity is related to the uptake of cate-cholamines by the chromaffin granules.     

A characterization of the nucleotide uptake by chromaffin granules of bovine adrenal medulla

Chromaffin granules isolated from bovine adrenal gland were incubated with (3)H-labelled nucleotides and [(14)C]noradrenaline to study the uptake of these substances. [(3)H]ATP, [(3)H]ADP and [(3)H]AMP are taken up by these organelles by the same temperature-dependent mechanism. The apparent K(m) for ATP and ADP is 1.4mm, and for AMP it is 2.9mm. The uptake of ATP has a flat pH optimum, whereas the catecholamine uptake increases with more alkaline pH. Atractyloside and carboxyatractyloside are competitive and specific inhibitors of nucleotide uptake, whereas reserpine inhibits only that for catecholamines. Mg(2+) ions activate uptake of both catecholamine and nucleotides, whereas EDTA and N-ethylmaleimide inhibit these processes. Nucleotide and catecholamine uptakes are inhibited by uncouplers of oxidative phosphorylation and by two ATP analogues. NH(4) (+) ions and nigericin in the presence of KCl inhibit only catecholamine uptake. It is concluded that nucleotide uptake, as proposed previously for catecholamine uptake, depends on an electrochemical proton gradient produced by a proton-translocating adenosine triphosphatase localized in the membrane of chromaffin granules. Furthermore, as suggested by the effect of NH(4) (+) and nigericin, catecholamine uptake apparently depends on the chemical part of this gradient, whereas the results for nucleotide uptake are consistent with its dependence on the electrical component.     

The amine transporter from bovine chromaffin granules. Partial purification

We have partially purified the amine transporter from bovine adrenal chromaffin granules in a single step utilizing affinity chromatography. A 5-hydroxytryptamine moiety has been coupled to a Sepharose 4B matrix in a position ortho to the hydroxyl group. When membranes solubilized with sodium cholate are chromatographed on the above matrix a 45,000 Mr polypeptide is highly enriched. The enrichment is dependent on the presence of the proper ligand on the matrix and is inhibited if the column is previously equilibrated with a soluble ligand. Enrichment of the above polypeptide is accompanied by an increase in the specific activity of the transporter as measured by its labeling by 4-azido-3-nitrophenylazo(5-hydroxytryptamine). The ability of reserpine, a competitive inhibitor of binding and transport, to inhibit labeling of the purified transporter correlates well with its known kinetic constants in the native membranes. The polypeptide purified is identical to the one previously identified as the putative transporter based on specific labeling by a photoaffinity label (Gabizon, R., Yetinzon, T., and Schuldiner, S. (1982) J. Biol. Chem. 257, 15145-15150). The results clearly support the contention that the 45,000 Mr peptide is the amine transporter or one of its subunits.     

Characterization and purification of the monoamine transporter of bovine chromaffin granules.

The monoamine transporter of the chromaffin granule membranes can be specifically labeled by the photoaffinity reagent 7-azido-8-[125I]iodoketanserin. The characteristics of the labeled protein have been investigated. Two-dimensional gel electrophoresis of the labeled membranes indicated a MW of about 70,000 and an isoelectric point ranging from 3.8 to 4.6. No clear protein spot was associated with the radioactive material, which migrated between glycoproteins GPII and GPIV. The diffuse aspect of the radioactive material indicated a heterogeneity, which was not modified after a second electrophoresis. This heterogeneity was, at least partially, due to glycosylation of the transporter; neuraminidase treatment increased the protein pI up to 6.3, whereas digestion with N-glycopeptidase markedly decreased the apparent MW, from 70,000 to 50,000. SDS-polyacrylamide gel electrophoresis showed that, at low acrylamide concentrations, the labeled material migrated more rapidly than predicted from the mobility of the markers of molecular weight, a behavior which indicated a marked hydrophobicity of the transporter. The labeled protein was purified to homogeneity by a combination of chromatography on DEAE-cellulose at pH 4.5, on immobilized wheat germ agglutinin, and on hydroxylapatite in the presence of SDS. During this purification, the specific radioactivity was increased by a factor of 300-500, with a yield of 10-20%.     

Solubilization and reconstitution of the catecholamine transporter from bovine chromaffin granules.

The catecholamine transporter from bovine chromaffin granules has been solubilized by using low concentrations of sodium cholate in the presence of phospholipids. The functional solubilized protein has been incorporated into liposomes after removal of the detergent either by gel filtration or by dialysis. Reserpine-sensitive accumulation against a concentration gradient is achieved by artifically imposing a pH gradient across the membrane. In the reconstituted system adenosine 5'-triphosphate (ATP) serves as an energy source only at higher detergent concentrations. The proton-translocating adenosine triphosphatase (ATPase) is solubilized in parallel with the increasing efficiency of ATP as an energy source. Several criteria are proposed to distinguish between carrier-mediated (reserpine sensitive) and unmediated transport in the reconstituted system. The reserpine-sensitive process shows affinity and ss presented in this communication provide further support for the contention that concentrative uptake in biogenic amine storage vesicles is driven by a transmembrane pH gradient, which, in the native system, is generated by a proton-translocating ATPase. Moreover, the assays described provide a tool for the isolation and purification of the transport protein.     

Identification and purification of a functional amine transporter from bovine chromaffin granules

The amine transporter from bovine chromaffin granules has been purified in a functional state. Two isoforms with different pI values have been separated and shown to be active. One with an unusually acidic pI (approximately 3.5) has been shown to be a glycoprotein with an apparent Mr of 80,000. The purified polypeptide catalyzes transport of serotonin upon reconstitution with an apparent Km of 2 microM and a Vmax of 140 nmol/mg/min, 150-200-fold higher than the one determined in the native system. Transport is inhibited by reserpine and tetrabenazine, ligands which bind to two distinct sites on the transporter. These findings suggest that the binding sites for both drugs reside on a single polypeptide. The reconstituted purified transporter binds [3H]reserpine with a biphasic kinetic behavior, KD values of 0.3 and 30 nM and Bmax of 310 and 4200 pmol/mg protein, respectively. In addition, binding of [3H]reserpine is accelerated upon imposition of a pH gradient across the proteoliposome. From these findings it is evident that a single polypeptide catalyzes the various functions of the transporter.     

NADH: (acceptor) oxidoreductase from bovine adrenal medulla chromaffin granules

The NADH:(acceptor) oxidoreductase from membranes of bovine adrenal medulla chromaffin granules has been purified by column chromatography. After solubilization of the membranes with emulphogen, a nonionic detergent, the enzyme was purified by dye-ligand chromatography and gel filtration. The oxidoreductase appeared essentially homogeneous on two gel electrophoretic systems. On polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, the enzyme revealed a dimeric structure with a combined molecular weight of about 55,000. The enzyme eluted as a detergent-lipid-protein aggregate with a Stoke's radius of 43 Å on gel filtration columns in the presence of emulphogen. The amino acid composition of the oxidoreductase was found to be distinct from that of similar enzymes from other organelles. Topographical experiments indicated that the enzyme is a transmembrane protein.     

A soluble lipid.protein complex from bovine adrenal medulla chromaffin granules

A unique soluble lipoprotein has been isolated from aqueous lysates of bovine adrenal medulla chromaffin granules by DEAE-cellulose chromatography and gel filtration. Chloroform/methanol extracts of this complex contain sphingomyelin, lecithin, and cholesterol. Gel filtration in aqueous media indicate an approximate molecular weight of 900,000 for the complex. Incubation with sodium dodecyl sulfate causes dissociation to a low molecular weight polypeptide; prolonged treatment with guanidine HCl does not promote dissociation at all. Amino acid analysis revealed a high content of hydrophobic amino acids. Analysis of the tryptic fingerprint indicates that a single type of polypeptide chain is present. The complex appears to contain approximately five copies of polypeptide per aggregate.     

Kex2-like proteolytic activity in adrenal medullary chromaffin granules.

This study demonstrates the presence of boc-Gln-Arg-Arg-MCA cleaving activity in bovine chromaffin granule membranes that resembles yeast Kex2 proteolytic activity. The chromaffin granule boc-Gln-Arg-Arg-MCA cleaving activity, like Kex2 proteolytic activity, shows calcium dependence, optimum activity at pH 7.5-8.2, inhibition by serine protease inhibitors, and preference for cleavage at the COOH-terminal side of Arg-Arg and Lys-Arg, over Lys-Lys, paired basic residues. Potent inhibition by the active-site directed inhibitor [D-Tyr]-Glu-Phe-Lys-Arg-CK (20 microM) provided further evidence for dibasic residue cleavage site specificity. These results are the first report of endogenous mammalian Kex2-like proteolytic activity that may be related to PC1/PC3 and PC2 enzymes, the newly discovered mammalian homologues of Kex2 protease. It will be important to determine the role of this Kex2-like proteolytic activity in processing the precursors of adrenal medullary neuropeptides.     

The molecular shape of dopamine beta-hydroxylase from chromaffin granules of bovine adrenal medulla

The structural features of the soluble dopamine beta-hydroxylase from chromaffin granules of bovine adrenal medulla were studied using negative staining and platinum shadowing electron microscopic methods. The enzyme was shown to be highly asymmetric as suggested in earlier hydrodynamic studies. The tetramer of the enzyme appeared as four subunits arranged in the shape of a planar rose with an estimated width of 15 nm. A minimum thickness of 3.0 nm for the enzyme monomer was calculated from the shadow length of unidirectionally shadowed molecules. A model composed of four oblate ellipsoid monomers in a tetrameric rose arrangement is proposed for the shape of the dopamine beta-hydroxylase molecule. Two monomers associate edge to edge to form an in-plane dimer and two dimers associate side-by-side with their respective long axes at a slight angle to form a tetramer. Theoretical calculations based on the model are consistent with previous hydrodynamic studies.     

Ascorbic acid regulation of norepinephrine biosynthesis in isolated chromaffin granules from bovine adrenal medulla

The effect of ascorbic acid on the conversion of dopamine to norepinephrine was investigated in isolated chromaffin granules from bovine adrenal medulla. Ascorbic acid was shown to double the rate of [3H]norepinephrine formation from [3H]dopamine, despite no demonstrable accumulation of ascorbic acid into chromaffin granules. The enhancement of norepinephrine biosynthesis by ascorbic acid was dependent on the external concentrations of dopamine and ascorbate. The apparent Km of the dopamine beta-hydroxylation system for external dopamine was approximately 20 microM in the presence or absence of ascorbic acid. However, the apparent maximum velocity of norepinephrine formation was nearly doubled in the presence of ascorbic acid. By contrast, the apparent Km and Vmax of dopamine uptake into chromaffin granules were not affected by ascorbic acid. Norepinephrine formation was increased by ascorbic acid when the concentration of ascorbate was 200 microM or higher; a concentration of 2 mM appeared to induce the maximal effect under the experimental conditions used here. The effect of ascorbic acid on conversion of dopamine to norepinephrine required Mg-ATP-dependent dopamine uptake into chromaffin granules. In contrast to ascorbic acid, other reducing agents such as NADH, glutathione, and homocysteine were unable to enhance norepinephrine biosynthesis. These data suggest that ascorbic acid provides reducing equivalents for hydroxylation of dopamine despite the lack of ascorbate accumulation into chromaffin granules. These findings imply the functional existence of an electron carrier system in the chromaffin granule which transfers electrons from external ascorbic acid for subsequent intragranular norepinephrine biosynthesis.     

Selective regulation of carboxypeptidase peptide hormone-processing enzyme during enkephalin biosynthesis in cultured bovine adrenomedullary chromaffin cells

Bovine adrenomedullary chromaffin cells in culture were incubated with reserpine or forskolin, two agents acting through different mechanisms, which increase cellular [Met]enkephalin levels by 2-fold after 72 h. Cells were harvested and chromaffin granules were purified on a linear sucrose gradient. After reserpine treatment, carboxypeptidase-processing enzyme specific activity in chromaffin granule fractions was stimulated 1.9-fold, and Co2+-stimulated carboxypeptidase specific activity was stimulated 3-fold. The increase in enzyme activity was dependent on the time of reserpine treatment. Forskolin, on the other hand, had no significant effect on carboxypeptidase activity. The differential effects of reserpine and forskolin suggest that the carboxypeptidase-processing enzyme may be selectively regulated during periods of elevated enkephalin formation. Kinetic studies revealed that in cells exposed to reserpine, the Km value for [Met]enkephalin-Arg6 for the Co2+-stimulated carboxypeptidase activity was lowered to 0.136 from 0.447 mM, but there was no change in the Km values of the non-Co2+-stimulated carboxypeptidase activity from reserpine and control groups. Cellular levels of immunoreactive carboxypeptidase-processing enzyme, measured by a radioimmunoassay method, were not altered after reserpine treatment. These data suggest that while the total number of carboxypeptidase enzyme molecules remained constant, there may be a conversion of existing enzyme molecules to a more active form which displays a higher affinity for [Met]enkephalin-Arg6 in the presence of Co2+.     

Purification and characterization of a cathepsin D protease from bovine chromaffin granules.

Purification and potential tachykinin and enkephalin precursor cleaving enzymes from bovine chromaffin granules was undertaken using as substrates the model precursors 35S-(Met)-beta-preprotachykinin [35S-(Met)-beta-PPT] and 35S-(Met)-preproenkephalin [35S-(Met)-PPE]. Purification by concanavalin A-Sepharose, Sephacryl S200, and chromatofocusing resulted in a chromaffin granule aspartyl protease (CGAP) that preferred the tachykinin over the enkephalin precursor. CGAP was composed of 47-, 30-, and 16.5-kDa polypeptides migrating as a single band in a nondenaturing electrophoretic gel system, and coeluting with an apparent molecular mass of 45-55 kDa by size-exclusion chromatography. These results suggest that two forms exist: a single 47-kDa polypeptide and a complex of 30 + 16.5-kDa-associated subunits. CGAP was optimally active at pH 5.0-5.5, indicating that it would be active within the acidic intragranular environment. Cleavage at basic residues was suggested by HPLC and HVE identification of 35S-(Met)-NKA-Gly-Lys as the major acid-soluble product generated from 35S-(Met)-beta-PPT. Neuropeptide K was cleaved at a Lys-Arg basic residue site, as determined by identification of proteolytic products by microsequencing and amino acid composition analyses. Structural studies showed that the three CGAP polypeptides were similar to bovine cathepsin D in NH2-terminal sequences and amino acid compositions, indicating that CGAP appears to be a cathepsin D-related protease or cathepsin D itself. The 47- and 16.5-kDa polypeptides of CGAP possessed identical NH2-terminal sequences, suggesting that the 16.5-kDa polypeptide may be derived from the 47-kDa form by proteolysis.(ABSTRACT TRUNCATED AT 250 WORDS)