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.     

The presence of lysophosphatidylcholine in chromaffin granules

Lysophosphatidylcholine is thought to be a characteristic component of the chromaffin granules in adrenal glands. By the use of a t.l.c. system that resolves minor phospholipids satisfactorily, this subcellular location was confirmed in the present study in bovine glands. However, phospholipid degradation was demonstrated in homogenates of the adrenal medulla and cortex under conditions similar to those of subcellular fractionation (incubation at 4°C for 90min). Phosphatidylethanolamine and cardiolipin were hydrolysed, but the concentration of lysophosphatidylcholine did not change, indicating that the latter was present in the medulla before this treatment. Attempts were made to decrease the time between death of the animal and the extraction of lipids. Lysophosphatidylcholine was easily demonstrable in lipid extracts of the dissected medulla and even in those of the whole bovine gland. For practical reasons it is not possible to decrease further the time lapse before extraction in the case of this animal. Adrenal glands were obtained from anaesthetized and untreated rabbits. These were frozen immediately in liquid N₂ and the lipids were extracted. In a control experiment, the glands from rabbit were dissected and treated in the same manner as with those of ox, and then the lipids were extracted. No lysophosphatidylcholine was detected in the extracts from glands frozen in liquid N₂ but lysophosphatidylcholine was observed in the controls. These results suggest that lysophosphatidylcholine is not a component of chromaffin granules, but is produced if the period between death of the animal and lipid extraction is unduly prolonged. To discover whether lysophosphatidylcholine affected the permeability barrier properties of chromaffin granules, sonicated liposomes of egg phosphatidylcholine alone or with lysophosphatidylcholine (15mol/100mol) were prepared. Both types were shown by electron microscopy to be largely made up of single bilayer vesicles. The exchange diffusion of [¹⁴C]dopamine was measured across their membranes. Both types of liposomes had similar capture volumes (0.5μl/μmol of phospholipid), and the activation energies of the exchange diffusion of dopamine were also similar (31kJ/mol). These results indicate that the presence of this proportion of lysophosphatidylcholine in chromaffin-granule membranes is not likely to influence their barrier properties towards catecholamines.     

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.     

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.     

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.     

Peptidylglycine alpha-amidating monooxygenase from bovine heart atrium secretory granules and adrenal chromaffin granules

About 40-60% of the peptidylglycine alpha-amidating amonooxygenase activity in the lysates of secretory granules from bovine atria and adrenal medulla isolated and lyzed in the presence of pepstatin, phenylmethylsulfonyl gluoride, N-ethylmaleimide and catalase, was found to be in the soluble form. The remaining part bound to the membrane fraction was extracted with Triton X-100. The procedure of purification of the soluble form of peptidylglycine alpha-amidating monooxygenase from both atrial and chromaffin granules in electrophoretically homogeneous enzyme preparations was developed. The enzyme is made up of a single subunit with a molecular mass of 68 kDa and contains one copper atom per molecule. The EPR spectra of peptidylglycine alpha-amidating amonooxygenase and dopamine beta-monooxygenase were found to be practically identical, thus indicating that the copper environment in the both enzymes is the same. Both peptidylglycine alpha-amidating monooxygenase and dopamine beta-monooxygenase are inhibited by the neurocuprein apoform, an extremely acidic protein isolated from brain and secretory granules of different endocrine tissues.     

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)     

A unique proenkephalin-converting enzyme purified from bovine adrenal chromaffin granules

A unique proenkephalin converting enzyme specifically generating enkephalin was partially purified from lysates of adrenal chromaffin granules. The enzyme, whose molecular weight is estimated as ca. 220,000, is thiol-dependent protease, with optimal pH at around 5.5. The enzyme converts proenkephalin to enkephalins by cleaving specifically at the sites of consecutive basic amino acid residues. The enzyme also converts BAM-12P, an adrenal “big” Met-enkephalin, to Met-enkephalin in a similar manner. During the enzyme reaction, formation of [Arg⁶]-Met-enkephalin was not observed. Additionally, [Arg⁶]-enkephalins were not converted to enkephalins by the enzyme. Consequently, the enzyme was proved to be a unique converting enzyme distinct from either trypsin-like or carboxypeptidase B-like proteases.     

Specificity and properties of the nucleotide carrier in chromaffin granules from bovine adrenal medulla.

1. The influence of various substances on the uptake of [3H]ATP and [14C]-noradrenaline into isolated bovine chromaffin granules was investigated. The carrier-mediated [3H]ATP uptake is specifically inhibited by SO42-, PO43- and phosphoenolpyruvate. Compounds with carboxylic acid or sulphonic acid groups had no significant inhibitory effects on either uptake. 2. 35SO42-, 32PO43- and phosphoenol[14C]pyruvate are taken up into chromaffin granules by a temperature-dependent process that is inhibited by atractyloside, uncouplers of oxidative phosphorylation and lipid-permeant anions. The apparent Km of 35SO42- uptake is 0.4 mM. 3. These results indicate that the nucleotide carrier in chromaffin granules has a broad specificity, transporting compounds with two strong negative charges. 4. Amino acid probes influence the uptake of ATP and catecholamines differently. Pyridoxal phosphate inhibits both uptake processes, 4,4'-di-isothiocyanostilbene-2,2'-disulphonic acid preferentially blocks ATP uptake, whereas phenylglyoxal blocks only ATP transport. It is suggested that the nucleotide carrier possesses arginine residues in a functionally important position. 5. The significance of these results obtained on isolated granules for the function of chromaffin granules within the cell is discussed.     

Electron probe microanalysis of the subcellular compartments of bovine adrenal chromaffin cells. Comparison of chromaffin granules in situ and in vitro

The elemental and water content of cultured bovine adrenal chromaffin cells and their secretory chromaffin granules have been measured and compared with isolated chromaffin granules using quick freezing, ultracryomicrotomy, and electron microprobe analysis methods. In units of millimole/kilogram dry weight (+/- S.E.) granules in situ contained: P, 523 +/- 32; K+, 124 +/- 9; S, 82 +/- 3; Cl-, 74 +/- 9; Ca2+, 13 +/- 2; Mg2+, 6 +/- 2; and Na+, -2 +/- 2. Following routine isolation in isotonic sucrose buffer, granule K and Cl- had decreased while granule Na+ increased. Cl- exhibited a consistent decrease to 35-40 mmol/kg dry weight. Granule Na+ and K+ concentrations ranged from 43 to 12 mmol/kg and 28 to 60 mmol/kg dry weight, respectively, depending on the Na+ and K+ content of the buffer. Despite the redistribution of monovalent ions, granule Ca2+, granule P, being in the form of ATP, and granule S, being in the form of protein, were not significantly changed. The stability of these elements is consistent with the existence of a stable storage complex for Ca2+, ATP, and protein. Using the granule as an internal standard with a water content of 66%, the water contents of external space, nucleus, cytoplasm, and mitochondria were estimated to be 89, 88, 82, and 70%, respectively. Wet weight concentrations for each element were calculated for granules and cytoplasm from which the transgranular concentration gradients for K+, Cl-, and Na+ were determined. Cl-, a permeant anion, was 2-fold higher in the granule than in the cytoplasm while K+, a slightly permeant cation, had an opposite distribution ratio slightly less than two. Together, the K+ and Cl- data suggest the presence of an inside-positive granule membrane potential of approximately 10-16 mV. The surprising lack of Na+ from the granule matrix suggests a hugh inward gradient for Na+ even though the Na+ content of chromaffin cell cytoplasm is low at 5 mmol/kg water. The lack of an outward Na+ gradient is important in that it indicates that the previously described electroneutral Na+-Ca2+ exchange system, by which isolated granules accumulate Ca2+, does not operate in mature granules in situ. Consequently, if chromaffin granules regulate internal calcium during stimulus secretion coupling, a mechanism other that Na+-Ca2+ exchange is necessary.     

Ion permeability of isolated chromaffin granules.

The passive ion permeability, regulation of volume, and internal pH of isolated bovine chromaffin granules were studied by radiochemical, potentiometric, gravimetric, and spectrophotometric techniques. Chromaffin granules behave as perfect osmometers between 340 and 1,000 mosM in choline chloride, NaCl, and KCl as measured by changes in absorbance at 430 nm or from intragranular water measurements using 3H2O and [14C]polydextran. By suspending chromaffin granules in iso-osmotic media of various metal ions and selectively increasing the permeability to either the cation or the anion by intrinsically permeable ions or specific ionophores, it was possible to determine by turbidity and potentiometric measurements the permeability to the counterion. These measurements indicate that the chromaffin granule is impermeable to the cations tested (Na+, K+, and H+). Limited H+ permeability across the chromaffin granule membrane was also shown by means of the time course of pH re-equilibration after pulsed pH changes in the surrounding media. The measurement of [14C]methylamine distribution indicates that a significant deltapH exists across the membrane, inside acidic, which at an external value of 6.85 has a value of 1.16. The deltapH is relatively insensitive to changes in the composition of the external media and can be enhanced or collapsed by the addition of ionophores and uncouplers. Measurement at various values of external pH indicates an internal pH of 5.5. Use of the ionophore A23187 indicates that Ca++ and Mg++ can be accumulated against an apparent concentration gradient with calcium uptake exceeding 50 nmol/mg of protein at saturation. These measurements also show that Ca++ and Mg++ are impermeable. Measurement of catecholamine release under conditions where intravesicular calcium accumulation is maximal indicates that catecholamine release does not occur. The physiological significance of the high impermeability to ions and the existence of a large deltapH are discussed in terms of regulation of uptake, storage, and release of catecholamines in chromaffin granules.     

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.