Protein phosphorylation and the exocytosis-like interaction between isolated adrenal medullary plasma membranes and chromaffin granules

The interaction between isolated adrenal medullary plasma membranes and chromaffin granules has been proposed as a cell-free model for exocytosis. Phosphorylation experiments showed that isolated chromaffin granules as well as isolated plasma membranes contain protein kinases and phosphate accepting membranous proteins. Upon joint incubation however, the chromaffin granule-located proteins are preferentially phosphorylated. β-ν-methylene-ATP, a non-hydrolysable analogue, was able to reduce both the plasma membrane-induced release of the soluble chromaffin granular content and the phosphate incorporation into the protein fraction. The results of these experiments on a cell-free model system fit in the hypothesis originating from work on several types of intact cells that the exocytotic event is linked with protein phosphorylation.     

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.     

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.     

A putative processing enzyme for proenkephalin in bovine adrenal chromaffin granule membranes. Purification and properties

A putative processing enzyme for proenkephalin, with activity directed toward basic residues, was purified over 2000-fold from washed bovine adrenal medullary chromaffin granule membranes. The molecular mass of this membrane-bound adrenal trypsin-like enzyme (mATLE) is 31 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the enzyme is extremely basic, binding to carboxymethyl-Sephadex at pH 8.5. The pH optimum of mATLE using t-butoxycarbonyl-Glu-Lys-Lys-aminomethylcoumarin as a substrate is 8.5-8.7, and its Km value for this substrate is 2.2 mM. mATLE activity was inhibited by soybean trypsin inhibitor, lima bean trypsin inhibitor, and aprotinin but not by metal chelators or thiol-directed reagents. Sequencing of cleavage products released from Peptide B revealed that the enzyme preferentially cleaves between and following the paired basic residues at positions 23 and 24 of Peptide B (thus generating [Met-enkephalin]-Arg-Phe and Arg-[Met-enkephalin]-Arg-Phe). Dynorphin A was cleaved following a single lysine at position 11 but not at the paired arginine site. Our results suggest that mATLE is a trypsin-like serine protease with the specificity appropriate to that of a proenkephalin processing enzyme.     

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.     

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.     

Purification and properties of an acidic protein from chromaffin granules of bovine adrenal medulla

1. A soluble protein has been purified from an aqueous extract of bovine adrenal chromaffin granules by chromatography on Sephadex G-200. This protein comprises 25% of the total protein of the granules and gave a single band on gel electrophoresis. 2. The protein is unusually rich in acidic amino acids, notably glutamic acid (26.0%, w/w); it is also relatively rich in proline (8.6%, w/w) but poor in cystine (0.35%, w/w). 3. A molecular weight of 77000 was obtained from sedimentation and diffusion measurements on the protein, and approach-to-equilibrium measurements gave apparent molecular weights of the same order. 4. A molecular weight 7 times that given above was estimated from the results of chromatography on a column of Sephadex G-200 that had been calibrated with globular proteins. However, good agreement between the ultracentrifuge and Sephadex experiments was obtained on the assumption that Sephadex chromatography depends on the effective hydrodynamic radii of proteins and not on their molecular weights. 5. The hydrodynamic properties of the protein differed from those of a typical globular protein. Thus the protein had a high intrinsic viscosity, a high frictional ratio and a large effective hydrodynamic volume. 6. The hydrodynamic properties of the protein, but not its molecular weight, were dependent on the ionic strength of the solvent. Increasing the ionic strength caused an increase in the sedimentation and diffusion coefficients, but a decrease in the intrinsic viscosity and in the frictional ratio of the protein. 7. Optical-rotatory-dispersion measurements indicated that only a small part of the polypeptide chain was in an alpha-helical conformation. 8. These results are compatible with the protein's having a conformation approaching that of a random-coil polypeptide, the volume occupied by the molecule being determined by electrostatic repulsion between the excess of negative charges.     

An improved method for the large-scale isolation of chromaffin granules from bovine adrenal medulla

Chromaffin granules essentially free of contamination from mitochondria, lysosomes and fragments of endoplasmic reticulum have been isolated in a large scale from bovine adrenal medulla. The homogeneity was judged by electron microscopy and assays of various ‘marker’ enzymes.     

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.     

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.     

3-O-methyl-D-glucose uptake in isolated bovine adrenal chromaffin cells

The characteristics and regulatory nature of sugar transport in freshly isolated bovine adrenal chromaffin cells were investigated. Transport was measured by following the cell/medium distribution of non-metabolizable glucose analogue, 3-O-methyl-D-glucose. The uptake of 3-O-methyl-D-glucose was was mediated by a saturable transport system with a Km of 8.2 mM and a Vmax of 0.69 nmol/mg protein per min. Basal 3-O-methyl-D-glucose transport was competitively inhibited by D-glucose and a countertransport effect was demonstrated. Cytochalasin B and phloretin, which are specific inhibitors of carrier-mediated glucose transport, significantly decreased basal 3-O-methyl-D-glucose uptake. Basal transport was stimulated by 50 mU/ml insulin, an effect associated with an increase in Vmax. The stimulatory effect of insulin was depressed in medium lacking external Ca2+, or containing the Ca2+-antagonistic ion, La3+, or the Ca2+ channel blocker, methoxyverapamil (D-600). The data suggest that the uptake of 3-O-methyl-D-glucose in freshly isolated bovine adrenal chromaffin cells is mediated by a specific facilitated diffusion mechanism, and is subject to regulation by insulin, thus resembling sugar transport in muscle. In addition, the insulin effect appears to depend on the presence of extracellular Ca2+.     

Flux of catecholamines through chromaffin vesicles in cultured bovine adrenal medullary cells

Primary cultures of bovine adrenal medullary chromaffin cells were pulse-labeled with [3H]dopamine or [3H]norepinephrine and examined for radioactive and total catecholamine contents by high performance liquid chromatography after additional incubations of 15 min to 10 days. [3H]Dopamine was rapidly taken up by chromaffin vesicles in situ and converted to norepinephrine with a half-time of approximately 6 h. [3H] Norepinephrine taken up by the cells was metabolized in three phases. 1) During its brief transit through the cytoplasm, 20 to 35% of this amine was converted to [3H]epinephrine. 2) Following vesicular accumulation, 65 to 70% of the remaining [3H]norepinephrine was methylated to form [3H]epinephrine with a half-time of approximately 30 h, corresponding to the rate of vesicular catecholamine loss from reserpine-treated cells. 3) The residual [3H]norepinephrine decreased with a half-time of 5 days, probably representing loss from norepinephrine-storing cells. [3H]Epinephrine formed endogenously had a half-life in the cultures of approximately 15 days. These data suggest that leakage of norepinephrine from chromaffin vesicles into the cytoplasm limits the rate of dopamine conversion to epinephrine in the adrenal medulla. The kinetic data indicate that approximately 18% of the endogenous norepinephrine and 73% of the endogenous dopamine are present in epinephrine cells.     

The uptake of calcium by isolated chromaffin granules of the adrenal medulla

Bovine chromaffin secretory granules were purified by isopycnic Metrizamide gradient centrifugation and their Ca²⁺ sequestration pathways were characterized. The rate of Ca²⁺ sequestration at 37°C was first order, with a maximal uptake of 26.9 ±0.46 (mean ± S.D., n = 3) nmol Ca²⁺/mg protein and a first order rate constant (k) of 0.046 ± 0.002 min^–1. At 4°C the rate of uptake was substantially attenuated, with only 2.47 ± 0.2 (mean ± S.D, n = 3) nmol Ca²⁺/mg protein sequestered in 60 min. Ca²⁺ sequestration was 93% inhibited by 180 mM NaCl [I_50% of 78.7 ± 9.3 mM NaCl (mean ± S.D., n = 11)] but only slightly inhibited by KCl or MgCl₂. Ca ²⁺ sequestration was not stimulated by incubation with MgATP but was inhibited by 57% after incubation with 30 M monensin. Ca ²⁺ sequestration was dependent on extravesicular Ca ²⁺ with half-maximal sequestration at pCa²⁺ 6.81 ± 0.028 (mean ± S.D., n = 3). Sequestered Ca²⁺ could be exchanged with external ⁴⁵Ca²⁺, the exchange rate was first order (k of 0.042 ± 0.004: mean ± S.D., n = 3) and saturated at 27.7 ± 1.1 nmol Ca²⁺/mg (mean ± S.D., n = 3). The Ca²⁺/Ca²⁺ exchange system was totally inhibited by NaCl or KCl but only slightly by MgCl₂. About 75% of sequestered ⁴⁵Ca²⁺ could be released by incubation with NaCl, but only 8% was released by incubation with KCI. Half-maximal release of sequestered ⁴⁵Ca²⁺ required 69.3 ± 12.2 mM NaCl (mean ± S.D., n = 3). The Na⁺-induced release of sequestered ⁴⁵Ca²⁺ was rapid, t_0.5 of 2.80 ± 0.63 min (mean ± S.D., n = 3) and inhibited at 4°C. The concurrent incubation of chromaffin granules with ⁴⁵Ca²⁺ and either annexin proteins V or VI resulted in attenuated uptake of ⁴⁵Ca²⁺. These results suggest that Ca²⁺ uptake in adrenal chromaffin granules is regulated by Na⁺ and Ca²⁺ gradients and also possibly by annexins V and VI.Abbreviations EGTA ethylene glycol bis (-aminoethyl ether)-N,-N,N,N-tetraacetic acid - SDS Sodium dodecyl sulphate - PAGE Polyacrylamide gel electrophoresis - BSA bovine serum albumin - AI Annexin I - AIIt Annexin II tetramer - AIII Annexin III - AIV Annexin IV - AV Annexin V - AVI Annexin VI - k first order rate constant - AT total extent of Ca²⁺ uptake (nmol) - BufferA 300 mM sucrose, 10 mM potassium phosphate (pH 7.0), 5 mM EGTA - Buffer B 300 mM sucrose, 10 mM potassium phosphate (pH 7.0) and 1 mM EGTA - Buffer C 300 mM sucrose, 10 mM potassium phosphate (pH 7.0) - Buffer D 300 mM sucrose, 10 mM potassium phosphate (pH 7.0), 0.5 mM EGTA and 0.65 MM CaCl₂ - Buffer E 300 mM sucrose, 10 mM potassium phosphate (pH 7.0), 0.25 mM EGTA and 0.325 mM CaCl₂     

The chromaffin granule proton pump and calcium-dependent exocytosis in bovine adrenal medullary cells

Summary Calcium-dependent exocytosis in leaky bovine adrenal medullary cells has a requirement for Mg-ATP. One possibility is that exocytosis depends in some way on the operation of the ATP-dependent proton pump that serves to maintain the core of the secretory vesicles both acid and at a positive potential with respect to the cytosol. This possibility has been tested in leaky cells by monitoring exocytosis under conditions where the secretory vesicle pH and potential gradients are measuredin situ. The results show rather clearly that exocytosis can persist, with unchanged Ca-activation kinetics, in the virtual absence both of a difference in pH between the cytosol and secretory vesicle core and also of a difference in potential across the vesicle membrane. The results do not, however, exclude a small modulating effect of vesicle pH or potential on exocytosis and shed no light on whether or not the plasma membrane potential, which is maintained close to zero in these experiments, influences exocytosis.     

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.     

Presence of proenkephalin in chromaffin cells of the frog adrenal gland

Using a specific antiserum to bovine proenkephalin 1–77 (synenkephalin), the distribution of this peptide in the frog adrenal gland has been studied by means of the indirect immunofluorescence technique. Proenkephalin immunoreactivity was found in all chromaffin cells, which also demonstrated enkephalin- and vasoactive intestinal peptide-like immunoreactivity. No nerve endings containing proenkephalin-, enkephalin-, or vasoactive intestinal peptide-like material could be detected. These data suggest a precursor-product mode of biosynthesis for enkephalins in amphibian chromaffin cells. On a phylogenic point of view, they further indicate a high stability of the structure of proenkephalin during the evolution process.