Characterization of tyrosine hydroxylase from bovine adrenal medulla

Tyrosine hydroxylase purified to apparent homogeneity from the soluble fraction of bovine adrenal medulla had an apparent Mr of about 280,000 by Bio-Gel A-1.5m chromatography, and gave a single band with a Mr of 60,000 by sodium dodesyl sulfate polyacrylamide gel electrophoresis. The enzyme is considered to be composed of four identical subunits. Isoelectric point of purified enzyme was pH 6.0. The amino acid composition of the enzyme was characterized by fairly high contents of glutamic acid and alanine residues. The N-terminal amino acid was determined to be glutamic acid.     

Diphosphoinositide metabolism in bovine adrenal medulla.

(1) A phosphatidylinositol kinase (EC 2.7.1.67) of a chromaffin vesicle membrane preparation isolated from bovine adrenal medulla was characterized. Its activity towards endogenous and exogenous phosphatidylinositol was very similar to the kinase activity of the microsomal fraction prepared from the same tissue. (2) Phosphomonoesterase (EC 3.1.3.36) and diesterase activity hydrolysing membrane bound phosphatidylinositol 4-phosphate was located mainly in the microsomal fraction. No hydrolytic activity was present in the vesicle membrane. (3) Phosphorylation of chromaffin vesicle membrane phosphatidylinositol did not increase calcium-binding by the membranes.     

Purification and characterization of dopamine beta-hydroxylase from bovine adrenal medulla.

A new purification procedure that permits large-scale purification of dopamine beta-hydroxylase from bovine adrenal medulla was developed. Whole adrenal medullas were extracted with 0.1% Triton X-100, and the enzyme was purified by precipitation with polyethylene glycol, chromatography on DEAE-cellulose, and adsorption to concanavalin A linked to agarose. The yield of protein and the specific activity were high compared with previously published methods. The enzyme appeared essentially homogenous by the criteria of polyacrylamide gel electrophoresis in the presence or absence of dodecylsulfate, and sedimentation velocity analysis. The purified protein was subjected to amino acid and carbohydrate analyses, and the results were compared with previously published data. We found about 3 mol of copper per mol of protein (tetramer of 290000 daltons). No free sulfhydryl groups could be found. Analysis for NH2-terminal amino acids with [14C]dansyl chloride revealed 2 residues of alanine and 2 residues of serine per tetramer. We found the NH2-terminal amino acid of chromogranin A to be leucine. The results of our analysis for amino acid composition and NH2-terminal amino acids do not support the suggestion that dopamine beta-hydroxylase and chromogranin A contain identical peptide chains.     

Purification and characterization of kinesin from bovine adrenal medulla

Kinesin was purified from bovine adrenal medulla. The sedimentation coefficient was 8.8 S. Sedimentation equilibrium ultracentrifugation studies showed the molecular weight of kinesin to be 300,000. The calculated axial ratio was 1:16. The Stokes radius was estimated to be 8.9 nm by gel filtration. Circular dichroism showed the alpha-helix content to be about 50%. Purified kinesin preparation contained a major polypeptide with a molecular weight of 120,000 and minor ones with molecular weights of 71,000, 68,000, and 65,000. Bovine adrenal kinesin had an ATPase activity which was stimulated severalfold by microtubules to a specific activity of about 0.1 mumol/min.mg. Kinesin molecules adsorbed to a glass slide promoted the movement of microtubules on the glass surface at a rate of about 0.5 micron/s. Immunostaining of EBTr (bovine embryonic trachea fibroblast) cells and bovine adrenal chromaffin cells in interphase with an affinity-purified antibody against the major polypeptide of kinesin showed that some kinesin was located on microtubules and the rest distributed throughout the cytoplasm in a diffuse manner. EBTr cells in mitotic phase gave a staining pattern showing that kinesin was present throughout the cytoplasm with higher concentration in the region of mitotic apparatus.     

Putative enkephalin precursors in bovine adrenal medulla.

Extracts from bovine adrenal medulla and adrenal medullary chromaffin granules were found to contain three proteins, 20,000, 10,000 and 5,000 approximate molecular weights which yield tryptic peptides with opioid activity. The opioid activity of these peptides was demonstrated with a radioreceptor assay and two radioimmunoassays. The three proteins yield the same active peptides all of which are chromatographically distinct from the tryptic opioid nonapeptide β-LPH 61–69, generated by trypsin digestion of pituitary endorphins and their precursors. Furthermore, these endorphins and their precursors do not appear to be present in the adrenal medulla. These findings further support the hypothesis that the enkephalin biosynthetic pathway is distinct from that leading to β-endorphin.     

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.     

Isolation and characterization of the trisialogangliosides from bovine adrenal medulla

Trisialogangliosides were isolated from bovine adrenal medulla by DEAE-Sephadex A-25 and Iatrobeads column chromatography. Their structures were elucidated by sugar analysis, neuraminidase digestion, and permethylation studies. The complete structures of trisialogangliosides, A to D, were identified as follows. A: GT1b, IV3NeuAc, II3 (NeuAc)2-GgOse4Cer. B: GT1b(NeuAc/NeuAc-NeuGc-); IV3NeuAc, II3 (NeuAc alpha 2-8 NeuGc-)GgOse4Cer. C: GT1b (NeuGc/NeuAc-NeuAc-); IV3NeuGc, II3 (NeuAc alpha 2-8 NeuAc-)GgOse4Cer. D: GT1b (NeuAc/NeuGc-NeuGc-); IV3NeuAc, II3 (NeuGc alpha 2-8 NeuGc-)GgOse4Cer. Gangliosides B, C, and D, which contain N-glycolylneuraminic acid, have not previously been reported in the literature.     

An improved method for the purification of kinesin from bovine adrenal medulla.

A method has been developed for the purification of bovine adrenal kinesin combining ion exchange chromatography on phosphocellulose and Mono-Q (FPLC), affinity binding to microtubules in the presence of tripolyphosphate and gel filtration on Superose 6 (FPLC). From 100 g of tissue this procedure yields 200 micrograms of a remarkably pure kinesin as assayed by SDS-PAGE and electron microscopy of rotary shadowed specimens. The enzyme has a Ca++ ATPase of 0.4 mumol/min per mg and a Mg++ ATPase of 0.03 mumol/min per mg in the absence of microtubules. The addition of microtubules (5 microM) activates the Mg++ ATPase activity by almost 70-fold to a value of 1.9 mumol/min per mg. This purification procedure results in a fairly large amount of a remarkably pure adrenal kinesin with high specific activity which is an important improvement over the method previously available.     

Dopamine beta-hydroxylase from bovine adrenal medulla contains covalently-bound pyrroloquinoline quinone

Treatment of homogeneous dopamine beta-hydroxylase (DBH) preparations from bovine adrenals with the inhibitor phenylhydrazine (PH) changed the structureless absorption spectrum of DBH into spectra with a maximum at 350 nm. A product with this absorption spectrum could be detached with pronase, enabling its isolation. It appeared to be the C(5) hydrazone of pyrroloquinoline quinone (PQQ) and PH, as judged from its properties and the fact that it could be transformed into PQQ itself. From the yield obtained a ratio of 0.85 PQQ per enzyme subunit was calculated. In contrast to copper-quinoprotein amine oxidases (EC 1.4.3.6), hydrazone formation in DBH did not require saturation of the mixture with O2. DBH is the first copper-quinoprotein hydroxylase found so far. The implications of this finding for the current views on mechanism of action and inhibition by hydrazines are discussed. The success of the recently developed 'hydrazine method' [(1987) FEBS Lett. 221, 299-304] for all different types of amine oxidoreductases, suggest that the method could also be applied to other enzymes for which hydrazines are inhibitors and where the identity of the cofactors has not been established or the presence of PQQ is suspected.     

Muscarinic receptor subtypes in bovine adrenal medulla

Catecholamine secretion in the bovine adrenal medulla is evoked largely by nicotinic receptor activation. However, bovine adrenal medulla also contain muscarinic receptors that mediate several cell responses. To understand the physiological role of muscarinic receptors in the bovine adrenal medulla it is important to identify the pharmacological subtypes present in this tissue. For this, we analyzed the abilities of differnt selective muscarinic antagonists in displacing the binding of the non-selective antagonist [3H] quinuclidinyl benzylate to an enriched plasma membrane fraction prepared from bovine adrenal medulla. All the selective antagonists bind at least two bindings sites with different affinities. The binding profile of the sites with high proportion is similar to the M2 subtype and those present in low proportion have a M1 profile. However, some variation in the proportion of the sites for the different ligands suggest the presence of the third pharmacological subtype (M3). We conclude that the sites in high proportion (60–80%) correspond to M2 muscarinic subtypes, and the rest is constitute by M1 plus M3 subtypes. The presence of multiplicity of subtypes in the adrenal medulla membranes suggests a diversity of functions of muscarinic receptors in the adrenal gland.Abbreviations [3H]QNB [3H]quinuclidinyl benzylate - HHSiD hexahydro-siladifenidol-hydrochloride - AF-DX 116 11-[[2-(diethylamino)methyl]]-1-piperidinyl]-5,11-dihydro-6H-pyrido[2,3,-b][1,4]benzodiazepin-6-one - 4-DAMP 4-diphenylacetoxy-N-methyl piperidine methobromide     

Tissue specificity of nuclear acidic proteins isolated from bovine brain and adrenal medulla.

Nuclear acidic proteins from bovine brain and adrenal medulla demonstrated dissimilar electophoretic and chemical profiles. The amino acid analysis of the acidic nuclear protein fraction isolated from these tissues revealed some variation in the ratio of acidic to basic amino acids. The estimation of free carboxylic acid groups confirmed the more acidic nature of the brain proteins. In comparing the acrylamide gels either visually or optically, several electrophoretically specific bands were apparent. Although the total number of protein bands from each tissue was approximately the same, the adrenal medulla contained a larger proportion of the more positively charged proteins. These observations are interpreted to indicate that the nuclear acidic protein from brain and adrenal medulla may show functional variation.     

Localization and characterization of aminopeptidase P in bovine adrenal medulla.

Aminopeptidase P (EC 3.4.11.9) is demonstrated for the first time in the cytosolic fraction of chromaffin cells of the bovine adrenal medulla. The enzyme is inhibited by metal chelators and by sulfhydryl-reactive agents, which suggests that both a tightly bound metal ion and a cysteine residue are necessary for enzymatic activity. Aminopeptidase P might be important for the modulation of the biological activity of neuropeptides. Its occurrence in the adrenal chromaffin cells provides a useful tool for studying the function of this unique proline-specific peptidase in neuropeptide processing and secretion.     

Morphological behavior of cultured bovine adrenal medulla capillary endothelial cells.

Bovine adrenal medulla capillary endothelial cells were isolated and cloned, and their morphological behaviors in vitro were examined. In the culture of primary or early passage, one type of colony formed intracellular lumina both on the dish and in the three dimensional collagen gel. Another type proliferated well and showed morphology ranging from slender-shape to cobblestone shape, and were easily cloned. Cloned cells which showed slender-shapes formed tubular network on plastic dish after addition of PMA, OAG or vanadate, and these cells also formed multicellular tubules in the three dimensional collagen gel. However, the formation of diaphragmed fenestrae by these slender-shape clones was rare. One clone which showed cobblestone shape formed diaphragmed fenestrae, when cultured on collagen gel for more than one month. Isolated colonies or clones showed heterogeneity of cell shape, angiogenic behaviors and fenestrae formation.     

Rapid purification and properties of protein kinase C from bovine adrenal medulla

Protein kinase C was purified from bovine adrenal medulla using a rapid procedure which resulted in an approximate 1500-fold increase in specific activity. The characteristics of the enzyme are reported and for the first time it is possible to compare the effects of TPA on secretion from intact and permeabilized cells with the effect of TPA on protein kinase C purified from the same secretory tissue.     

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.