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D Aunis

Publications and source records attributed to D Aunis.

213 records · Page 12Linked to original sources
PubMed1975

[Structural study of dopamine beta hydroxylase purified from human serum].

Dopamine-beta-hydroxylase (DBH) has been purified from human serum. The ensyme appeared to be pure as judged by the criterion of polyacrylamide gel electrophoresis. The native protein has a molecular weight of 250,000. When treated with dithiothreitor, the native protein could be dissociated into species with molecular weight of 64,500 and 32,000. It is suggested that the human circulating DBH is composed of four subunits and that each subunit may be composed of two polypeptide chains.

Dithiothreitol↗
PubMed1975

Human serum dopamine-beta-hydroxylase: purification, molecular weight, presence of sugars and kinetic properties.

1) Dopamine-beta-hydroxylase has been purified from human serum. Ammonium sulphate fractionation was used as the first step, the enzyme being precipated between 30 and 50 percent saturation. For the second step, the enzyme was adsorbed onto, and then eluted from a column of Concanavalin A covalently bound to Sepharose 4B. In the third step the enzyme was further purified by passage through two successive columns of Sephadex G-200. The final step, chromatography on DEAE-cellulose, gave a preparation of the enzyme with a specific activity of 36 mumoles of octopamine formed/30 min per mg of enzyme, representing a purification from the starting serum of 3,000 fold. 200 mug of enzyme could be obtained from 200 ml of serum. 2) The enzyme preparation was found to be pure or, at most, only slightly contaminated (depending on the starting serum) as judged by the criterion of polyacrylamide gel electrophoresis. 3) No cross-reaction could be observed between the human serum enzyme and rabbit antibody against bovine adrenal dopamine-beta-hydroxylase. 4) The molecular weight of the enzyme was found to be 250,000, i.e. less than the bovine adrenal enzyme. 5) Kinetic properties of the enzyme were studied. The mechanism of action of the enzyme was found to be similar to that of the bovine adrenal enzyme. Real kinetic parameters for both tyramine and ascorbate were calculated and found to be the same as those described for the bovine adrenal enzyme. 6) Interaction with Concanavalin A strongly suggests that the serum dopamine-beta-hydroxylase is a glycoprotein.

Ascorbic Acid↗
PubMedDate not supplied

The hypophysis controls expression of SNAP-25 and other SNAREs in the adrenal gland.

SNAP-25 (Synaptosomal Associated Protein of 25 kDa), in association with two other SNARE (soluble NSF attachment protein receptor) proteins, syntaxin and Vesicle Associated Membrane Protein, VAMP, is implicated in regulated and constitutive exocytosis in neurones and neuroendocrine cells. Our previous studies have shown that it is expressed more by noradrenergic than adrenergic chromaffin cells in the rat adrenal gland. Since certain hormones under hypophyseal control play an essential role in determining chromaffin cell phenotype, the present study examined the effect of hypophysectomy on SNAP-25 expression. Hypophysectomy was found by immunoblotting and RT-PCR analysis to increase adrenal gland SNAP-25, syntaxin-1 and VAMP-2 levels, without modifying the relative expression of SNAP-25 isoforms: immunocytochemistry showed a dramatic increase in SNAP-25 expression in former adrenergic chromaffin cells. Since adrenal glucocorticoids are considerably reduced by hypophysectomy, the effect of corticosterone replacement therapy was investigated. This did not change levels of SNAP-25, syntaxin-1 or VAMP-2. SNARE expression was also unmodified in pheochromocytoma cells treated with a synthetic glucocorticoid. In contrast, subcutaneous injection of hypophysectomized rats with thyroid hormone decreased adrenal SNAP-25, demonstrating the potential importance of the pituitary-thyroid axis. The current data thus demonstrate that the hypophysis exerts an inhibitory control on adrenal gland SNARE proteins. They suggest that glucocorticoids are unlikely to be directly responsible for this but provide evidence that thyroid hormones are implicated in this phenomenon. The putative role of hormonal regulation on SNARE function is discussed.

Adrenal Medulla↗
PubMedDate not supplied

Reorganization of alpha-fodrin induced by stimulation in secretory cells.

Spectrin is an ubiquitous protein composed of heterodimers with alpha and beta subunits. It was first described in erythrocyte cell membranes (see ref. 1 for review) and subsequently in brain, intestinal brush borders, kidney, liver and adrenals. Brain spectrin (fodrin) alpha-subunit, responsible for actin binding, has a relative molecular mass (Mr) of 240,000, whereas the beta-subunit, involved in membrane attachment, has an Mr of 235,000 (refs 1, 3, 9-13). The membrane of secretory granules from adrenal chromaffin cells membrane of secretory granules from adrenal chromaffin cells increases the viscosity of F-actin solution, and spectrin-like protein is associated with storage granule and plasma membranes. Here, we report the localization of fodrin in secretory cells using monospecific antibodies against the alpha-subunit of fodrin using indirect immunofluorescence. We find that the alpha-subunit forms an intensely stained continuous ring in the subplasmalemmal region of resting chromaffin cells. On stimulation of the cell with nicotine, high potassium or ionophores in the presence of calcium, fodrin forms patches. This aggregation is inhibited by trifluoperazine, hence the entrance of calcium into cells following cell depolarization seems to be the calmodulin-dependent stimulus initiating patch formation.

Adrenal Medulla↗
PubMedDate not supplied

Anti-alpha-fodrin inhibits secretion from permeabilized chromaffin cells.

Chromaffin cells release catecholamine- and peptide-containing granules by exocytosis, by a mechanism involving movement of secretory granules towards the cell membrane, their apposition to it and the fusion of the granule membrane with the plasma membrane. One of the two subunits of membrane-associated brain spectrin, alpha-fodrin is an actin-binding protein which is found at the periphery of chromaffin cells and may be involved in secretion. Because cultured chromaffin cells can be permeabilized with detergents, giving pores large enough to permit the entry of immunoglobulin molecules, we used permeabilized cells to test the effect of specific antibodies on secretory mechanisms. Incubation of permeabilized cells with polyclonal immunoaffinity-purified monospecific anti-alpha-fodrin antibody or its Fab fragments did not modify basal release but did specifically inhibit Ca2+-induced catecholamine release by exocytosis. Our observations indicate that fodrin and the cytoskeleton participate in the release mechanism.

Adrenal Medulla↗
PubMedDate not supplied

Comparative study of plasma dopamine-beta-hydroxylase activities in noradrenaline-secreting and adrenaline-secreting phaeochromocytomae.

1. Circulating dopamine-beta-hydroxylase activities were measured in two hypertensive patients, with a phaeochromocytoma tumour. Tumours were found to differ by their secretory properties, one secreting noradrenaline, the other adrenaline. After removal of the tumour, the plasma dopamine-beta-hydroxylase activities in both patients gradually decreased to reach a stable value in correlation with urinary, excreted catecholamine levels. The only difference was the rate of the plasma enzyme activity decrease. 2. Thus, it appeared that some phaeochromocytomae are able to secrete dopamine-beta-hydroxylase in addition to catecholamines. Therefore, dopamine-beta-hydroxylase activity measurements may be of interest: (1) in determining secretory properties of phaeochromocytoma tumours, and (2) in following the evolution during the postoperative period.

Adrenal Gland Neoplasms↗