Effect of periodate oxidation on the properties of dopamine-β-hydroxylase

Portugal, María Teresa Miras; Ruiz, Angel Santos

doi:10.1016/s0300-9084(77)80251-4

Cited by 8 publications

(5 citation statements)

References 21 publications

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“…Chromogranin A was purified by the isolation of the protein band from polyacrylamide gels after electrophoresis (Aunis et al, 1975). Antisera against chromogranin A (prepared by Dr. Miras-Portugal, Madrid, Spain) have been shown to be specific to bovine chromogranin A by biochemical (Miras-Portugal and Santos-Ruiz, 1977) and immunocytochemical (Aunis et al, 19806) techniques. The immunoglobulin fraction was isolated by ammonium sulphate fractionation (as described above for antitubulin antiserum) and used for indirect immunofluorescence study at I :250 dilution in phosphate-buffered saline.…”

Section: Antiserum To Chromogranin Amentioning

confidence: 99%

Immunocytochemical Study of Microtubules in Chromaffin Cells in Culture and Evidence That Tubulin Is Not an Integral Protein of the Chromaffin Granule Membrane

Bader

Ciesielski-Treska

Thiersé

et al. 1981

Journal of Neurochemistry

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Bovine adrenal chromaffin cells were maintained in culture in Dulbecco's modified Eagle's medium containing 20% foetal calf serum and 10 units per ml of Nerve Growth Factor. Under these conditions, chromaffin cells developed up to five neurites per cell. The neurites showed lateral branches and varicosities along their trunk which ended with thick growth cone-like structures. Cultures of chromaffin cells were stained by indirect immunofluorescence with antibodies against (a) chromogranin A to follow the distribution of chromaffin granules, the catecholamine-storing organelles, and (b) tubulin, to study the microtubular system during outgrowth of neurites. Chromogranin A antibodies showed a very intensely staining punctate pattern, not randomly distributed but localized in neurites. Chromaffin granules were found to migrate from the cell body to reach neurite endings where they were densely packed. Intense staining was also observed in varicosities; a linear arrangement of granules was evident along neurite trunks. Tubulin antibodies decorated a complex network, clearly visible at the cell periphery and also in the growth cone-like structures, in the palm region of the growth cone. Colchicine treatment effected retraction of neurites and disappearance of organized microtubule networks; chromaffin granules were found in the perinuclear region of the cell. Some tubulin (0.2% of total membrane proteins) was found in the purified chromaffin granule membrane preparation; however, this tubulin is probably associated with contaminating plasma membranes. By the criteria of morphology and staining with antitubulin antibodies, adult bovine chromaffin cells in culture display characteristics similar to those of sympathetic neurones. In addition, they showed an exaggerated transport of granules. Adult bovine chromaffin cells in culture offer an excellent model for studying the role of microtubules and the contractile apparatus in relation to cell morphological changes and neurosecretion.

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Section: Antiserum To Chromogranin Amentioning

confidence: 99%

Immunocytochemical Study of Microtubules in Chromaffin Cells in Culture and Evidence That Tubulin Is Not an Integral Protein of the Chromaffin Granule Membrane

Bader

Ciesielski-Treska

Thiersé

et al. 1981

Journal of Neurochemistry

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“…It has also been reported that breakdown of CgA occurs at room temprature [24] due to the presence of acetylcholinetransferase, tyrosine like enzymes in theadrenal medulla and the contamination of lysosomal enzymes, and endogenous proteases in chromaffin granules [11,23]. Therefore, the inactivation of these enzymes and the removal of DBH and other proteases were necessary by boiling the homogenate prior to applying it to column chromatography and the rest of the process was carried out at room temprature.…”

Section: Discussionmentioning

confidence: 99%

“…In the present study we report a simple and highly reproducible method of purification of CgA, a protein of Mr 67 from human and bovine adrenal glands which is stored in catecholamine vesicles of the adrenal medulla. Although CgA does not have any known AB C enzymatic activity [13], the removal of dopamine ~ hydroxylase (D~H) was necessary due to its potential confusion for the properties of CgA and seems to be a more potent immunogen than CgA [19][20][21][22][23]. It has also been reported that breakdown of CgA occurs at room temprature [24] due to the presence of acetylcholinetransferase, tyrosine like enzymes in theadrenal medulla and the contamination of lysosomal enzymes, and endogenous proteases in chromaffin granules [11,23].…”

Section: Discussionmentioning

confidence: 99%

“…Although CgA does not have any known AB C enzymatic activity [13], the removal of dopamine ~ hydroxylase (D~H) was necessary due to its potential confusion for the properties of CgA and seems to be a more potent immunogen than CgA [19][20][21][22][23]. It has also been reported that breakdown of CgA occurs at room temprature [24] due to the presence of acetylcholinetransferase, tyrosine like enzymes in theadrenal medulla and the contamination of lysosomal enzymes, and endogenous proteases in chromaffin granules [11,23]. Therefore, the inactivation of these enzymes and the removal of DBH and other proteases were necessary by boiling the homogenate prior to applying it to column chromatography and the rest of the process was carried out at room temprature.…”

Section: Discussionmentioning

confidence: 99%

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Purification and characterization of chromogranin‐A from the adrenal gland of human and bovine

Kumar

1996

IUBMB Life

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SUMMARYIn the present study, we report a simple, highly reproducible procedure for the purification of chromogranin A. A three step column chromatography of supernatant from human and bovine provided a major single band at Mr 67 and two minor bands at low molecular mass. Identity of the major and minor bands of CgA was confirmed by western blotting using LK2H10, a monoclonal antibody against chromogranin A. Recognition of smaller fragments in the final preparation in both species by antibody to chromogranin A may suggest that these are the proteolytic breakdown product of the main protein. Bovine adrenal gland was found to be many fold richer in chromogranin than human adrenal gland. The simplification of the purification procedure for CgA may now help in elucidating further physiological function of this protein.

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“…Back in Spain, María Teresa also undertook to characterise human DBH present in serum [5,6] and to study its modifications in stress conditions such as hypoglycaemia and cold [7] or in streptozotocin-induced diabetes [8,9]. The fact that this enzyme is released into the bloodstream together with catecholamines was one of the first biochemical proofs that this release takes place by the mechanism of exocytosis [10] and led at that time to investigate whether circulating levels of the enzyme were a good indicator of the activity of the adrenal medulla and peripheral adrenergic nerves [11][12][13].…”

Section: In the Beginning Was Dopamine Beta-hydroxylasementioning

confidence: 99%

María Teresa Miras Portugal: a pioneer in the study of purinoceptors in chromaffin cells

Artalejo

Arribas-Blázquez

Barahona

et al. 2023

Purinergic Signalling

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María Teresa Miras Portugal devoted most of her scientific life to the study of purinergic signalling. In an important part of her work, she used a model system: the chromaffin cells of the adrenal medulla. It was in these cells that she identified diadenosine polyphosphates, from which she proceeded to the study of adrenomedullary purinome: nucleotide synthesis and degradation, adenosine transport, nucleotide uptake into chromaffin granules, exocytotic release of nucleotides and autocrine regulation of chromaffin cell function via purinoceptors. This short review will focus on the current state of knowledge of the purinoceptors of adrenal chromaffin cells, a subject to which María Teresa made seminal contributions and which she continued to study until the end of her scientific life.

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Effect of periodate oxidation on the properties of dopamine-β-hydroxylase

Cited by 8 publications

References 21 publications

Immunocytochemical Study of Microtubules in Chromaffin Cells in Culture and Evidence That Tubulin Is Not an Integral Protein of the Chromaffin Granule Membrane

Immunocytochemical Study of Microtubules in Chromaffin Cells in Culture and Evidence That Tubulin Is Not an Integral Protein of the Chromaffin Granule Membrane

Purification and characterization of chromogranin‐A from the adrenal gland of human and bovine

María Teresa Miras Portugal: a pioneer in the study of purinoceptors in chromaffin cells

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