Maria-Teresa Miras-Portugal

Bovine adrenal medullary dopamine-β-hydroxylase: Studies on the structure

Abstract 1. 1. Amino acid analysis is presented for dopamine-β-hydroxylase (3,4-dihydroxyphenylethylamine, ascorbate :O2 oxidoreductase (hydroxylating), EC 1.14.17.1) isolated from bovine adrenal medulla either by affinity chromatography or by the use of N- cetylpyridinium chloride. It is shown that the amino acid compositions are similar for the enzyme isolated either from the soluble part of the chromaffin granules or from the membranes of the granules. The amino acid composition of dopamine-β-hydroxylase is closely identical to that of chromogranin A. 2. 2. Dansyl end-group analysis suggests that dopamine-β-hydroxylase is composed of polypeptide chains containing leucine residue as the only amino terminal residue. Quantitative estimations showed close to two N-terminal residues per 77 000 subunit. 3. 3. Dopamine-β-hydroxylase was treated with dithiothreitol, and in the presence of 6 M guanidine·HCl, the 77 000 subunit could be dissociated in species with a molecular weight of 37 500. 4. 4. Tryptic and CNBr patterns were obtained. The number of peptides were in agreement with the number of lysine and arginine and methionine, respectively. These results support the view that dopamine-β-hydroxylase is composed of similar or identical subunits, and, along with analysis of the dithiothreitol-treated enzyme, that each subunit could be composed of two similar or identical polypeptide chains.

BOVINE ADRENAL MEDULLARY DOPAMINE‐β‐HYDROXYLASE: STUDIES ON INTERACTION WITH CONCANAVALIN A

Bovine adrenal medullary dopamine‐β‐hydroxylase binds with concanavalin A and forms an enzymically active precipitate. The formation of the insoluble complex is pH‐dependent and can be inhibited by α‐methyl‐D‐mannoside, D‐mannose and D‐glucose. The insoluble complex can be dissociated into two species with α‐methyl‐D‐mannoside. From the results, it appears that the interaction between dopamine‐β‐hydroxylase and concanavalin A is due to the carbohydrate moiety of dopamine‐β‐hydroxylase. This property was used to purify the enzyme from a soluble lysate of chromaffin granules. Of all the proteins contained in the soluble lysate, dopamine‐β‐hydroxylase was the only one to be retained on a column of concanavalin A covalently bound to Sepharose 4B. The preparation of pure dopamine‐β‐hydroxylase exhibits a very high specific activity of 320 μmol of octopamine formed per 30 min per mg of protein.

Bovine adrenal medullary chromogranin A: Studies on the structure and further evidence for identity with dopamine-β-hydroxylase subunit

1. Chromogranin A was purified by the use of polyacrylamide gel electrophoresis. The amino acid composition of chromogranin A appeared to be nearly identical to that reported by other investigators and, moreover, was confirmed to be similar to that of dopamine beta-hydroxylase. 2. Dansyl-end group analysis revealed the presence of leucine as the only amino-terminal residue and quantitative estimations showed the presence of two leucine residues per molecule of 77 000 molecular weight. 3. Tryptic and CNBr patterns were obtained. Data are in good agreement with the concept of two nearly identical polypeptide chains per chromogranin A molecule of mol. wt 77 000. Patterns were compared with those obtained in parallel dopamine beta-hydroxylase and support the idea that chromogranin A and the dopamine beta-hydroxylase subunit are identical. Digestion with leucine amino peptidase gave further additional evidence for this suggestion. 4. Chromogranin A appeared to be free of carbohydrates. No cross-reaction was detected between chromogranin A and rabbit antibody against bovine adrenal dopamine beta-hydroxylase.

Human serum dopamine-β-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.

Human circulating dopamine-Β-hydroxylase and epilepsy

The activity of circulatory dopamine-Β-hydroxylase (DBH) in humans is shown to be lower in some epileptic subjects than in normal subjects. The activity of the enzymes was found to be dramatically low in subjects who experienced an epileptic seizure 24 hrs before DBH activity was determined. The activity varied through the course of epileptic seizures induced by a convulsant drug and these variations might be due to the “en masse” changes of the sympathetic nervous system.

CHANGES OF HUMAN PLASMA DOPAMINE-β-HYDROXYLASE ACTIVITY AFTER INTRAVENOUS ADMINISTRATION OF THEOPHYLLINE

The intravenous administration of theophylline to ten healthy human subjects produced either an increase of circulating plasma dopamine‐β‐hydroxylase or no change. The rise of plasma enzyme activity may reflect the increased peripheral catecholamine release induced by theophylline.

pH-dependent deuterium solvent isotope effects on bovine adrenal medullary dopamine-β-hydroxylase

Abstract The effect of p 2 H upon the dopamine-β-hydroxylase was studied. 2 H 2 O inhibited the enzyme (50 % inhibition in 99.7 % 2 H 2 O. A deuterium solvent isotope effect is observed which seems to correspond to an upward shift of 0.5 unit in both pK a s of an active site group. Our present data confirm the assumption that this group is a histidyl residue. The results are consistent with the interpretation that a proton transfer step is mediated by this group. The proton was demonstrated to come from an ascorbate molecule. Attempts to demonstrate the presence of serine in the active centre were unsuccessful.

Studies on the interaction of dopamine β-hydroxylase from various sources with phytohaemagglutinins

1. Bovine adrenal medulla dopamine beta-hydroxylase, a glycoprotein with terminal mannose residues in carbohydrate moiety, did not precipitate with any lectins tested except concanavalin A. After digestion with neuraminidase, the enzyme was shown to interact with ricin, and a good correlation was found between the amount of liberated sialic acids and the extent of agglutination. This finding show either that more than one type of carbohydrate unit occurs on the protein or that three are multibranched chains in the carbohydrate moiety. 2. Dopamine beta-hydroxylase from human serum, pheochromocytoma and normal adrenal were incubated with concanavalin A, ricin, wheat germ agglutinin and lectins from Dilochos biflorus and Robinia pseudoacacia. 83% of dopamine beta-hydroxylase from pheochromocytoma was precipitated by ricin, whereas the enzyme from human serum precipitated to a lesser extent (5-15%). Neuraminidase digestion of human serum dopamine beta-hydroxylase led to an increase of precipitation with ricin. The low extent of native human serum dopamine beta-hydroxylase precipitation with ricin can be explained by the attack of plasma membrane sialidases of liver cells, whereas the greater ricin precipitation of pheochromocytoma and normal adrenal dopamine beta-hydroxylases could be due to post-mortem effects. The clinical implications of possibility of difference concerning the carbohydrate moiety structures of pheochromocytoma and normal enzymes is discussed.

Plasma dopamine β-hydroxylase in a noradrenalin-secreting pheochromocytoma

Abstract Circulating dopamine β-hydroxylase activity was studied in a case of a hypertensive patient bearing a pheochromocytoma. The tumour secreted noradrenalin. The enzyme activity was found to decrease gradually after removal of the tumour, and to reach a stable value, in correlation with the decrease of urinary catecholamines. It is concluded that some pheochromocytoma tumours are able to secrete dopamine β-hydroxylase.