V. M. Trikojus

Thyroid acid proteinase Properties and inactivation by diazoacetyl-norleucine methyl ester

Abstract A method is described for the purification of thyroid acid proteinase from extracts of pig thyroid glands. Weight-average molecular weight determinations are used to show that the enzyme undergoes a pH- and temperature-dependent dimerization reaction in 0.10 ionic strength buffers, involving monomers of molecular weight 21 000. The extent of dimerization is maximal at the isoelectric point of the enzyme (pH 7.5) and decreases as the net charge borne by the protein increases. Also, at pH 7.5 an increase of temperature from 1–25° favours dimer formation. The amino acid composition of the proteinase is reported, and it is shown that glycine is the only major component evident in N-terminal analysis. The enzyme is active against haemoglobin as substrate (37° and pH 3.6) and is inhibited to the extent of 90–100% by the pepsin inhibitor, diazoacetyl- dl -norleucine methyl ester. As with pepsin, only one dicarboxylic acid residue appears to be involved in reaction with the inhibitor. Studies with radioactively labelled thyroglobulin (alone and in thyroid extracts) as substrate indicate that the inhibitor is partially effective in preventing proteolysis by acid proteinase of its natural substrate.

Darstellung, Eigenschaften und biologische Wirkungen von Derivaten (Äthern) des Thyroxins, Dijodthyronins und Dijodtyrosins

Den zahlreichen Untersuchungen fiber die Wirkungen des Thyroxins und verwandter Verbindungen stehen nur wenige gegenfiber, die sich mit den Beziehungen zwischen der chemischen Konstitution und dem physiologischen Effekt dieser Substanzen befassen. An erster Stelle sind hier die bekannten Beobachtungen 1 (iber den Einflul~ des Jodatoms bzw. yon Halogen im Thyroxinmolekiil ffir seine Aktivit~t zu nennen. Aus ihnen geht fibereinstimmend hervor, dab das halogenfreie Thyroxin keine thyroxinartige Wirkung mehr besitzt. Ebenso soll auch das decarboxylierte Thyroxin, das Thyroxamin, unwirksam sein ~. Das gleiche gilt yon der dem Thyroxin isomeren Aminoss tiff-all-(3, 5-dijod-4-hydroxyphenyl) -Alanin a. Nach G a d d u m 4 u.a . zeigefi die Thyroxinderivate Glycylund Alanylthyroxin sowie N-Azetylthyroxin und Diazetylthyroxin eine ~ihnliche Wirkung auf den Stoffwechsel yon Rat ten wie Thyroxin selbst. Wird die Aminogruppe der Seitenkette dutch eine Ketogruppe ersetzt, dann nimmt die Wirksamkeit des Thyr0xins ab 5.

Purification and properties of adrenal acid proteinase.

Abstract The proteinase activities at pH 3.5 (haemoglobin substrate) of extracts of a number of bovine tissues, including endocrine glands, have been compared. All endocrine extracts tested were highly active at this pH. Extracts of thyroid and of adrenal glands, when tested for proteinase activity (haemoglobin substrate) over a range of pH values, showed two pH optima, one in the acid and one in the alkaline range, thus resembling extracts of pituitary glands. A proteinase optimally active between pH 3.5 and pH 4.0 has been purified approx. 70-fold from extracts of bovine adrenal glands and some of its properties are reported. The enzyme had no action on any of the synthetic peptide substrates tested.

Studies with purified pig thyroglobulin and thyroid enzymes

Abstract The course of the hydrolysis of pig thyroglobulin by pig thyroid acid proteinase has been followed by estimating the increase and nature of newly exposed N-terminal amino acids. Bonds containing alanine, leucine, tyrosine and phenylalanine have been shown to be most susceptible. Only traces of free amino acids could be detected. By contrast, with [131I]thyroglobulin as substrate, the enzyme has been shown to liberate an average of 25–30% of the total iodinated amino acids in the protein during a 24-h incubation period. N- Acetyl - l - phenylalanyl - l - tyrosine hydrolase—also from pig thyroids—has been shown to act on intermediate peptides, formed during the hydrolysis of the labelled protein by the proteinase, releasing further amounts of mono- and di-iodotyrosines. A similar synergistic action occurs, as regards mono-iodotyrosine, when carboxypeptidase is substituted for N- acetyl - l - phenylalanyl - l - tyrosine hydrolase. The possible relevance of these findings to the hydrolysis in vivo of thyroglobulin is discussed.

A metal-dependent peptidase from thyroid glands

Abstract 1. 1. An enzyme capable of hydrolysing the dipeptide l -cysteinyl- l -tyrosine has been purified 60-fold from bovine-thyroid tissue by means of acetone fractionation, precipitation with zinc acetate and chromatography on calcium phosphate and diethylaminoethyl-cellulose. 2. 2. The purified “cysteinyltyrosinase” ( l -cysteinyl- l -tyrosine hydrolase) was found to hydrolyse a wide range of peptides but was substantially free from proteinase activity as tested against haemoglobin. 3. 3. Preliminary studies with cysteinyltyrosine, leucyltyrosine and leucylglycine suggest that only one enzyme is involved in the hydrolysis of these peptides. 4. 4. Enzymic activity can be inhibited by ethylenediamine tetraacetate and restored by the addition of Zn 2+ and to a lesser extent by Mn 2+ . 5. 5. No evidence was found for the hydrolysis of thyroglobulin by cysteinyltyrosinase leading to the formation of iodinated amino acids, although an enzyme preparation showing high proteinase and low cysteinyltyrosinase activities did effect such release.

Interactions of phospholipids with thyroglobulin and their influence on the enzymic hydrolysis of this protein

Abstract The interactions between purified [125I]thyroglobulin and various phospholipids were examined. Addition of either pea or thyroid phosphatidylinositol (pH range 2.7–4.0) caused precipitation of both interactants; however, pea phosphatidylinositol appeared to be more effective in complex formation. With limiting quantities of phosphatidylinositol, the degree of interaction was maximal at pH 4.0. Phosphatidylserine and phosphatidic acid formed complexes with thyroglobulin over a pH range 3.5–4.0, but the interaction decreased as the pH was lowered. Phosphatidylethanolamine was less effective than the above lipids in precipitating thyroglobulin, while phosphatidylcholine did not interact within the pH range studied. The formation of insoluble complexes with phosphatidylserine and phosphatidylinositol significantly increased the hydrolysis of [125I]thyroglobulin by purified thyroid acid proteinase at pH4.0. Complexes of [125I]thyroglobulin with phosphatidylinositol (formed at pH 4.0) were hydrolysed by mitochondrial/lysosomal particles at pH 5.0 to the same extent as thyroglobulin alone. By contrast, phosphatidylinositol not complexed prior to incubation inhibited the degree of hydrolysis of [125I]thyroglobulin by mitochondrial/lysosomal particles when mixed and incubated at pH 5.0, whereas phosphatidylserine tended to increase hydrolysis, but this effect was neutralized by simultaneous addition of phosphatidylinositol. The possible physiological significance of the results in relation to the hydrolysis of thyroglobulin in vivo is discussed.

Zur Pharmakologie des Thyroxamins

ZusammenfassungEs wurde der Einfluß von Thyroxamin auf das Körpergewicht, auf den Energiestoffwechsel und auf den Kohlehydratstoffwechsel der Leber untersucht und die erhaltenen Ergebnisse mit der Wirkung von Thyroxin verglichen. Dabei zeigte sich, daß zwischen der Wirkung beider Substanzen nur quantitative, aber keine qualitativen Unterschiede bestehen.Die Untersuchungen wurden mit Mitteln der Freiburger Wissenschaftlichen Gesellschaft ausgeführt

Der Einfluß des thyreotropen Hormons der Hypophyse auf den C-Vitamingehalt der Nebennieren und der Leber von Meerschweinchen

ZusammenfassungDer C-Vitamingehalt der Nebennieren vom Meerschweinchen sinkt bei Einwirkung von thyreotropem Hormon ab, gleichzeitig nehmen Ascorbinsäurekonzentration und Reduktionsvermögen der Leber zu. Die Veränderungen sind jedoch gering und gleichen sich bei Dauerzufuhr von thyreotropem Hormon wieder aus. In den Nebennieren sind die Vitaminwerte dabei nachher höher als bei den Kontrolltieren.Die bei der experimentellen Hyperthyreose auftretende Größen- und Gewichtszunahme der Nebennieren werden durch zusätzlich zugeführte Ascorbinsäure nicht unterdrückt.Beziehungen zwischen dem C-Vitamingehalt der Nebennieren und dem Auftreten von Hemmungsstoffen gegen das thyreotrope Hormon konnten nicht nachgewiesen werden.Die Versuche werden fortgesetzt.Ausgeführt mit Hilfe der Deutschen Forschungsgemeinschaft.