Isolation and characterization of three lethal and hemolytic toxins from the sea anemone Actinia equina L.

Lethal and hemolytic toxins were purified by acetone precipitation, Sephadex G-50, CM-cellulose and CM-Sephadex column chromatography from the tentacles and bodies of the sea anemone Actinia equina. The isolated toxins, with a mol. wt of 19,000 determined by SDS-polyacrylamide gel electrophoresis, differed only slightly in amino acid composition and had a high tryptophan content. The isoelectric points were estimated to be 9.8 for equinatoxin I and 10.5 for equinatoxins II and III. The pure toxins exhibited high lethal potency; the acute i.v. LD50 in mice of equinatoxins I, II and III were 23, 35 and 83 micrograms/kg, respectively. The sigmoidal time course of hemolysis is characteristic of toxins.

Isolation and partial characterization of three lethal and hemolytic toxins from the sea anemone Actinia cari.

Three lethal and hemolytic toxins, caritoxin I, II and III were isolated from the sea anemone Actinia cari. Following controlled autolysis, tentacles were strained through a nylon sieve and thus a crude extract was obtained. The toxins were further purified by Sephadex G-75 gel chromatography. All three toxins which were eluted at the same peak, were separated by ion-exchange chromatography on CM-cellulose. Disc-PAGE revealed electrophoretic homogeneity of caritoxin II. Caritoxin I was obtained in pure form following rechromatography on CM-cellulose. Purified toxins were stable in the pH range 3 - 8. The preliminary determined molecular weight of the toxins was 20 - 25000 daltons. All three toxins are basic proteins. Isoelectric point of caritoxin I was determined as 10.7. The isolated toxins are lethal (LD 5o of caritoxin I is 22 microgram/kg mice, i.v.) and indicate high hemolytic activity with a pH optimum between 8 and 9. Sphingomyelin specifically and irreversibly inhibits the hemolytic activity of caritoxins as well as some other sea anemone cytolytic toxins.

Proteolytic activity in liver cells from mouse, rat, Ehrlich ascites carcinoma bearing mouse and in Ehrlich ascites carcinoma cells.

The activity of intracellular proteinases from Ehrlich ascites carcinoma bearing mice were compared with that from liver cells of normal mice and rats. The activity of intracellular proteinases was measured in the supernatant of Ehrlich ascites carcinoma cells homogenate. The activity of intracellular proteinases in normal mouse and normal rat liver were different at pH 3.5, pH 6.0 and pH 7.5. The activity in liver cells from Ehlrich ascites carcinoma bearing mouse at pH 3.5 was not significantly changed from normal mouse or rat liver cells, however at pH 6.0 and pH 7.5 the activity in the affected liver significantly decreased. The proteolytic activity in the supernatant of Ehrlich ascites tumor cell homogenate was 0.110 E750 mmu/mgN at pH 7.5, 0.154 E750 mmu/mgN at pH 3.5. The proteolytic activity at pH 6.0 was not detected in any experiment.

Intracellular distribution of neutral proteinases and inhibitors in pig leucocytes. Isolation of two inhibitors of neutral proteinases.

Granule and post-granular-supernatant fractions were obtained from pig leucocyte cells by differential centrifugation in 0.34 M sucrose. Granule extract possesses proteinase activity at acid and at neutral pH. Three groups of neutral and a group of acid proteinases were isolated from granule extracts by chromatography on DEAE-cellulose. In the first group are present elastase-like and plasminogen-activator proteinases, that are inhibited by diisopropylphosphorofluoridate, alpha1-antitrypsin, intracellular leucocyte inhibitor and partly with p-aminomethylbenzoic acid and Trasylol. The second group of neutral proteinases is unstable under the conditions of isolation used the third group of neutral proteinases comprises collagenases that are inhibited by ethylenediamine tetraacetic acid disodium salt, alpha1-antitrypsin and leucocyte inhibitor. The acid proteinases are inhibited only with pepstatin, up to 90%. In the post-granular supernatant was found the acid proteinase activity towards hemoglobin and casein, and non-stable neutral proteolytic activity towards bovine serum albumin and serum gamma globulin. In the post-granular supernatant also the inhibitors of neutral proteinases were found. By gel filtration on Sephadex G-100 and ion-exchange chromatography on CM-cellulose two inhibitors of neutral proteinases were isolated. The majority of the inhibitor capacity (about 80%) of post-granular supernatant was eluted together with ovalbumin (Mr 43000) and the remainder with cytochrome c (12300). These inhibitors inhibit the granule neutral proteinases, acting on all substrates used, but do not inhibit granule acid proteinase. Inhibition effects of post-granular-supernatant inhibitors on trypsin and chymotrypsin were obtained only when bovine serum albumin was used as substrate. Inhibitors of post-granular supernatant are stable at pH 6-8, but unstable in the pH rnage 2-5 and are thermolabile.