[Isolation and characterization of an amatoxin-binding protein from wheat germ (author's transl)]. / Isolierung und Charakterisierung eines Amatoxin-bindenden Proteins aus Weizenkeimen.

The preparation and characterization of a protein from wheat germ showing strong affinity to amatoxins (ABP) but differing from RNA polymerase B (II) is described. The purification, traced by [3H]amatoxin (O-methyldehydroxymethyl-alpha-amanitin), comprises 4 chromatographic steps, on Biogel A 1.5, DEAE-Sephadex, phosphocellulose, and again Biogel A 1.5. The protein exhibited in dodecyl sulfate polyacrylamide gel electrophoresis one single band with a molecular mass of 29,000 Da. Its isoelectric point is 4.9. The dissociation constant of the complex ABP-[3H]amatoxin is KD20 = 5 X 10(-7)M as determined by equilibrium dialysis against alpha-amanitin. By cyanogen bromide the protein is split into two fragments with molecular masses of 22,000 and 7,000 Da, respectively whose amino acid analyses, on summation, give the amino acid composition of ABP.

Endocrine GEP-cells in primary testicular teratoma.

Differentiated teratomas frequently contain the apparent equivalent of gastrointestinal mucosa. 53 testicular teratomas were investigated for the incidence of entero-endocrine cells. Enterochromaffin(EC)-cells were demonstrated by formaldehyde induced fluorescence (FIF), while the other endocrine cells were identified by immunohistochemistry. 11 of 53 teratomas contained endocrine cells associated with the gastrointestinal epithelium. The most frequently found cell type was the EC-cell, followed by somatostatin-, glucagon- and pancreatic polypeptide-immunoreactive cells. The teratoma tissue blocks (20 of 53) also frequently exhibited normal testicular tissue which did not contain any EC-cell or other entero-endocrine cells. The results are of interest in considering the cytogenesis of entero-endocrine cells and the histogenesis of testicular carcinoids, indicating that the entero-endocrine cells derive from the intestinal carcinoids, indicating that the entero-endocrine cells derive from the intestinal epithelium arising from undifferentiated stem cells. Furthermore, it seems probable that primary testicular carcinoids can develop from pre-existent teratomas by proliferation of their entero-endocrine cells.

Identification of the amatoxin-binding subunit of RNA polymerase B by affinity labeling experiments. Subunit B3-the true amatoxin receptor protein of multiple RNA polymerase B.

Crude calf thymus DNA-dependent RNA polymerase, RNA polymerase B (ribonucleoside triphosphate: RNA nucleotidyltransferase, EC 2.7.7.6), was incubated with the tritium labeled, potent inhibitor [3H]amanin, in order to form the enzymatically inactive [3H]amanin-polymerase complex ([3H]A-P complex). Subsequent purification procedures for the [3H]A-P complex were based on radioactive assays. Phosphocellulose chromatography separated two radioactive components: PCI, the previously reported amatoxin binding protein, ABP (Brodner and Wieland, 1976), and PC II, the [3H]A-P complex. Sodium dodecyl sulfate gel electrophoresis of the complex showed the presence of a new heavy band very close to subunit B 1 and a decreased intensity of subunit band B 3. These were the only differences noted in the subunit structure of RNA polymerase B. [3H]Amanin was covalently coupled to the enzyme, affinity labeling, by a water-soluble carbodiimide and the resultant conjugate submitted to sodium dodecyl sulfate gel electrophoresis. The profile of radioactivity showed one main peak (greater than 2000 cpm) coinciding with the 550-nm absorption peak of subunit B 3 on a stained parallel gel. Since no other protein band contains any significant radioactivity, the binding site for [3H]amanin and most probably for all amatoxins is localized on the B 3 subunit SB 3.

A carcinoid occurring in the testis.

Carcinoids of the testis are rare tumours developing in three different ways: 1. It may differentiate within a teratoma, 2. it may be a metastasis of a "loco alieno" seated carcubiud abd 3. it may represent a real primary carcinoid. The observation of a primary testicular carcinoid in a man aged 55 years afforded the opportunity to study such a tumor for the first time by electron microscopic and fluorescence microscopic methods. Thereby, it could bw shown, that this testicular carcinoid corresponds to the carcinoids of the lower small gut. According to the specific ultrastructure of the intracytoplasmic granules it must derive histogenetically from an EC-cell. At the moment it cannot be decided whether the primary testicular carcinoid represents an autochthonous tumor of the male gonad or solely a teratoma with one-sided differentiation in the sense of a simplified teratoma.

[Isolation of an amatoxin binding protein (ABP) different from RNA-polymerase B and C ((author's transl))]. / Die Isolierung eines Amatoxin-bindenden Proteins, das von der RNA-Polymerase B und C verschieden ist

During the isolation of the amatoxin RNA-polymerase B-complex from calf thymus tissue we also isolated a protein (ABP) which shows such strong affinity to [3H)amanin that significant binding occurs at low concentrations (10-7M) of the label. The presence of a new amatoxin-complex is demonstrated by coprecipitation of amatoxin and ABP with ammonium sulphate and the common chromatography on phosphocellulose and Sephadex G-25. The new protein ABP is characterized by denaturating sodium dodecylsulphate-gel electrophoresis. The molecular masses of both main bands - possibly subunits of ABP - are determined as 100000 and 10000 - 15000 Dalton and different from the subunit pattern of RNA-polymerases B and C.