The small-scale isolation and characterization of adrenocorticotrophin.

Several methods for isolating adrenocorticotrophin from small quantities of porcine and bovine pituitary tissue are compared. Initial extraction of the hormone by an acid-acetone technique was simpler and more efficient than one employing acetic acid extraction and ether precipitation. Subsequent purification procedures utilizing adsorption of the peptide on to oxycellulose realized the highest yields. CM-cellulose-column chromatography followed by Sephadex-gel filtration were suitable final steps for obtaining highly purified adrenocorticotrophin. The purity of the hormone was demonstrated by determining its amino acid composition, C-terminal analysis, polyacrylamide-gel electrophoresis, chymotrypsin digestion and paper electrophoresis and by radioimmunoassay and bioassay. Adrenocorticotrophin was found to be rapidly destroyed in intact and especially in homogenized glands kept at room temperature. At 4 degrees the rate of destruction was less rapid and at -20 degrees losses were minimal.

Synthesis of malformin by an enzyme preparation from Aspergillus niger.

Yukioka, M. (University of Hawaii, Honolulu), and T. Winnick. Synthesis of malformin by an enzyme preparation from Aspergillus niger. J. Bacteriol. 91:2237-2244. 1966.-An enzyme fraction derived from disrupted Aspergillus cells was able to utilize each of the component labeled amino acids of malformin for the synthesis of this cyclic pentapeptide. The process was stimulated by adenosine triphosphate, K(+), and Mg(++), and was optimal at approximately pH 8.5. It was not affected by inhibitors of protein synthesis (ribonuclease, chloramphenicol, puromycin). There is evidence that cysteine, rather than cystine, was incorporated into peptide linkage, so that the disulfide bridge of malformin was formed subsequently. Although only the d isomers of cysteine and leucine occur in the malformin molecule, the l, as well as the d form of these amino acids, was readily utilized by the enzyme preparation. As in the case of several other microbial peptide systems, it appears that the d enantiomorph can arise from the l isomer at an intermediate stage of polypeptide synthesis.