Studies on the pituitary "Fettstoffwechselhormon". VIII. Radioimmunoassay of the lipolytic factor P-LF II D.

A radioimmunoassay of the lipolytic peptide P-LF II D from porcine pituitaries is described. The assay is performed with 125-iodine labeled P-LF II D and with antisera either from guinea pigs or from rabbits. Bound antigen is separated from the free by double antibody technique. No cross reaction is observed with gamma lipotropin, peptide B, secretin, glucagon, isoproterenol. Due to contamination P-LF II C, beta lipotropin and human growth hormone displace the tracer when added at large doses. Complete cross reaction is observed between porcine 1-39 ACTH and P-LF II D. Specificity of this reaction is demonstrated by the increase of cross reaction, when ACTH fragments of increasing length of the polypeptide chain are used (1-23 ACTH, 1-24 ACTH and 1-28 ACTH).     

"Joining peptide" of pro-opiomelanocortin. I. Radioimmunoassay and extraction of related peptides from pituitary glands

In order to immunoassay the specific region of bovine pituitary pro-opiomelanocortin (POMC) between ACTH and gamma-MSH, referred to as "joining peptide," antisera were prepared against the synthetic amidated decapeptide Val-Ala-Val-Gly-Glu-Gly-Pro-Gly-Pro-Arg-NH2. The non-amidated peptide represents residues -23 to -14 of bovine POMC. An NH2-terminal tyrosine analog of the decapeptide was used as the radioligand. Under optimal conditions, immunoassay with selected antisera exhibited a sensitivity (50% displacement of the radioligand) toward the decapeptide in the range of 31-55 pg. Immunoreactivity found in extracts of fresh or lyophilized bovine pituitary glands displaced the iodinated Tyr-decapeptide in the RIA in a parallel manner. The amount of immunoreactive (ir)-material was dependent upon the state of preservation of the tissue, the method of extraction, and the particular antiserum used. Extractable immunoreactivity was separated into low (Mr 1,500) and high (Mr 17,000) molecular weight peptides using gel chromatography (G-75). Additional ir-material appeared in the void volume (Mr greater than 22,500). Thus, these antisera have the capacity to interact not only with a region of the joining peptide but also with its larger, and apparent precursor forms. The immunoassay developed should be valuable in understanding the disposition and processing in this specific region of POMC.     

Studies on lysophospholipases. VI. The action of two purified lysophospholipases from beef liver on membrane-bound lysophosphatidylcholine.

The action of two lysophospholipases purified from beef liver on lysophosphatidylcholine in microsomal membranes has been studied. Enzyme I, which has been shown to be localized in the soluble fraction of the beef liver cell, has a higher specific activity on microsomal lysophosphatidylcholine than Enzyme II, which originates from the microsomal cell fraction. This trend is also observed with phosphatidylcholine liposomes and single bilayer vesicles in which lysophosphatidylcholine has been incorporated. At low mol fractions of lysophosphatidylcholine in liposomes, the maximum enzymatic rate is proportional to this mol fraction. Similar results are obtained with mixed micelles of lysophosphatidylcholine and Triton X-100. The results are explained in terms of a model in which the two-dimensional substrate density in the membrane surface controls the rate of enzyme action.     

Preparation of highly purified momordin II without ribonuclease activity.

Momordin II, a ribosome-inactivating protein from Momordica charantia seeds, was purified by a procedure involving a series of chromatographies on S-Sepharose, Sephadex G-50, CM-Sepharose, and Red Sepharose columns. Highly purified momordin II inhibited cell-free protein synthesis, released adenine from rat liver ribosomes and from DNA, and had no RNase activity.     

Preparation of highly purified 3 alpha- and 3 beta-hydroxysteroid dehydrogenases from Pseudomonas sp.

A method is described for preparing highly purified 3α- and 3β-hydroxysteroid dehydrogenases (EC 1.1.1.50 and EC 1.1.1.145, respectively), essentially uncontaminated with one another, from extracts of a steroid-induced Pseudomonas species. These enzymes are suitable for the microanalysis of 3α-hydroxy-, 3β-hydroxy-, and 3-ketosteroids.