Henry D. Moon

Partial purification of RNA Polymerase from bovine thymus

Abstract A method is presented for obtaining large quantities of highly purified, stable, mammalian nucleolar and nucleoplasmic RNA polymerase from bovine thymus. The thymus glands were homogenized in a Waring Blendor, and the nuclei were separated by centrifugation, then lysed by incubation at 37 °. The two RNA polymerase enzymes were purified by streptomycin treatment, ammonium sulfate fractionation, DEAE-cellulose chromatography (which separated the two activities), and glycerol gradient centrifugation. The nucleolar enzyme was 400 × purified over the nuclear lysate, while the nucleoplasmic enzyme was 1500 × purified. Glycerol and very high levels of thioglycerol (0.05 m ) were necessary for stability of the enzymes as well as for maximal activity. Phosphate-cellulose chromatography separated from the catalytic portion of the nucleoplasmic (but not the nucleolar) enzyme a protein factor which is necessary for the enzymes stability and which stimulates the activity of the catalytic portion several-fold. The anatomic sites of origin of the two RNA polymerases eluted from the DEAE-cellulose column were established by comparing them with RNA polymerase activities isolated from the nucleoli and nucleoplasm. The two enzymes otherwise behaved like similar enzymes obtained from rat liver nuclei.

Effect of hydrocortisone on human cells in tissue culture.

Summary Hydrocortisone inhibited the growth of cells in a synthetic medium. Degree of inhibition was proportional to concentration of the hormone and duration of exposure. The inhibition in nuclear multiplication was relatively greater than the inhibition of protein synthesis. The cells grown in media with hydrocortisone had larger, more variable nuclei, increased amounts of acid phosphatase and nonspecific esterase and decreased amounts of succinic dehydrogenase and glucose-6-phosphate dehydrogenase. The authors gratefully acknowledge the technical assistance of Miss Virginia Jentoft and Miss Barbara Jennings.

Purification of human elastase.

Summary Elastase was obtained from human pancreas by extraction with sodium acetate buffer, pH 4.5, fractionation with ammonium sulfate, and adsorption on powdered elastin. The enzyme was eluted from elastin with dilute acetic acid and precipitated with ammonium sulfate. An orcein–elastin substrate was used for assay of the enzymatic activity. Maximum activity occurred at pH 8.6 to 8.9. Proelastase was not present in the purified preparations, i.e., there was no enhancement of enzymatic activity upon treatment with trypsin.

ISOLATION OF IMMUNOLOGICALLY COMPETENT LYMPHOCYTES FROM SENSITIZED MOUSE SPLEENS.

Summary Highly purified suspensions of living, immunologically competent lymphocytes were obtained from splenic tissue of mice by destroying the red blood cells with hypotonic saline and separating the nucleated cells by centrifugation. The procedure yielded approximately 40 million lymphocytes from each spleen. Usually 96% (range 90–99%) of the cells were lymphocytes; the rest were other nucleated cells. The viability of lymphocytes was demonstrated by their motility and the exclusion of dye from their cytoplasm. The immunologic competence of these cells was shown by their cytolytic effect on homologous cells in vitro.

Dose-Response of Lymphocytes to Purified, Protein-Free Phytohemagglutinin: Lack of Metabolic Inhibition with Increasing Concentrations

Summary An essentially protein-free preparation of PHA prepared by the method of Goldberg et al. from Phaseolus vulgaris was tested for its ability to stimulate RNA-and DNA-synthesis in lymphocytes. It was noted that there is an increase in RNA- and DNA-synthesis with an increase of the dose of PHA until a plateau is reached. Even increasing the amount of PHA by many-fold does not lead to a decrease in RNA- and DNA-synthesis, which is in contrast to the properties of previously described protein-containing preparations of PHA. It is concluded that the depression of metabolic activity by more than optimal doses of protein-containing PHA preparations is due to toxic contaminants and not due to the RNA- and DNA-synthesis-stimulating principles themselves.

Effect of Follicle-Stimulating Hormone on Gonads of Immature C57 Black Mice.

Summary The responsiveness of mice to injections of a highly purified FSH preparation has been investigated. At a total dose level of 12.5 mg no microscopic changes in the testes of male mice were observed, although some weight increment was evident. In female mice the uterus showed greater sensitivity to hormone injections than did the ovaries.