Melvin L. Goldberg

The glucose effect: carbohydrate repression of enzyme induction, RNA synthesis, and glucocorticoid activity -- a role for cyclic AMP and cyclic GMP.

Abstract Feeding a variety of carbohydrates (but not all carbohydrates) to mammals results in blocking the induction of many enzymes involved in amino acid metabolism as well as stimulation of some enzymes which participate in glucose utilization. In addition, glucocorticoid activity, both catabolic and anabolic, is inhibited; alterations in nuclear morphology become apparent, and m-RNA synthesis is greatly depressed. Evidence clearly opposes the mediation of the glucose effect by insulin. In bacteria, similar events following glucose feeding are caused by a 90% drop in cyclic AMP levels. But only a relatively small (20%) reduction occurs in mammals; however, the concentration of the antagonist of cyclic AMP — cyclic GMP — is considerably increased, thereby producing a functional decrease in the activity of cyclic AMP. Some, not all, of the glucose effect can be reproduced by the administration of bromo-cyclic-GMP, indicating that part of the glucose effect is mediated by elevation of the guanosine cyclic nucleotide.

Quantitative assay for submicrogram amounts of protein

Abstract An ultrasensitive protein assay, linear between 0.01 and 0.2 μg protein, is described. In this assay, copper is complexed to protein, excess copper is removed by adsorption to a small Sephadex column, and the copper-protein complex is destroyed by digestion with hydroperoxide. Phenol and chloramine-T are added to the reaction mixture and the copper catalyzes the production of a color-producing reaction between the two compounds. The assay is not affected by low levels of phosphate, Tris, metals, or a tenfold excess of nucleic acid over protein. Reducing agents, sucrose, certain anions, high salt concentrations, and EDTA seriously interfere. The method is about 500 times as sensitive as that of Lowry et al. [ Lowry, O. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951) J. Biol. Chem.193, 265]. Like the biuret procedure, the assay measures copper complexed to the peptide bonds but is probably influenced by other factors, since equivalent amounts of different proteins give similar but not identical amounts of color.

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.

The effects of glucose and cyclic GMP on RNA synthesis and nuclear morphology in starved rats

Feeding rats in diet high in glucose has been demonstrated to inhibit the induction of many enzymes, block the action of glucocorticoids, and, in general, appears to result in decreased cyclic AMP activity. We found that glucose feeding depresses both messenger RNA (mRNA) and non-mRNA synthesis. Electron microscopic examination of the nucleus revealed that glucose feeding decreases the granular component of liver cell nucleoli. It only slightly decreases liver cyclic AMP levels, but produces a sixfold elevation in levels of the cyclic AMP antagonist, cyclic GMP. Administration of bromocyclic GMP, like glucose feeding, depresses mRNA synthesis, but does not simulate the effect of the carbohydrate on nuclear morphology. In addition, glucose feeding halves liver inorganic phosphate and triples ATP levels. Phosphorylation of nuclear proteins, however, remains unaltered. Despite the antagonism between glucose feeding and glucocorticoid activity, the former compound did not change the binding of dexamethasone to liver nuclei.

Cyclic AMP mediates the activity of steroid hormones

Abstract There are two classes of hormones, those derived from amino acids and those with a steroid nucleus. Those from amino acids are said to act by raising intra-cellular levels of cyclic AMP. Because the glucocorticoids (and, in general, all steroid hormones) do not raise tissue levels of cyclic AMP, their activity is not considered to be mediated by this nucleotide. I hypothesize that the role of glucocorticoids is to sensitize the cell to the effector molecule, cyclic AMP. This concept is founded on the following, oft-repeated observations from the literature:- Cyclic AMP can always mimic glucocorticoid activity. Tissues from adrenalectomized animals show greatly diminished sensitivity to cyclic AMP. Hormones which act by elevating cyclic AMP levels, and exogenously added cyclic AMP, exert their effect only at levels much higher than are required in normal tissue. If the ability of cyclic AMP and glucocorticoids to initiate a particular activity is compared, cyclic AMP is the more rapid effector. A system maximally stimulated by cyclic AMP is but slightly enhanced by the addition of a glucocorticoid, whereas a system maximally stimulated by glucocorticoids is strongly stimulated by adding cyclic AMP.

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.

Differences in nuclear ribonucleic acid of human neoplastic and normal tissue.

Summary We observed definite and consistent differences between RNA isolated from neoplastic and normal human tissue by electrophoresis on polyacrylamide gels. The major differences were in the nuclear RNA preparations; here the preparations from neoplastic tissue contained several fractions not found in those from normal tissue. Only minor differences were detected in the RNA patterns of cytoplasmic preparations.