Shaul Itzhaki

Regulation of uracil utilization for RNA synthesis in Ehrlich ascites tumour cells by glucose metabolism

Abstract The incorporation of [14C]uracil and [14C]orotic acid into RNA of Ehrlich ascites tumour cells in vitro has been studied. Glucose added to the medium enhances the incorporation of uracil progressively to about 35-fold when its concentration is increased to 3–5 mM; the increase in orotic acid incorporation under similar conditions is very small. It is suggested that this is possibly due to the existence of different transport mechanisms operating for the two precursors. Although no label from either precursor has been found in DNA under the in vitro conditions, [14C]thymidine is extensively utilized for DNA synthesis.

Evidence for inhibitors of the cell surface protease guanidinobenzoatase.

Guanidinobenzoatase is a protease present on the surface of tumour cells. The present study describes the isolation of a protein inhibitor of guanidinobenzoatase obtained from extracts of liver and pancreas and purified by affinity techniques. Pancreatic acinar cells have been shown to possess a latent form of guanidinobenzoatase and this latency is due to complex formation with the inhibitor. A fluorescent marker has been employed to demonstrate the presence or absence of the inhibitor on sections of pancreatic tissue. The inhibitor has been shown to be exchangeable with liver and pancreatic inhibitors obtained from different species. It is postulated that these inhibitors may play a role in enzyme control.

Inhibition properties of Sepharose-bound trypsin and a protease on the surface of Ehrlich ascites tumour cells.

Ehrlich ascites cells have been shown to possess a protease with beta-naphthylamidase activity located on the surface of these cells. This enzyme is protected from the inhibitory action of protein inhibitors of trypsin (EC 3.4.21.4) in free solution, but is inhibited by high concentrations of active site-directed inhibitors of trypsin. We believe the protection against inhibition is provided by the location of this protease on the cell surface. We employed a model system of trypsin coupled to Sepharose to demonstrate the protective action of an inert surface, resulting in a marked reduction in inhibition of trypsin-Sepharose, compared to trypsin in free solution, when exposed to both high and low molecular weight inhibitors. This cell protease has been shown to play a role in activation of the zymogen of collagenase exported by tumour cells. This role may have important implications for tumour cell invasion of the intercellular matrix.

Biochemical studies on the nuclei of rat salivary glands stimulated with isoproterenol: Phosphorylation of chromatin proteins

Abstract 1. The phosphorylation in vitro of chromatin proteins was determined in nuclei isolated from submaxillary and parotid glands of rats after injection of isoproterenol. 2. A greater incorporation of radioactivity was observed in the non-histone proteins than in the histones. 3. The phosphorylation of non-histone proteins was diminished during the first 14 hr after isoproterenol injection with a return to control values by 48 hr. 4. A reduction in the non-histone protein/DNA ratio was observed during the period of 24 hr following isoproterenol injection and was particularly marked in the case of the parotid.

The phosphorylation of salivary gland chromatin proteins following treatment of rats with dibutyryl cyclic AMP and dibutyryl cyclic GMP.

Abstract 1. The phosphorylation and protein content of salivary gland chromatin were determined in rats 2 hr after injection of theophylline, dibutyryl cyclic AMP and dibutyryl cyclic GMP. 2. The non-histone protein/DNA ratio was decreased by about 40% in the parotid gland after dibutyryl cyclic AMP injection, but no change was observed in the submaxillary. 3. Both dibutyryl cyclic AMP and dibutyryl cyclic GMP stimulate the phosphorylation of protein above the theophylline control values. However, it appears from combined treatment experiments that both nucleotides exert their action independently.

EVIDENCE FOR THE CARRIAGE OF SILVER BY SULPHADIMIDINE: INHIBITION OF PROTEOLYTIC ENZYMES

1 Trypsin in free solution and trypsin‐sepharose were shown to be inhibited by Ag+ in the absence of Cl‐. 2 In the presence of Cl‐ and absence of a suitable carrier, Ag+ has no inhibitory action on trypsin or chymotrypsin. 3 Sulphadimidine bound Ag+ in the presence of Cl‐, and carried the Ag+ to both trypsin and chymotrypsin in free solution as well as to trypsin‐sepharose leading to the inhibition of all these enzyme systems. 4 The neutral protease of tumour cell surfaces was inhibited by Ag+ transported by sulphadimidine in the presence of Cl‐. 5 Kinetic data demonstrated the requirements for both Ag+ and carrier to effect inhibition, the degree of inhibition being directly related to the molarity of each of these reagents. 6 The known inhibition of trypsin by Ag+ binding to histidine in the active site has been defined in mechanistic terms employing the sulphonamide drug, sulphadimidine, to illustrate this exchange mechanism.