The mammalian G protein rhoC is ADP-ribosylated by Clostridium botulinum exoenzyme C3 and affects actin microfilaments in Vero cells.

Clostridium botulinum C3 is a recently discovered exoenzyme that ADP-ribosylates a eukaryotic GTP-binding protein of the ras superfamily. We show now that the bacterially-expressed product of the human rhoC gene is ADP-ribosylated by C3 and corresponds in size, charge and behavior to the dominant C3 substrate of eukaryotic cells. C3 treatment of Vero cells results in the disappearance of microfilaments and in actinomorphic shape changes without any apparent direct effect upon actin. Thus the ADP-ribosylation of a rho protein seems to be responsible for microfilament disassembly and we infer that the unmodified form of a rho protein may be involved in cytoskeletal control.

Functional modification of a 21-kilodalton G protein when ADP-ribosylated by exoenzyme C3 of Clostridium botulinum.

Exoenzyme C3 from Clostridium botulinum types C and D specifically ADP-ribosylated a 21-kilodalton cellular protein, p21.bot. Guanyl nucleotides protected the substrate against denaturation, which implies that p21.bot is a G protein. When introduced into the interior of cells, purified exoenzyme C3 ADP-ribosylated intracellular p21.bot and changed its function. NIH 3T3, PC12, and other cells rapidly underwent temporary morphological alterations that were in certain respects similar to those seen after microinjection of cloned ras proteins. When injected into Xenopus oocytes, C3 induced migration of germinal vesicles and potentiated the cholera toxin-sensitive augmentation of germinal vesicle breakdown by progesterone, also as caused by ras proteins. Nevertheless, p21.bot was immunologically distinct from p21ras.

The possible role of tyrosinase in malignant growth.

The sera of patients who suffer from melanoma and mammary carcinoma show higher tyrosinase activity than normal sera. The enzyme tyrosinase oxidizes tyrosine to DOPA and also catalizes the oxidation of DOPA to Melanin. The catalytic oxidation might also occur in tyrosine while it is conjugated to other amino acids in a polypeptide or a protein molecule. It is theorized here that gamma globulins and interferons are vulnerable to this catalytic oxidation which eventually denaturates them and diminishes their immunological properties.

A minimal toxicity approach to cancer therapy: possible role of beta-glucuronidase.

Most cancer cells differ from normal cells in that they show higher beta-glucuronidase activity and lower pH of their cytoplasm. Anti-cancer drugs can be designed which take advantage of these gradients to deliver maximal toxicity to tumors and minimal toxicity to normal tissue. Many design criteria are suggested here, the most basic of which is the use of the glucuronide structure, in which glucuronic acid acts as a protective carrier of a toxic fragment which becomes active when split off by the beta-glucuronidase at the tumor site. The high beta-glucuronidase activity in cancer cells is also discussed here as a possible explanation for some of the pathognomonic features of a malignant growth: the automatic proliferation of tumor tissue, the invasion of tumors into adjacent tissue, the metastases to remote sites, and the weak response of the immune system.