Human colony-stimulating factor-1 (CSF-1) was purified from the serum-free conditioned medium of a human pancreatic carcinoma cell line. The four-step procedure included chromatography on DEAE Sepharose, Con A Sepharose and HPLC on phenyl column and reverse-phase C-3 column. The purity of human CSF-1 was demonstrated by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS—PAGE) as a single diffuse band with a molecular weight (Mr) of 42,000-50,000 and was further confirmed by a single amino-terminal amino acid residue of glutamate. Under reducing conditions, purified CSF-1 appeared on SDS-PAGE as a single protein band with a Mr of 21,000-25,000 and concurrently lost its biological activity, indicating that human CSF-1 consists of two similar subunits and that the intact quaternary structure is essential for biological activity. When treated with neuraminidase and endo-8~D~N—acetylglucosaminidase D, the Mr of CSF-1 was reduced to 36,000-40,000 and to a Mr of 18,000-20,000 in the presence of mercaptoethanol.

Bacterial toxins have been shown to modify animal cell proteins in vivo with ADPR. Animal cells also contain endogenous enzymes that can modify proteins. Indirect evidence for the existence in vivo of rat liver proteins modified by ADPR on arginine residues has been reported previously. Presented here is direct evidence for the existence of ADP-ribosylarginine in rat liver proteins. Proteins were subjected to exhaustive protease digestion and ADP-ribosyl amino acids were isolated by boronate chromatography.

In this study, the production of monoclonal antibodies directed against the activating enzyme for an S6 kinase is examined and described. Evidence is presented for the association of an Mr. 55,000 abd Mr. 95,000 protein with the s6 kinase. These proteins are phosphorylated in the presence of Activating Enzyme. A sequence of regulatory events for insulin-stimulated phosphorylation of ribosomal protein S6 in cells is postulated as follows: insulin activates the receptor tyrosine kinase, which phosphorylates the Mr 116,000 subunit of Activating Enzyme. The Activating Enzyme then activates the S6 kniase by phosphorylation, and phosphorylation of the ribosomal protein s6 is promoted.