The isolation of carcinogen-binding protein from livers of rats given 4-dimethylaminoazobenzene

Abstract

1. Three azo-dye-binding proteins were identified in the soluble cell supernatant fraction from livers of rats that had received 4-dimethylaminoazobenzene by intraperitoneal injection. 2. One is basic and was highly purified. It has an isoelectric point of pH8.4 in barbital-sodium chloride buffer, I0.1, an S(20,w) value of 3.5s and a molecular weight determined by Sephadex chromatography of 45000. 3. It does not have N-terminal amino acids with free alpha-amino groups. 4. Digestion with Pronase gives rise to a single azo-dye-bound peptide, which on hydrolysis is shown to contain glycine, alanine, serine, threonine, glutamic acid and aspartic acid. The amino acid that binds the azo-dye was not identified. 5. On starch-gel electrophoresis the basic protein separates into a double band, indicating microheterogeneity. 6. The other two proteins were partially purified and occur in a fraction together. They have isoelectric points near neutrality and a molecular weight as determined by Sephadex chromatography of 13800. 7. The absorption spectra in formic acid of both the basic and the low-molecular-weight proteins are similar. The azonium ion has an absorption maximum at 518mmu and another adsorbed chromogen is present with an absorption maximum at 395mmu.

A simple procedure for the purification of porcine ligandine (Y‐protein)

“…The results of Ketterer [4] and Morey [7] of the amino acid analysis of rat liver ligandine are in close agreement. Proportionally the amino acid composition of porcine ligandine is different: almost equal quantities of serine, glycine, alanine, histidine, and lysine, more threonine, while the other amino acids are present in a lesser amount.…”

“…Chromatography on CM-Sephadex G-50 [4] or TEAE-cellulose [ 131 was not satisfactory in our hands. For further purification we tried the DEAEcellulose and cellulose phosphate columns as described by Morey [7].…”

“…In rats in vivo binding has been described of bromosulphthalein, bilirubin, indocyanine green [2], hydrocortisone metabolites [3], azodye carcinogens and some of their conjugates [4,51, and in vitro binding of radiographic contrast materials [61.…”

“…Isolation methods of rat ligandine have been reported in detail by Ketterer [4] and Morey [7], and in brief by Arias et al [8]. Based on these techniques a simplified method is described for the purification of porcine ligandine.…”

Ligandin in steroidogenically active cells of rat gonads

“…Preparation of monospecific anti-ligandin antibodies has been described in detail (Bannikov et al, 1973). Highly purified preparations of ligandin were isolated according to the method of Ketterer et al, (1967). Sera obtained by immunization of rabbits with these preparations were absorbed by spleen extracts under the control of double immunodiffusion in gel.…”