A factor in animal tissues that causes sialyl residues of mucoproteins to react as free sialic acid in the Warren assay.

Abstract

1. The mucin of the Cowper's gland of the boar is a sialomucoprotein similar to submaxillary-gland mucin. When a solution of either mucin has been incubated for 5min or less with a particulate fraction from homogenized uterine endometriumplus-myometrium of the rabbit, 10-20% of sialyl residues (N-acetylneuraminic acid) give a positive Warren reaction for free N-acetylneuraminic acid. The particulate fraction is devoid of neuraminidase and no free (diffusible) N-acetylneuraminic acid appears during incubation. The factor that catalyses the formation of directreading non-diffusible N-acetylneuraminic acid occurs also in liver, kidney and intestinal mucosa of the rabbit. The factor is present in very small (;microsomal') particles and has not yet been solubilized. Homogenates of boar Cowper's gland contain both factor and mucin; thus direct-reading non-diffusible N-acetylneuraminic acid appears when such homogenates are stored. 2. Under optimum conditions 1mg of uterine protein catalyses the formation of 0.05-0.1mumol of direct-reading non-diffusible N-acetylneuraminic acid/min. This activity is considerably higher than the neuraminidase activities of comparable homogenates of animal tissues or of liver lysosomes. The factor is thermostable and its activity shows little variation within (i) the pH range 3-10, (ii) the temperature range 20-37 degrees C. Activity is inhibited strongly by 2,2'-bipyridyl and by ammonium pyrrolidine dithiocarbamate but is unaffected by EDTA. Its action can be simulated by low concentrations of Fe(2+). From this it may be inferred that the factor is a protein-bound from of bivalent iron. A number of pure iron-containing proteins and haemoproteins were completely inactive. The following substrates were not sources of direct-reading non-diffusible N-acetylneuraminic acid: methoxyneuraminic acid, sialyl-lactose, brain gangliosides, and sialoproteins in which N-acetylneuraminic acid is linked to galactose residues. 3. It is proposed that the factor (or Fe(2+)) reacts with the mucin in a manner that renders the C-4-C-5 bond of sialyl residues susceptible to periodate oxidation.