MULTIPLE FORMS OF 5'-NUCLEOTIDASE FROM LACTATING RAT MAMMARY-GLAND RESULTING FROM THE ASSOCIATION OF THE ENZYME WITH DIFFERENT MEMBRANE-FRACTIONS

Microsomes isolated from rat mammary gland can be separated into two fractions by flotation on a discontinuous sucrose density gradient. The lighter fraction (F1) exhibits by Lineweaver-Burk analysis two forms of 5′-nucleotidase (5′-ribonucleotide phosphohydrolase, EC 3.1.3.5), a low Km form (Km, 22 μM) and a high Km for (Km 133 μM). The heavier fraction (F2) shows only the low Km form. Digitonin treatmetn of F1 followed by reflotation permits an additional fractionation (Huggins, J.W. and Carraway, K.L. (1976) J. Supramol. Struct. 5, 59-63). The digitonin-shifted heavier fraction (F1DF3) is enriched in plasma membrane markers, is depleted of galactosfyltransferase and contains the high Km form of the nucleotidase. The unshifted fraction (F1DF3) is enriched in galactosyltransferase, depleted in plasma membrane markers and contains the low Km form of the nucleotidase. Digitonin treatment and reflotation of F2 permits isolation of a density-shifted fraction (F2DF3) which is enriched in plasma membrane markers and galactosyltransferase and which contains the unchanged low Km form of the nucleotidase. Solubilization of either F1 or F1DF3 with deoxycholate or Triton X-100 results in conversion of essentially all of the nucleotidase to a low Km form. Arrhenius plots of all forms of the enzyme (high Km, low Km, digitonin treated, solubilized) exhibit a temperature transition in the 15-20°C range. In addition membranes exhibiting high Km forms have a transition near 35°C which is abolished upon treatment with detergents. Inhibition of the low Km form of nucleotidase by concanavalin A is highly cooperative (Hill coefficient, 1.9-2.4). The high Km form shows less cooperativity (Hill coefficient, 1.4) in untreated membranes, but a pronounced increase in cooperativity is observed upon solubilization in detergents. We propose that the two enzyme forms are best explained by differences in the association of a single type of enzyme molecule with the different types of membranes. According to this proposal specific membrane enzyme-protein or enzyme-lipid interactions place restrictions on the nucleotidase which result in the higher Km, decreased cooperativity and new thermal transition observed for the enzyme of the F1 and F1DF3 fraction. © 1979.