Purification of plasma membranes of rat mammary gland : Comparisons of subfractions with rat milk fat globule membrane

Partially purified plasma membranes of rat mammary gland, obtained as light (F₁) and heavy (F₂) fractions by flotation on a discontinuous sucrose density gradient, were further fractionated by density perturbation flotation using digitonin to shift the density of the cholesterol-rich portion of the membranes. The shifted fraction (F₁F₃) of digitonin-treated F₁ was highly enriched in 5′-nucleotidase, cholesterol and sialic acid, but free of galactosyltransferase, suggesting that it contained highly purified plasma membranes. The unshifted fraction (F₁DF₁) was enriched in galactosyltransferase and depleted in nucleotidase, cholesterol and sialic acid, suggesting that it contained Golgi fragments. The F₂ fraction shows substantially different behavior. Part of it re-equilibrates to the F₁ position upon reflotation. When treated with digitonin, part of F₂ is shifted to a higher density (F₂DF₃). F₂DF₃ is enriched in 5′-nucleotidase, cholesterol, sialic acid and galactosyltransferase. These properties suggest that this subfraction comes from a plasma membrane containing galactosyltransferase.The sialoglycoproteins of the various fractions were compared with those of rat milk fat globule membrane, which is derived in part from the apical surface of the mammary secretory cell. Dodecyl sulfate (SDS) polyacrylamide gel electrophoresis reveals two major glycoprotein bands (GP-II and GP-III) in F₁DF₃. F₂DF₃ contains these and an additional band of lower mobility (GP-I). Both crude and purified MFGM contain all three bands. Comparisons of peanut lectin receptors by autoradiography of polyacrylamide gels run in SDS and then treated with ¹²⁵I]peanut lectin also suggest that F₂DF₃ is more similar to the milk fat globule membrane than is F₁DF₃. However, analysis of the membrane polypeptides and concanavalin A (ConA) receptors shows no obvious relationship between milk fat globule membrane and any of the isolated mammary membrane fractions. These results indicate that the relationship between the milk fat globule membrane and mammary membranes is complex, possibly involving components not associated with the mammary plasma membrane or only selected components of the plasma membrane.