Partial purification, characterization and localization of the membrane-associated invertase of yeast.

The membrane-associated isozyme of invertase (beta-D-fructofuranoside fructo-hydrolase, EC 3.2.1.26) -- precursor of the external glycoprotein invertase (Babczinski, P. and Tanner, W. (1978) Biochim. Biophys. Acta 538, 426-434) - has been purified 60-fold from deoxycholate extracts of derepressed yeast cells. The partially purified enzyme exhibits considerable stability as a salt-free lyophilized powder. Its molecular weight, in this precursor form, has been determined by by sodium dodecyl sulphate (SDS) gel electrophoresis to be 180 000 daltons. This correlates well with the presence of only the inner core carbohydrate parts of the external invertase. The enzyme can be split completely by treatment with endo-beta-N-acetyl-glucosaminidase H from Streptomyces griseus, demonstrating the presence of a di-N-acetylchitobiosyl-asparagine linkage. The proteinaceous split product is still active and has a molecular weight of approx. 120 000. The enzyme cannot be transferred into a supernatant fraction upon osmotic shock treatment of yeast membrane vesicles, indicating that it is strictly membrane-bound. After separation of yeast membranes on a sucrose density gradient, precursor invertase is predominantly associated with two gradient membrane fractions which most probably represent rough and smooth endoplasmic reticulum.