The specificity of induction of alpha-galactosidase from Saccharomyces carlsbergensis.

A number of sugars and derivatives have been tested for their ability to induce the synthesis of alpha-galactosidase from Saccharomyces carlsbergensis. Besides galactose and the substrates of the enzyme melibiose, raffinose and stachyose, D-galacturonic acid, L-arabinose, D-tagatose, methyl-alpha-D-galactoside, lactose and isopropyl-beta-D-thiogalactoside were able to act as inducers. Of these, methyl-alpha-D-galactoside, lactose, isopropyl-beta-D-thiogalactoside and L-arabinose have been shown to be gratuitous inducers with which kinetic studies of induction have been carried out. Lactose was the most efficient inducer, giving a maximal differential rate of synthesis of the enzyme of 110 mU/10(7) cells at a concentration of 180 mM, followed by L-arabinose (60 mU/10(7) cells at 40 mM), isopropyl-beta-D-thiogalactoside (43 mU/10(7) cells at 60 mM) and methyl-alpha-D-galactoside (25 mU/10(7) cells at 150 mM). The concentration of inducer required to obtain half-maximal induction was similar for lactose, L-arabinose and isopropyl-beta-D-thiogalactoside and about 5-fold higher for methyl-alpha-D-galactoside. The property of the compounds to act as inducers was compared to their ability to interact with the enzyme and the results discussed in terms of the molecular structures which are recognized by the enzyme and by the induction machinery.

alpha-Galactosidase from Saccharomyces carlsbergensis. Cellular localization, and purification of the external enzyme.

1. The alpha-galactosidase of Saccharomyces carlsbergensis in an inducible enzyme which is localized mainly outside the cell membrane and which is secreted into the culture medium in increasing amounts during the growth cycle. 2. The soluble form of alpha-galactosidase localized inside the cell appears to have the same characteristics as the external one, contrasting with the different forms found in the case of invertase. Although some activity is membrane-bound, this activity, when solubilized with detergent, has the same characteristics as the external form of the enzyme. 3. A procedure has been developed by which the enzyme has been purified using batch adsorption with DEAE-Sephadex and column chromatography in DEAE-Sephadex, DEAE-cellulose and Sephadex G-200, using the supernatant of a culture of Saccharomyces carlsbergensis grown in yeast/nitrogen base complemented with galactose. 4. The purified enzyme, which is homogeneous by chromatographic criteria and polyacrylamide gel electrophoresis, appears to be glycoprotein. 5. Invertase copurifies with the alpha-galactosidase but because of its lower stability, together with the fact that the synthesis of both enzymes can be controlled separately, it was possible to obtain preparations in which the contaminant activity was approximately 1%.

Molecular forms of yeast invertase.

The molecular forms of yeast invertase have been studied. It is shown that by gel filtration on Sephadex G-200 it is possible to demonstrate the presence not only of a light, carbohydrate-free, invertase, and a heavy invertase containing 50% carbohydrate, but also of a continuous spectrum of molecular forms that probably represent the sequential addition of mannose to the light form during the secretion process, which culminates in the formation on the heavy enzyme that is found outside the cytoplasmic membrane. The elution volume-void volume ratio in Sephadex G-200 varies from 1.75 of the light to 1.05 of the heavy invertase. The separation of invertase has also been achieved by ion-exchange chromatography and by isoelectric focusing and is facilitated by removal of the heavy form by ammonium sulphate precipitation. During the protoplasting process the removal of the cell wall is accompanied by the loss of most of the heavy form. Thintermediate forms are exclusively detected inside the protoplast, together with the light invertase and a small amount of heavy invertase. The effect of 2-deoxy-D-glucose and cycloheximide on the biosynthesis and distribution of molecular forms of yeast invertase has also been studied. In the presence of 10 mM glucose Saccharomyces 303-67 repressed cells readily synthesize invertase during the two-hour incubation period. Upon the addition of 2-deoxy-D-glucose, at a concentration of 75 mu g/ml, the observed inhibition in the cells is 60%, but if the activity is measured after breaking the cells, only a 31% inhibition is found, revealing an accumulation of invertase inside the protoplast. 2-Deoxy-D-glucose originates a pile-up of the light and intermediate forms at the expense of the formation of the heavy enzyme, showing that the inhibition of the glycosilation and, therefore, the secretion process, has taken place. In the absence of de novo invertase synthesis originated by cycloheximide, the glycosilation process still takes place as indicated by the accumulation of the heavy form at the expense of the light, carbohydrate-free, enzyme.