Subunit Structure of External Invertase from Saccharomyces cerevisiae

Because 50% of the mass of the external invertase of Saccharomyces cerevisiae consists of carbohydrate, it has been extremely difficult to obtain an accurate molecular weight of this enzyme by centrifugal or electrophoretic techniques. However, on removing almost all of the oligosaccharide chains of this enzyme with the endo-beta-N-acetyl-glucosaminidase H from Streptomyces plicatus, it has been possible to show that carbohydrate-free invertase is composed of two 60,000-dalton subunits. Terminal sequence analysis with carboxypeptidases A, B, and Y provided strong evidence that the subunits are identical.


Lampen
(1) from FH,C strain of Saccharomyces cerevisiae or was purchased from Boehringer Mannheim Corp. as a dry powder containing about 10% enzyme by weight.
No differences were observed between the final products from these sources. The following procedure was used to purify the commercial enzyme.
Step 1 -Three grams of the dry powder were dissolved in 50 ml of 10 mM sodium phosphate, pH 6.5, and dialyzed exhaustively at 4" against several changes of buffer. The specific activity of the dialyzed enzyme was 850 unit.s/mg of protein.
Step 2-The retentate from Step 1 (85 ml; 1.4 x 10" units) was passed over a column (1.5 x 16 cm) of Whatman microgranular DE52 cellulose equilibrated at 4" with 10 rnM sodium phosphate, pH 6.5. The column was washed with 150 ml of this buffer and developed with 1.2 liters of a linear NaCl gradient (0 to 0.2 M in 10 rnM sodium phosphate, pH 6.5) at a flow rate of 40 ml/h. Invertase activity eluted between 50 and 120 rnM NaCl. The column fractions containing invertase with a specific activity greater than 4500 unitsimg of protein were pooled and concentrated with an Amicon ultrafilter. The concentrated enzyme (8 to 10 mg of protein/ml) was dialyzed exhaustively against distilled water and stored frozen. The DE52 fraction represented 75% of the starting activity (1 x 10" units) with a specific activity of 4,700 unitslmg of protein.
Step 3 -When electrophoresed on SDS-5.25% acrylamide gels, the Step 2 enzyme migrated as a major diffuse protein band ranging in size from 90,000 to 160,000 daltons with a trace contaminant at 40,000 daltons.
To remove the latter, 3 ml of the Step 2 enzyme were chromatographed at room temperature on a Bio-Gel P-200 (100 to 200 mesh) column (1.5 x 114 cm) equilibrated with a solution of 10 rnM Tris.HCl, 50 rnM NaCl, pH 7.1. The enzyme eluted at the column void volume, while the contaminant was retarded. The pooled fractions were lyophilized, dissolved in 3 ml of distilled water, and dialyzed against distilled water.
Step 3 invertase migrated as a single, diffuse high molecular weight band on SDS-acrylamide gels and possessed a specific activity of 5100 units/mg of protein.

Enzyme
Purification-The DE52 gradient elution of commercial yeast invertase provides a rapid purification to near homogeneity of large quantities of enzyme. As Fig. 1 shows, the specific activity of invertase is constant through the peak fractions, but its mannose content decreases on the rear side of the elution profile. The heterogeneity of the carbohydrate content of invertase appears to affect its retention by DE52, indicates the sodium chloride concentration through the elution profile. Inset, invertase assays through the peak fractions revealed a constant specific activity (Cl) but the carbohydrate content of the enzyme, measured as the ratio (A) of phenolsulfuric acid color (A,,,) to protein (A,,,), declined. Fractions 35 to 49 (11 and 50 to 66 (II) were pooled as shown.
since carbohydrate-depleted enzyme was not eluted until 0.20 M NaCl (not shown), while the elution of carbohydrate-containing invertase was initiated at 0.05 M NaCl (Fig. 1).
The DE52 enzyme activity (Fig. 1) was collected as two pools consisting of Fractions 37 to 48 (Z) and 49 to 65 (II), which were then concentrated and dialyzed. A portion of each pool was passed separately through Bio-Gel P-200 (Step 3, see "Materials and Methods") to remove a trace of low molecular weight contaminant (not shown). Although the average mannose/protein ratios (w/w) of Z and ZZ as eluted from the P-200 column were 0.85 and 0.62, respectively, the amino acid and glucosamine analyses of each were identical. These results indicate that the carbohydrate fraction of invertase is composed of a heterogeneous population of oligosaccharides, in confirmation of the observations of Tarentino et al.
(3). Pool Z was used for the remaining studies described in this report.
Carbohydrate Removal -On SDS-acrylamide gels, native invertase migrates as a diffuse band between 90,000 and 160,000 daltons, making it impossible to assess either the size or the subunit structure of the protein moiety of this enzyme. We believed that by depleting invertase of its attendant oligosaccharide chains a sharper resolution of this protein on acrylamide gels would be obtained, thus enabling its molecular weight to be more accurately determined.
As shown in a previous study (31, this could be accomplished with endo-P-Nacetylglucosaminidase H from Streptomyces plicatus. Native invertase was, therefore, treated with this endoglycosidase for 16 h and passed through a Sepharose 6B column to separate the protein from the released oligosaccharide chains. Phenolsulfuric acid analysis revealed that the invertase was depleted of 85% of its mannose, but, surprisingly, the endoglycosidasetreated invertase migrated on SDS-acrylamide gels as two equally staining bands of about 65,000 and 68,000 daltons and a minor band of 63,000 daltons (not shown).
Although this result suggested that invertase may be composed of dissimilar subunits, an idea initially advanced by Secondly, exhaustive digestion of endoglycosidase-treated invertase with jack bean meal ol-mannosidase removed about 80% of its residual mannose, after which the resultant protein migrated as a single, somewhat diffuse band on SDS-acrylamide gels with a molecular weight centered at 63,000. Together these observations suggest that although carbohydrate depletion of invertase greatly improved its resolution on SDS-acrylamide gels, there was still sufficient carbohydrate present to impair the separation of distinct subunits. Efforts to remove additional oligosaccharide chains from carbohydrate-depleted invertase by retreatment with endoglycosidase alone or in the presence of either SDS or mercaptoethanol were unsuccessful, although these procedures were shown previously to aid greatly in the removal of oligosaccharide chains from hen ovalbumin (22) and immunoglobulin M (3). To enhance the susceptibility of potentially masked oligosaccharides in the native protein to the action of the endoglycosidase, invertase was denatured, and its sulthydryl groups were carboxymethylated with iodoacetate (71. The resultant protein, although soluble before treatment with the endoglycosidase, came out of solution during the course of digestion with this enzyme. By bringing the reaction mixture to 0.1% with respect to SDS, precipitation of the protein could be prevented without impairing the endoglycosidase. To compare the kinetics and extent of enzymatic oligosaccharide depletion from both native and (Cm)-invertase, separate endoglycosidase digestion mixtures were prepared, and at various times lo-p1 aliquots were withdrawn from each and mixed with 40 ~1 of electrophoresis sample buffer (16). Prior to electrophoresis, each sample was heated at 100" for 3 min and subjected to discontinuous electrophoresis in a flat plate system containing an SDS-lo% acrylamide resolving gel and an SDS-5% acrylamide stacking gel (16). As shown in Fig. 2, native and (Cm)-invertase, before endoglycosidase treatment, display identical diffuse bands ranging from 90,000 to 160,000 daltons. The time course of digestion suggests that oligosaccharide removal from the native enzyme is a slow but progressive process with a family of bands appearing initially between 65,000 and 80,000 daltons and eventually attaining a minimum of 63,000 daltons. In contrast, (Cm)-invertase is rapidly depleted of carbohydrate in only 30 min of endoglycosidase digestion, as indicated by the single sharp band at 63,000 daltons. After 6 h of incubation, the remaining 0.95 ml of each reaction was passed separately through a Sepharose 6B column and analyzed for protein and carbohydrate as depicted in Fig. 3. Comparison of the protein-containing regions clearly demonstrates that carboxymethylation enhances the ability of the endoglycosidase to remove oligosaccharide chains from invertase, as evidenced by the lesser quantity of phenolsul-fur&positive material present in Fig. 3B relative to that in 3A.

Sedimentation
Equilibrium Analysis -A portion of the carbohydrate-depleted (Cm)-invertase was dialyzed against 6 M guanidine . HCl and subjected to sedimentation equilibrium analysis. The results revealed a homogeneous species with a molecular weight of 62,800. Because of the limited solubility of (Cm)-invertase under nondenaturing conditions, its molecular weight could not be obtained. However, samples of native invertase, when treated with both endoglycosidase and (Ymannosidase, were soluble and yielded a molecular weight of 118,000 +-1500. The molecular weight of this preparation was reduced to 59,000 in 6 M guanidine~ HCl. Thus, the external I 2 3 4 5 6 7 8 9 IO II it is often possible to evaluate on SDS-acrylamide gels the number and type of subunits in an oligomeric protein (18). In the case of carbohydrate-depleted (Cm)-invertase, two bands were obtained, one migrating at 61,800 and the other at 123,000 daltons (not shown). This result is also consistent with invertase being composed of two subunits of identical size.

Residual
Carbohydrate Analysis -In order to assess more accurately the sugar composition of invertase fractions, mannose and glucosamine analyses were performed on the native and S-carboxymethylated enzyme before and after endoglycosidase treatment. The results in Table I show 36 glucosamine residues in both the native and (Cm)-invertase which, on the basis of two glucosamines/neutral oligosaccharide chain, indicates 18 chains/holoenzyme or S/subunit. Endoglycosidase treatment of the native enzyme removes seven oligosaccharide chains/subunit, which must be viewed as an average number because of the heterogeneity observed in Fig. 2. All but one oligosaccharide chain is removed by the endoglycosidase from S-carboxymethylated holoenzyme of 120,000 daltons, suggesting that half of the subunits still possess an oligosaccharide chain. By subtracting the weight of the SN-acetylglucosamine residues and the small amount of mannose remaining per -To determine whether the subunits are identical in amino acid composition, (Cm)-invertase was subjected to automatic sequence analysis. However, even after 15 coupling cycles, no amino acids were released, suggesting that the NH,-terminal ends of both subunits are blocked. To circumvent this problem, an analysis of the carboxyl end of invertase was attempted with carboxypeptidases A, B, and Y. Carboxypeptidase B released 1.94 mol of lysine/mol of invertase and no other detectable residues, indicating that each subunit contained lysine at its COOH terminus. A kinetic study with carboxypeptidase A indicated a terminal sequence of Lys-Val-Glu-(Ser or Gin)-Phe-which by the use of a mixture of carboxypeptidases A and B was extended to -Arg-Val-Tyr-. This sequence was confirmed by a timed digestion with carboxypeptidase Y, which yielded 4 additional residues. The final COOH-terminal sequence and the quantitative recovery of residues, in moles/mole of invertase, obtained with the carboxypeptidases are: The excellent quantitative recovery of 2 mol of each amino acid released/m01 of holoenzyme provides strong evidence for the identity of the invertase subunits.
In conclusion, this report demonstrates, as shown earlier (3, 23-25), that endo-/3-N-acetylglucosaminidase H from S. plicatus is a valuable tool for clarifying the structure and composition of glycoproteins.
With the aid of this enzyme it has been possible to demonstrate that the externa'l invertase from S. cerevisiae is composed of two identical 60,000-dalton subunits, to each of which is added an average of nine neutral oligosaccharide chains consisting (3) of a di-iV-acetylchitobiosyl core and 26 to 54 mannose residues.