a-galactosidase from Saccharomyces carlsbergensis

Transcription

1 Eur. J. Biohem. 77, (1977) agalatosidase from Saharomyes arlsbergensis Cellular Loalization, and Purifiation of the External Enzyme Pedro S. LAZO, Amparo G. OCHOA, and Santiago GASCON Departamento Interfaultativo de Bioquimia, Universidad de Oviedo (Reeived Deember 2, 1976) 1. The agalatosidase of Saharomyes arlsbergensis is an induible enzyme whih is loalized mainly outside the ell membrane and whih is sereted into the ulture medium in inreasing amounts during the growth yle. 2. The soluble form of agalatosidase loalized inside the ell appears to have the same harateristis as the external one, ontrasting with the different forms found in the ase of invertase. Although some ativity is membranebound, this ativity, when solubilized with detergent, has the same harateristis as the external form of the eniyme. 3. A proedure has been developed by whih the enzyme has been purified using bath adsorption with DEAESephadex and olumn hromatography in DEAESephadex, DEAEellulose and Sephadex G2, using the supernatant of a ulture of Saharomyes arlsbergensis grown in yeast/ nitrogen base omplemented with galatose. 4. The purified enzyme, whih is homogeneous by hromatographi riteria and polyarylamide gel eletrophoresis, appears to be glyoprotein. 5. Invertase opurifies with the agalatosidase but beause of its lower stability, together with the fat that the synthesis of both enzymes an be ontrolled separately, it was possible to obtain preparations in whih the ontaminant ativity was approximately 1 %. A number of enzymes have been desribed to be loalized outside the ell membrane in yeasts. These inlude invertase 111, agalatosidase (melibiase) [2], aid phosphatase 131, gluanases 141, and esterase 151. Considerable attention has been devoted to the problem of the seretion of these maromoleules aross the ell membrane. Sine two of these enzymes (invertase 161 and aid phosphatase [7]) have been purified and have been demonstrated to be glyoproteins, it has been onsidered that the glyoprotein moiety of these maromoleules is involved in the seretory proess, by analogy with the proesses desribed in animal [8] and plant ells [9]. Of speial interest is the ase of invertase, where two [lo] or more [ll, 121 isozymes have been desribed, and where speial emphasis has been plaed on the possible preursorprodut relationship of the internal and external forms of the enzyme. Very few studies of agalatosidase in yeast have been arried out. Nothing is known about its moleular Enzymes. adgalatoside galatohydrolase, agalatosidase or melibiase (EC ) ; BDfrutofuranoside frutohydrolase or invertase (EC ); orthophosphori monoester phosphohydrolase (aid optimum) or aid phosphatase (EC ); 1,3P~gluan gluanohydrolase or laminarinase (EC ). properties and the fators whih ontrol its synthesis, and few data have been reported about its loalization [2] and role in the whole ell [14]. We initiated the study of agalatosidase with the objetive of establishing its nature, its ellular distribution, and the differene, if any, between the internal and external enzyme. This paper desribes our studies on the ellular loalization of the enzyme in two strains of Saharomyes arlsbergensis as well as attempts to establish the identity or dissimilarity of the enzymes loalized inside and outside the ell membrane. We also desribe the proedure by whih it has been possible to obtain a homogeneous preparation of the enzyme using the ulture supernatant of S. arlsbergensis, A further paper desribing the kineti and strutural haraterization of the purified enzyme is in preparation. MATERIALS AND METHODS Materials pnitrophenyladgalatoside, peroxidase, gluose oxidase, odianisidine, pyridoxine, pantothenate and inositol were obtained from Sigma. Yeast/nitrogen

2 376 agalatosidase from Yeast base medium was purhased from Difo. Sephadex G2, Sepharose 6B and diethylaminoethyl Sephadex A5 were obtained from Pharmaia and DEAEellulose from Whatman. The snail juie was obtained from L'Industrie Biologique Franaise (Gennevilliers, Frane). Laminarin was purhased from Koh Light Laboratories and Aquaide I1 from Calbiohem. All the other hemials used were obtained from ommerial soures and were of the highest grade. Miroorganisms The strains used are referred to with their number in the Spanish olletion of yeast ultures (C.E.C.T., Department of Mirobiology, University of Salamana, Spain). Saharomyes arlsbergensis strain 3349 (C.E.C.T. 1323), isolated by Winge and Roberts [15], and Saharomyes arlsbergensis strain (3517 [I61 (C.E.C.T. 1317) were used. The former strain arries the genes Me and gs whih respetively allow it to utilize melibiose as the sole arbon soure and to ferment galatose slowly. The latter strain is able to utilize the sugar raffinose ompletely (and therefore to synthesize invertase and agalatosidase) and is auxotrophi for pantothenate, inositol and pyridoxine. When ultured in a syntheti medium, these ofators were added to ensure growth. To obtain agalatosidase for purifiation, strain 1317 was ultured at 28 "C in yeast/nitrogen base medium omplemented with 1 % galatose and the three ofators in a New Brunswik S. Co. fermentor. agalatosidase Assay agalatosidase was assayed in two different ways. Assay I. This assay was based on the hydrolysis of pnitrophenyladgalatoside. 25 pl of the diluted enzyme was added to 5 pl of.5 M aetate buffer, ph 4.5 and 25 pl of.1 M pnitrophenyladgalatoside. After 5 min of inubation at 3 "C, the reation was stopped by addition of 3 ml of.1 M Na2C3. The olor produed from the liberated pnitrophenol was measured at 41 nm. Assay II. This assay was based on the measurement of the gluose released after the hydrolysis of melibiose at 3 "C. In the first step 25 p1 of the diluted enzyme was added to 5 pl of.5 M aetate buffer, ph 4.5, and 25 p1 of.1 M melibiose. The reation was stopped after 5 min by addition of 3 pl of.2 M dibasi potassium phosphate. The gluose formed was assayed following the method desribed by Gasbn and Lampen [17]. In both ases one unit (U) of agalatosidase is defined as the amount of enzyme whih hydrolyses 1 pmol of substrate in 1 rnin at 3 "C in.25 M aetate buffer, ph 4.5, ontaining 25 mm substrate. In both ases the olor produed is a lineal funtion of the amount of enzyme up to an absorbane of.8. Invertase Assay Invertase ativity was assayed aording to Gason and Lampen [17] by measuring the liberated gluose from surose. AidPhosphatase Assay To 1 pl of enzyme preparation were added 2 pl of.1 M glyine/hcl buffer, ph 3.6, and 5 p1 of 4 mm pnitrophenyl phosphate. The reation was stopped by addition of 2 ml of.1 M NaOH and the olor produed was measured at 41 nm. Gluanase Assay,U(1 t 3)Gluanase ativity was assayed by measuring the liberation of reduing sugars from.5% laminarin in.5 M aetate buffer, ph 5.5. The reduing sugars were measured by the method of Somogyi [18] and Nelson [19]. In the ases of invertase and phosphatase, one unit (U) of enzyme was defined as the amount of enzyme whih atalyses the hydrolysis of 1 pmol of substrate in 1 min at 3 "C in the onditions of the assay. For gluanase the unit (U) was defined as the amount of enzyme able to release 1 pmol of reduing sugars in 1 min at 3 "C in the onditions of the assay. Formation of Protoplasts To 5 ml of yeast suspension ontaining 8 x 1' ells/ ml were added 1 ml of.5 M TrisHC1 buffer, ph 7.5, 6 ml of 1.8 M KC1 ontaining.2 M MgS4,.5 ml of snail juie and.2 ml 1 M 2meraptoethanol. The mixture was inubated with shaking at 35 "C until the protoplasts were formed. In 2 h of inubation 95 1 % of protoplasts was obtained. Polyarylamide Gel Eletrophoresis This was performed by the method of Davis [2] with the 7.5 % arylamide standard gel as desribed in the manual supplied by Shandon, at ph 6.6. The sample and spaer gels were omitted. After eletrophoresis, gels were stained for protein following the method of Wellner and Hayes [21] using 5% trihloraeti aid, 5 "/, sulphosaliyli aid, 18 o/, methanol and.2% brilliant blue for 5 h. The gels were destained with 7 % aeti aid. agalatosidase ativity was loated in gels by utting these in 2mm slies and eluting the enzyme by immersion in the assay buffer.

3 P. S. Lazo, A. G. Ohoa, and S. Gason 317 The arbohydrate was visualized in the gels using the Shiff reagent aording to the method desribed by Kapitany and Zebrowski [22]. Determination of Protein and Carbohydrate During the purifiation proess protein was measured either spetrophotometrially, aording to Warburg and Christian [23], or by proedure of Lowry et al. [24]. The total arbohydrate was measured by the orinol/hzso4 method [25]. RESULTS INDUCIBILITY AND CELLULAR DISTRIBUTION OF agalactosidase Melibiase (agalatosidase) was initially desribed as an adaptative enzyme [26] whih was synthesized only when melibiose was present in the ulture medium as the sole arbon soure. Simultaneously, the synthesis of galatosidase is also ontrolled through atabolite repression (unpublished data from our laboratory). The two Saharomyes arlsbergensis strains used throughout this work show this behavior but other sugars an also indue the synthesis when substituted as arbon and energy soures. In this way galatose has been systematially used as a good induer during this work. When galatose was used as induer, an important differene was found between the two strains. Although both strains an synthesize the same amount of enzyme, strain 1317 has an 812h lag phase while strain 1323 has a 36h lag phase, whih onfirms previous reports that the latter organism an ferment galatose at a slower rate than other sugars [15]. agalatosidase ativity an be found outside the ell membrane [2] and there is evidene that melibiose is split into its two monosaharide units before entering the ell [14]. We have studied the ellular distribution of agalatosidase ativity during the growth of S. arlsbergensis with galatose as induer. The results presented in Table 1 learly show that most of the ellbound ativity is found outside the plasma membrane and is probably between the membrane and the ell wall, sine 78% is solubilized during the formation of protoplasts. On the other hand, although in the earlier phases of growth the ativity is muh higher in the ells than in the supernatant, as the ulture reahes stationary phase the external ativity an be found in approxi,mately the same amount in both ells and supernatant. It an also be seen in Table 1 that the amount of internal ativity remains essentially onstant throughout the ulture at a very low level, whih indiates that the seretion of agalatosidase ours at a high rate. The snail juie used in this experiment ontained 2.8 U/ml agalatosidase whih represented approxi Table 1. Distribution of ogulutosiduse ativity in yeast ells and protoplusts Sahuromyes urlsbergensis was grown in a.3 % yeast extrat, 1 % galatose medium in a rotatory shaker at 28 "C. At different times the ells were harvested and washed twie with distilled water by entrifugation at 18OOxg for 5 min. The protoplasts, obtained as indiated in Materials and Methods, were olleted by entrifugation at 18 x g for 5 min and then resuspended in.9 M KC1 ontaining.1 M MgS4. For lysis, protoplasts were suspended in.5 M TrisHC1 buffer, ph 7.5, and the supernatant was obtained after entrifugation at 18 x g for 1 min. All the values are expressed as agalatosidase ativity per 1' ells, or the volume in whih this number of ells is ontained in the ase of the supernatants Fration agalatosidase ativity in 1' ells at early growth log phase U stationary phase Culture supernatant Cells Enzyme released during protoplast formation Protoplasts Supernatant of lysed protoplasts mately 2% of the total enzyme released during protoplast formation. The results ontained in Table 1 were obtained using S. arlsbergensis Results with S. arlsbergensis 1323 were the same exept for the time required for initiation of the synthesis whih was, as mentioned previously, muh longer. Charaterization of the Internal and External Ativities by Gel Chromatography In the ase of invertase, the most extensively studied sereted enzyme in yeast, two isozymes had been found whih were different in their loalization and strutural properties [13]. While the external enzyme was a glyoprotein (moleular weight 27 ), the one found inside the plasma membrane did not ontain arbohydrate and had a moleular weight of 135. These findings led to the idea that the internal enzyme was the preursor of the external one. Subsequently a wide spetrum of internal forms were found [12]. However, in view of the possibility that the apparent isozymes were due to the presene of degradation produts [27], the relationship of the internal form of the enzyme to the external form should be larified. We have investigated possible differenes between the internal and external agalatosidases. Gel hromatography with Sephadex G2 and Sepharose 6B were arried out with samples of ulture supernatants and ellfree extrats. In ontrast to the invertase, the internal agalatosidase elutes at the same position

4 378 agalatosidase from Yeast 1.5. E 3 1. G " m &.5 A I, I B O.1 t Fig. 1, Analytial gel hromatography of agalutosiduse on Sephadex G2 (A) and Sepharose 6B (B). 2ml samples of onentrated ulture supernatants ontaining 45 U of agdlatosidase () and 2ml samples of ellfree extrats ontaining 15 U () were applied to a 95 x 2.5m olumn of Sephadex G2 equilibrated with.5 M TrisHC1 buffer, ph 7.5, or to a 37 x 2.5m olumn of Sepharose 6B equilibrated with the same buffer. Both olumns were alibrated previously with blue dextran as the external enzymes (Fig.l), both in Sephadex G2 and Sepharose 6B. The Sepharose 6B hromatography exludes the existene of a lighter form whih would not be separated from the main form in Sephadex G2, sine the ativity elutes very lose to VO in this partiular gel. The enzyme released during the formation of protoplasts elutes with the same pattern (not shown). However, when an extrat of protoplasts (prepared as desribed in the legend of Fig. 2) is hromatographed, a partiulate fration an be visualized. This ativity, whih represents a small fration of the total, is probably formed by membranebound enzyme as an be inferred from the fat that suh ativity an be solubilized with Triton X1. But again this solubilized enzyme behaves in the same manner as the soluble one when hromatographed in Sephadex G2 or Sepharose 6B. So neither the internal soluble agalatosidase nor the partiulate form appear to have lower moleular weights than the sereted one and therefore the idea of the internal preursor appliable in the ase of invertase is not relevant to this system. It should be pointed out that the possibility that only the moleules ontaining all the arbohydrate exhibit enzymi ativity annot be exluded. If so, the preursors would not be visualized with the method we used. PURIFICATION OF agalactosidase Table 2 summarizes the purifiation of agalatosidase sereted by S. arlsbergensis in the ulture supernatant. The organism is grown in a proteinfree medium and agalatosidase represents about 5 of the total protein present. With the proedure desribed below, most of the proteins other than vewo I 2.5 Fig. 2. Gel hromatography on Sepharose 6B qf an extrat obtained from protoplasts. Protoplasts obtained as desribed in Methods were lysed by suspension in.5 M TrisHC1 buffer, ph 7.5, and entrifuged at 18 x g for 1 min. The preipitate was resuspended in 5 ml of the same buffer and after ultrasoni disruption for 3 min, entrifuged at 59 x g for 1 min. The supernatant was applied to a 37 x 2.5m olumn of Sepharose 6B equilibrated with.5 M Tris HCI buffer, ph 7.5 agalatosidase are disarded after DEAESephadex bath adsorption. After hromatography on DEAE Sephadex and DEAEellulose the enzyme is suffiiently pure so that the ativity elutes from Sephadex G2 with a single peak of protein. However, invertase opurifies with agalatosidase and only after its partial degradation, and subsequent hromatography in Sephadex G2, an the ontamination be redued to negligible levels. All the steps were arried out at 4 "C exept for some experiments where the invertase degradation was favored by storing the samples at room temperature. Choie of the Enzyme Soure To obtain large amounts of enzyme of a relatively high speifi ati\ ity, we ultured S. arlsbergensis in yeast/nitrogen base. Indued, logarithmially growing ells were inoulated aseptially into a fermentor, inubated at 28 "C for 48 h and the ph held above 3 by the addition of Na2C3 solution. Enough galatose was added to give a final onentration of 1 %. In this manner we were able to have between 22 and 25 1 of ulture supernatant ontaining between.6 and.8 U/ml with a speifi ativity that was always at least 5fold higher than that estimated in a ellfree extrat. DEAESephadex Bath Adsorption In a typial experiment, 2235 ml of ulture supernatant were olleted after 48h inubation and the ioni strength adjusted to less than of.1 M NaCl by dialysis against distilled water. Then the ph was adjusted to 7.5 with 1 M Tris, a suspension ontaining 3 g (dry weight) of DEAESephadex in

5 P. S. Lazo, A. G. Ohoa, and S. Gason 379 Table 2. Purifiation of agulatosidase from Saharomyes arlsbergensis Step Volume Total ativity Total protein Speifi Yield Purifiation agalatosiativity dase/invertase ml U mg U/mg protein % fold 1. Culture supernatant DEAESephadex bath adsorption DEAESephadex hromatography DEAESephadex hromatography DEAEellulose Chromatography Sephadex G2 hromatography " : 2 7 e. m J 8 5 E " 6 Volume (ml) Fig. 3. Frationation on DEAESephadex A5 offration 2 of Tabb 2. The omplete fration was applied to a olumn (38 x 3 m) equilibrated with.5 M TrisHCl buffer, ph 7.5, and a linear gradient was made up to 1 M NaCl in the same buffer with a total volume of 6 ml. The elution was followed with a LKB Uviord and reorded at 254 nm. ()Absorbane at 254 nm (), galatosidase ativity; ( ) NaCl onentration of the eluent. The frations under the bar were pooled and used in the further steps of purifiation.5 M TrisHC1 buffer, ph 7.5, was added and stirred gently for 12 h. The gel with the ativity adsorbed was olleted by filtration and resuspended in 6 ml of 1 M NaCl solution prepared in the same buffer. After 12 h the gel was separated and again treated with 1 M NaC1, leaving the suspension in this ase for 1 h. These two supernatants ontaining the agalatosidase ativity were pooled and dialyzed against first distilled water and then against.5 M Tris HC1 buffer, ph 7.5. DEAESephadex A5 Chromatography The 122 ml reovered from the DEAESephadex bath adsorption was onentrated to 29 ml by dialysis against the sodium salt of arboxymethylellulose (Aquaide 11) and, after ensuring that both the ph and the ondutivity were the same as that of.5 M TrisHC1 buffer, ph 7.5, the enzyme was adsorbed on a DEAESephadex A5 olumn (38 x 3 m) equilibrated with the same buffer. The olumn was eluted with a linear gradient to 1 M NaCl as shown in Fig. 3. The ative frations were pooled and dialyzed against.5 M TrisHC1 buffer, ph 7.5. The 15 ml, ontaining 6 U/ml whih were reovered from this hromatography were adsorbed on another DEAESephadex A5 olumn as before. In this ase a lower salt gradient of up to.5 ym NaClwas used for the elution. DEAEellulose Chromatography The most ative frations from the last hromatography in DEAESephadex were pooled, dialyzed against.5 M TrisHC1 buffer, ph 7.5, and adsorbed on a DEAEellulose olumn (22 x 2.5 m) equilibrated with the same buffer. The olumn was eluted with a linear gradient of NaCl up to.5 M and the protein profile monitored. The peak of protein assoiated with the agalatosidase ativity was the main peak

6 38 rgalatosidase from Yeast (not shown) and only two additional peaks of protein eluted from the olumn. The ative frations were pooled; 15 ml with 55 U/ml were reovered from this hromatography after dialysis against.5 M TrisHC1 buffer, ph 7.5. Sephadex G2 Gel Filtration The sample from the DEAEellulose hromatography was stored for 2 days at 4 "C. The sample was then onentrated to 1 ml dialysis against Aquaide 11 and loaded on a 95 x 2.5m olumn of Sephadex G2 equilibrated with.5 M TrisHC1 buffer, ph 7.5. The result of this hromatography is shown in Fig.4. It an be seen that a single peak of protein is present whih is oinident with the peak of CIgalatosidase ativity. It should be noted that the invertase has been resolved into two peaks, the bigger of whih has been displaed to higher V,/VO ratios. The ontribution of invertase to the total protein must be very small sine the larger peak of ativity ontains very little protein. The frations ontaining the minimum amount of invertase (under the bar in the figure) were pooled and examined for homogeneity. Copurifiation of Invertase Table 2 indiates that the ratio of galatosidase to invertase is maintained throughout the purifiation proedure, and only after the partial degradation of invertase ould the ontamination be redued. The degradation of invertase has two onsequenes : first, the total ativity is redued and seond, the remaining enzyme is degraded to lowermoleularweight forms whih an be separated from the native galatosidase 4 5., 6 T 3 2 7, C r + : 8o F t 9 5 I 1 I I 1.5 I UVO Fig.4. Frationation in Sephader G2 of fration 5 of' Tuble 2. The whole fration was kept at 4 "C for 2 days, onentrated with Aquaide I1 to 1 ml and applied to a Sephadex G2 olumn equilibrated with.5 M TrisHC1 buffer, ph 7.5. The elution was followed with a LKB Uviord at 254nm (..) and the txgalatosidase () and invertase (A) ativities were assayed in the frations by gel filtration. Although the opurifiation is an indiation that the properties of the two enzymes are very similar, the differential degradation of invertase an be related to the fat that invertase is a muh more thermosensitive enzyme than agalatosidase (Lazo, Ohoa and Gason, unpublished results). An important way of reduing invertase ontamination in the agalatosidase preparations is by taking advantage of the ontrols under whih the synthesis of both enzymes take plae. Sine galatose an repress the synthesis of invertase, it is possible to obtain ulture supernatants in whih the invertase represents only 5 % of the agalatosidase ativity, by keeping the galatose at a onstantly high onentration during growth. To demonstrate the importane of the growth onditions and the degradation proesses to the suesful purifiation of agalatosidase, an experiment was arried out in whih the synthesis of invertase was favored and its degradation avoided. The initial ulture supernatant ontained invertase whih omprised about 5% of the agalatosidase. In this ase the Sephadex G2 hromatography was arried out immediately after the DEAEellulose step. The preparations, whih were homogeneous by the usual riteria, still ontained the same proportions of agalatosidase and invertase. Criteria of Purity When the purified enzyme was rehromatographed in DEAESephadex or Sephadex G2, no further purifiation was obtained. Fig. 5 shows a hromatography in DEAESephadex at ph 7.5 of the enzyme reovered from the Sephadex G2 gel hromatography. It an by seen that the single peak of protein oinides exatly with that of the ativity. It is also important to note that the remaining invertase ativity elutes in the same frations. When arbohydrate was measured in the frations it was found that one peak overlapped exatly with the protein and galatosidase peaks. This was the first indiation that agalatosidase is a glyoprotein. On polyarylamide gel eletrophoresis, the purified agalatosidase gave a single band of protein whih ontained the enzymati ativity (Fig. 6). In addition, when the arbohydrate was assayed in the gel a major band was also found in this position (not shown). Other external enzyme ativities were assayed in the purified frations and neither gluanase nor aid phosphatase ould be found. Alternative Methods of Purifiation When experiments were arried out with ell extrats, it was found that, by preipitation with (NH4)2S4 at 7% of saturation, it was possible to obtain a good purifiation. After entrifugation, 7 %

7 P. S. Lazo, A. G. Ohoa, and S. Gason , I Po I I I I I E %.6 1 fa a,,".4 L a 4.2 Volume (ml) Fig. 5. Chromatography on DEAESephadex A5 ofthe purified aguiutosiduuse. Fration 6 of Table 2 was applied to a olumn equilibrated with.5 M TrisHC1 buffer, ph 7.5. A linear gradient was established to 1 M NaCl in the same buffer with a total volume of 5 ml. () rgalatosidase ativity; () absorbane at 28 nm; (A) total arbohydrate; (A) invertase ativity; ( ) NaCl onentration of the eluent J I I I I I I Migration from the origin (rnm) Fig. 6. Polyurylumide gel eletrophoresis oj t/iepurfied rguiutosiduse. Samples ontaining 2 pg of protein were run in.2 M phosphate buffer, ph 6.8. (A) Densitometer traing at 56 nm showing the protein present in the gel. (B) Histogram showing the ativity found in a gel whih was run in parallel with the first. x Galatosidase ativity was assayed as desribed in Materials and Methods of the protein but more than 8 % of the total ativity remained in the supernatant. DISCUSSION The existene of external hydrolyti enzymes in yeast an be attributed to the impermeability of the ell membrane to their orresponding substrates. Among them, agalatosidase is an unusual enzyme sine it is induible and its synthesis is ontrolled by atabolite repression. + A The signal leading to the indution of the synthesis of agalatosidase, in the absene of gluose from the ulture medium, ould result from three possible events: (a) the binding of substrate to the ell membrane, (b) the appearane of substrate in the ytoplasm and () the appearane in the ytoplasm of hydrolysis produts of the substrate resulting from the presene of onstitutive levels of agalatosidase on the ell surfae. The third possibility is in agreement with the fat that galatose, whih is a part of the melibiose moleule, among other related sugars (unpublished data from our laboratory) is able to indue the synthesis. In addition, some ativity an be found in ells growing in gluose after the sugar has been utilized. As soon as the ells are indued the enzyme is brought to the ell surfae. While the internal enzyme level remains essentially onstant during the whole ulture, it is possible to see that the external enzyme is first mainly loalized in the ell wall and then is released to the ulture supernatant. It is interesting to note that when Friis and Ottolenghi [2] first desribed melibiase as an external enzyme in yeast, they reported that during the preparation of protoplasts all the ellbound enzyme was released and that 8 of the ativity was lost. In our ase some ativity is also lost during the proedure, but in ontrast we an find some ativity whih remains on the protoplasts. They suggested that the loss of ativity ould be due to the ation of the enzymes present in the snail juie. We heked this possibility with soluble enzyme and after 5 h, in the presene of the same onentration of snail juie, there was no loss of ativity. Besides, the enzyme is stable for 2 h at 3 "C in the presene of 2meraptoethanol up to.25 M.

8 382 P. S. Lazo, A. G. Ohoa, and S. Gason: agalatosidase from Yeast The possibility that the membranebound enzyme in the gel filtration experiments is the one whih remains bound to the protoplasts should not be disarded. But in any ase, this would not hange the fat that in this partiular enzyme there is no internal form (free or membranebound) whih one ould postulate to be the preursor of the external one. In order to purify the enzyme, either strain of S. arlsbergensis an be used. However, growing S. arlsbergensis 1323 in yeastlnitrogen base plus 1 % galatose, 1.6 U/mg of protein are obtained after 72 h, whereas when growing S. arlsbergensis 1317 under the same onditions, 4.5 U/mg of protein are obtained after 48 h. Furthermore, when galatose is present ontinuously in high levels, 67 U/mg of protein are obtained with the latter strain. S. arlsbergensis 1323 has the advantage of not produing invertase. In the ase of the strain 1317, whih is able to produe invertase, galatose an repress its synthesis. Under these onditions we deided to use the latter strain, sine the yield is muh higher and the small amount of invertase in the purified preparation does not interfere with the results. On the other hand, invertase has been used as a marker enzyme in several experiments. Thus, it was in general advantageous to use the strain 1317 whih produes invertase and agalatosidase. During the purifiation proedure it was advantageous that agalatosidase was the first protein eluting from either DEAESephadex or DEAEellulose olumns (at an ioni strength of.175 M NaC1). This meant that all the proteins other than agalatosidase present in the preparations ould be disarded simply by repeating the hromatographi steps with gradients of different slopes. It is of interest to point out the similarities between invertase [6], aid phosphatase [7] and agalatosidase. The reported moleular weights of 27 for invertase and 29 for aid phosphatase are very similar. Both moleular weights must be similar to that of xgalatosidase, sine they elute so losely in gel hromatography experiments. But in addition, they are also similar beause both invertase and aid phosphatase have been reported to be glyoproteins. In experiments reported in this paper there are lear indiations that agalatosidase is indeed a glyoprotein sine either in ioni hromatography or polyarylamide gel eletrophoresis, a peak of arbohydrate an be found together with those of protein and ativity. In onnetion with the fat that invertase opurifies with agalatosidase, it is worthwile mentioning that agalatosidase was found as a ontaminant ativity in highly purified preparations on invertase [28]. Part of this work was arried out in the Department of Mirobiology, University of Salamana and Consejo Superior de Investigaions Cientifias. The authors wish to thank Professor Julio R. Villanueva for his help and enouragement. We are also indebted to Dr P. Stroobant for his ritial reading of the manusript. P.S. Lazo was a reipient of a fellowship from the Ministerio de Eduaibn y Cienia. REFERENCES 1. Friis, J. & Ottolenghi, P. (1959) C. R. Trav. Lab. Carlsberg, 31, , 2. Friis, J. & Ottolenghi, P. (1959) C. R. Trav. Lab. Carlsberg, 31, MLellan, W. L. & Lampen, J.. (1963) Biohim. Biophys. Ata, 67, Phaff, H. J. (1966) Abstr. 9th Int. Congr. Mirohiol., p Wheeler, G. E. & Rose, A. H. (1973) J. Gen. Mirohiol. 74, Neumann, N. P. & Lampen, J.. (1967) Biohemistry, 6, Boer, P. & Steyn Parve, B. (1966) Biohim. Biophys. Ata, 128, Siekevitz, P. & Palade, G. E. (196) J. Biophys. Biohem. Cytol. 7, Northote, D. H. (1968) Br. Med. Bull. 24, Gason, S. & Ottolenghi, P. (1967) C. R. Trav. Lab. Carlsberg, 36, Hoshino, J. & Momose, A. (1966) J. Gen. Appl. Mirobiol. 12, Moreno, F., Ohoa, A. G., Gason, S. & Villanueva, J. R. (1975) Eur. J. Biohem. 5, Gason, S., Neumann, N. P. & Lampen, J.. (1968) J. Biol. Chem. 243, De la Fuente, G. & Sols, A. (1962) Biohim. Biophys. Ata, 56, Winge, 8. & Roberts, C. (1957) C. R. Trav. Lab. Carlsberg, 25, Gilliland, R. R. (1969) Antonie van Leewenhoek J. Mirohioi. Serol. 35, Gason, S. & Lampen, J.. (1968) J. Biol. Chern. 243, Somogyi, M. (1952) J. Bid. Chem. 1Y5, Nelson, N. (1944) J. Biol. Chem. 153, Davis, B. J. (1964) Ann. N. Y. Aad. Si. 121, Wellner, D. & Hayes, M. B. (3973) Ann. N. Y. Aad. Si. 29, Kapitany, R. A. & Zebrowski, E. J. (1973) Anal. Chem. 56, Warburg,. &Christian, W. (1941) Biohem. Z. 31,384421, 24. Lowry,. H., Rosebrough, N. J., Farr, A. L. & Randall, R. J. (1951) J. Biol. Chem. 193, Vinzler, R. J. (1955) Methods Biohem. Anal. 2, Lindegren, C. C., Spiegelman, S. & Lindgren, G. (1944) Pro. Natl Aad. Si. U.S.A. 3, Lampen, J. O., Kuo, S. C., Cano, F. R. & Tkaz, J. S. (1972) in Fermentation Tehnology Today(Terni, G., ed.) pp , Soiety of Fermentation Tehnology, Japan. 28. Adams, M., Rihtmyer, N. K. & Hudson, C. S. (1943) J. Am. Chem. So. 65, P. S. Lazo, Rohe Institute of Moleular Biology, 34 Kingsland Road, Nutley, New Jersey, U.S.A. 711 A. G. Ohoa and S. Gason *, Departamento Interfaultativo de Bioquimia, Faultad de Mediina, Universidad de Oviedo, Oviedo, Spain * To whom orrespondene should he addressed.