THE JOURNAL OF BIOLOGICAL CISEA~ISTRY Vol. 243, No. 7, Issue of April 10, pp. 1567-1.572, 1968

Printed in U.S.A.

Purification of the Internal Invertase of Yeast*

(Received for p[thlication, November 20, 1967)

SANTIAGO GASC~N~ AND J. OLIVER LAMPEN

From the Institute of Microbiology, Rutgers. The State LT~aivemity, New Brunswick, New .Jelxey 08903

SUMMARY

Most of the studies on yeast invertase have been con- cerned only with the external enzyme (a mannan protein which is localized in the cell wall), but recently it has been reported that the invertase inside the cytoplasmic membrane is a smaller form. This paper presents a comparative study of the distribution of invertase isozymes in two stains of yeast and, after the selection of the best conditions and source, the purification of the internal or small enzyme. The major purification is obtained by chromatography on diethyl- aminoethyl Sephadex, although ammonium sulfate precipita- tion and gel filtration in Sephadex G-200 are also used to separate the large and small enzymes. The purified internal invertase is homogeneous on the basis of chromatographic criteria and its behavior in the analytical ultracentrifuge. The molecular weight of the enzyme is approximately 135,000, and the specific activity is 2,900 units per mg of protein.

-4 considerable amount of information has accumulat,ed on the characteristics, localization, and secretion of yeast invertase (P-n- fructofuranoside fructohydrolase, EC 3.2 1.26). It has been established (2-5) that in derepressed cells most of the invertase is external (i.e. cell-bound but outside t,he cytoplasmic mem- brane) ; only a small proportion of the enzyme is located inside the membrane and is not accessible to the substrate. In fully repressed cells all the invertase is intracellular (4). Recently invertase isozymes have been found in Neurospora (6-9) and bakers’ yeast (10-13). Gas& and Ottolenghi (14) have found that internal invertase of yeast is considerably smaller, as judged from its behavior on Sephadex G-200 columns, than is the external invertase.

Purificat-ion of the ext,ernal invertase has been carried out, in several different laboratories (for a comparison, see “Discussion” in Reference 15). Neumann and Lampen (15) have obtained

* This work was supported in part by United States Prtblic Health Service Grant AI-04572 from the National Institute of Allergy and Infectious Diseases. A preliminary report of part of this work has been given (1).

t On leave of absence from the Institute de Biologia Celular, VelLzquez 138, Madrid 6, Spa,in. Supported dining part of this work by a fellowship from the Flmdaci6n Juan March, Spain.

a homogeneous preparation of the external enzyme from yeast strain FH4C. A characteristic of the external invertase relevant to this study is its glycoprotein nature. The enzyme contains 50 y0 mannose and 30/, glucosamine. Eylar (16) has compiled B considerable amount of evidence that most mammalian extra- cellular proteins contain carbohydrate. Soodak (17) believes that, in order for protein secretion to occur, covalently bound carbohydrate must be present in the protein in question. In our laboratory we are studying the secretion of invertase in a proto- plast system (1, 18). In this context it is desirable to know the properties of the internal invertase.

This communication is concerned with (a) the distribution of invertase isozymes in yeast strain 303-67 and the repression- resistant mutant FH4C when grown in high or low levels of glucose and (b) the purification of bhc internal invertase from strain FH4C. The properties of the purified external and internal enzymes are compared in a companion paper (19).

EXPERIMINTAL PROCEDURE

,l”laterials-Glucose oxidase (pure) was obtained from Nutri- tional Hiochemicals; peroxidase (POD-II) and lactate dehydro- genase, from Boehringer; and o-dianisidinc, from Sigma. Yeast ext,ract (Ardamine Z) was purchased from Yeast Products, Inc., Pat,erson, New ,Jersey. Sephades G-200, diethylaminoethyl Sephadex A-50, sulfotthyl Sephadex C-50, and blue destran were obtained from Pharmacia. Protaminc sulfate, Aquacide 11, and dithiothrrit,ol were purchased from Calbiochem; 2- mercaptoethanol, from Eastman Organic Chemicals; and the snail enzyme (SW digest$’ &Helix pomatia stabilis~), from L’ industrie Biologiquc Francaise, Seine, France.

Invertuse .?ssay-Before the assay, the enzyme was diluted in 0.02 M Tris-HCI, pH 7.5, and incubated at 30” for 10 min to ensure maximum activity (19). Hydrolysis of sucrose and assay of the glucose formed were carried out in two steps at 30”. In the first step, 50 ~1 of the dilut’ed enzyme were added to 100 ~1 of 0.1 sf acetate buffer, pH 5.0, and 50 ~1 of 0.5 M sucrose. The reaction was stopped after 10 min, unless otherwise indicated, by addition of 300 ~1 of 0.2 RI dibasic potassium phosphate, and the tubes were immediately placed in a boiling water bath for 3 min. The glucose formed was assayed by a method similar to that of Keston (20) by addition of 2 ml of 0.1 RI phosphate buffer, pH 7.0, containing 200 pg of glucose oxidase, 10 pg of peroxidase, and 600 pg of o-dianisidine. After 30 mill of incubation, the reaction was stopped by addition of 2.5 ml of 6 N HCl. The

1567

1565 Internal Invertase of Yeast ‘\l’ol. 243, No. 7

color produced was read at 540 171~. For fractions containing bot,h glucose and invertase, an extra sample was run, in which t,he invert,ase was first’ inactivated at, 100” for 5 min. This sample provided a measure of glucose preserrt, which was sub- stracted from t,he assay. One unit, of invertase is defined as the amount of enzyme which hydrolyzes I pmole of sucrose in I min at 30” in 0.05 M sodium acetate buffer, pH 5.0, cont’aining 0.125 M sucrose.

Total carbohydrate was measured by t,he phenol-sulfuric method of I>ubois et al. (21). Total protein was est#imated by the procedure of Lowry et al. (22) or by absorption at 280 mp, with Imrified internal invertase as standard, The amount of enzyme in the standard was determined from the amino acid analysis data (19).

Analytical Gel PUtration-The quantitative analysis of external and internal invertasrs and determination of the relative propor- tions of the two forms were performed according to Gascon and Ottolenghi (14) in an analytical column of Sephadex G-200 (2 X 47 cm) equilibrated with 0.05 M Tris-HCl buffer, pH 7.5. The same column was used for the estimation of the molecular weight of t,he internal enzyme by the method of Andreus (23).

lJltracentri&gal Analysis-A Reckman/Spiuco model E ultra- centrifuge equipped with a constant temperature device and schlieren optics was used. Plates were read with the aid of a Kikon shadowgraph. A partial specific volume of 0.72 was used. This value was obtained from the amino acid composition (19) by the method of Cohn and Edsall (24).

Yeast Strains and Culture Conditions-Two yeast st’rains were used, Sacchurorr~yces strain 303-67 and mutant FH4C derived from it. The parent Sacchuromyces strain 303-67, in which the distribution of invertase isozymes has been studied previously (14), is diploid and homozygous for t,he Suz gene (Rs of Winge and Roberts (25)). It forms invert,ase in appreciable amounts only after t,he glucose has disappeared from the medium (4), and does not, hydrolyze maltose (25). The mutant strain FH4C was obtained by Symingt,on and Lampen (26) by ultraviolet, irradia- tiorr of strain 303-67. It, produces high levels of external invert- ase even when growing in the presence of glucose (26).

For growth of the FH4C Arain on a large scale, the following medium was used: glucose, 4%; ammonium sulfat,r, O.a(j/,; and yeast extract,, 1%. The pH was adjusted to 5.5. The yeast were normally grown in 500-lit,er lots. The fermenter was inoculated aseptically with 5 liters of logarithmically growing yeast. Growth was cont,inued for 16 hours at 28”, by which time all the glucose had disappeared from the medium. The yeast were harvested in a SharIlIes ccntrifugc. Approximately 14 kg of cell paste were obt,ained per run.

E’ormation of Protoplasts--To 5 ml of yeast suspension con- taining approximately 8 x 10’ cells per ml, the following additions were made: 0.05 hI l’ris-WC’1 buffer, pH 7.5, 1 ml; 1.2 M KCl, containiirg 0.02 M magnesium sulfate, 6 ml; snail enzyme, 0.5 ml; and 1 M 2-mercaptoethanol,10.2 ml. The mixture was incubated wit,h shaking at 35”, and after 3 hours more t’han gOOr, of the cells had been converted into protoplasts.

RESULTS

Distribution of Invertase Isozymes

A st,udy was undertaken of the distribution of t,he invertase isozymes in two strains of yeast. We were especially irrterested

1%Mercaptoethnnol (27) has been used rontinely for the pro-

in deter~mining whether mutant I?H4C would be a better source of int,ernal inrertase than the parent 303.67 strain. Some of t’he results are presented in Table I. Resides the known sensitivity of the level of external invcrtase of st,rairr 303-65 to the growth conditions (4) and the comparatively small effect of the glucose concentration on the production of external enzyme by the FH4C strain (26), it is worthwhile to not,e that there is ap- proximately the same proportion of internal invcrtase in the FH4C cells as in the low glucose cells of st,rain 303-67. The low glucose FH4C cells have approsimatelg twice as much external invertase as the high glucose ones. This is probably related to the finding of Lampen et al. (1) that, protoplasts of FH4C secrete twice as much invertase in the presence of 0.005 M glucose than at 0.05 M or greater concentrations of sugar.

Fig. 1 shows the behavior of large and small invertase on gel filtration in Sephades G-200. The examples chosen are extreme, but imermediate ratios of external and internal enzymes can be obt’ained. The enzyme released into t’hc medium by actively growing yeasts, as well as the invertase released during the forma- tion of protoplasbs, is of the large type. Also in the large form is the enzyme secreted by protoplasts (1). Predominantly small enzyme is released by lysis from protoplasts of Saccharomyces

strain 303-67 high glucose cells. This confirms the idea that the small invertase is internal with respect to t,he cytoplasmic membrane. Incomplete removal of the cell wall probably ac- counts for the presence of t,hc large form found in prot,oplast lysatrs (14). This is supported by observation that the invertase which is not solubilized during the lysis, but is subsequently rcleased by sonic treatment of the residue, is in the large form.

Internal invertase, determined as the difference between total act’ivity and the activity of osmotically stabilized protoplasts, represents only a minimal value. The recovery of small enzyme in the supcmatant of lysed protoplasts was equal to or greater t,han this (Table I); therefore, it is concluded that most of the internal invertase is in t,he small form. The possibility cannot be ruled out, however, that some of the large invertase revealed by lysis of the protoplast,s was imernal.

We have also studied t,he quantitative distribution of invertase isozymes in Succharomyces cerevisiae strain LK2G12, which has been used in our laboratory for extensive studies on invertase and its secretion (5, 15, 29). The results were similar to those described for strain 303-67 and we have not included these data in Table 1.

I’uriJicution of Internal Invertase

The high production of external invertase which characterizes strain FH4C is accompanied by a corresponding increase in t,he level of internal enzyme. Mutant FH4C is the richest available source of internal invertase and was used for preparation of the purified enzyme.

Table II summarizes the purification from this strain. Evalua- tion of the first steps in purification is quit’e complicated, since the ratio of est,ernal to internal invertase in the crude extract is about, 25 : 1. Estimation of internal invertase in a sample involves double precipitation with ammonium sulfate at 707; saturation and subsequent gel filtration of the precipitate in an

duction of protoplasts from our yeast strains with excellent results. We have observed that dithiothreitol works ecluall) well in the same concentration rarrge.

Issue of April 10, 1968 X. Gas&n and J. 0. Lampen 1569

TABLE I Distribulion of invertuse activity and invertuse isozynes in yeast cells and proloplasls

The yeasts were inoculated into flasks conlaining 100 ml of magnesium sulfstc. Rot oplasts were lysed by resnspensioiL in

Wickerham’s medium (28) and iilcubated on a rotary shaker at 28” 0.05 M Tris-TIC1 buffer, pH 7.5, atid t,he sLLperLiatant was obtained for 1G hours. The medium for high glucose cells contained 8% by ccntrifugation at 30,000 X y for 20 mitr. For estimation of in- glucose and had more than 3&jG left in t,he culture supernatant at verlase activity iti intact protoplasts, all solutions were supple-

the time of harvesting. For low glucose cells, lyh glucose was merited wit,h 0.6 M KCl. Total itlvertase is the value obtained used, and this disappeared before 14 hours of cullivatiou. The with protoplasts that had been frozen alid thawed twice; that with cells were harvested and washed twice with distilled water by ceil- iutact proloplnsts, extcrnnl iirvertase; the differelrce gave the

tr.ifugat,ion a,t 5,000 X 9 for 10 min. The protoplasts, obtaiired as value for internal iilvertnsc. The relative proportiotis of large indicated iti “fixperirnental Procedure,” were centrifuged at and small invert, rsc were estimated as indicated iti Fig. 1. All 10,000 X g for 10 mill and then resuspended and washed twice in values for activity are cxpresscd as ilivertase units per ci X lo9 cells

0.05 .M phosphate butfer, pH 6.0, containing 0.6 M KC1 and 0.01 M or protoplasts, or their equivalent in the case of the supernatants.

Source of enzyme

Cells.. ..,.

Cullure supernatant Enzyme released during protplusts f’or-

mation”.

Protoplasls B:xternal enzyme”. lllternal enaym@

Strpernatailt of lysed protoplasts’

303-6;

Invertase

uni1.s

1.5

0

0

0 .c1 2.0 2 .5

Small invertsse

FHK 303-67

units

700 200

730

56 14

29

SIlldl invertase

% 3 0

0

62

Imertase

15’2

11.5 G

12

SIlldl invertase

___-

5%

4

0

0

55

FIIK

lnvertase

mits

1320 500

1390

4x 37

50

0

58

0 Aft,cr protjoplast formation, the recovery of iilvcrlase was al-

ways greater than 100c~~ because of the synthesis atid secretion of

enzyme which occurred (5). h The values given for external enzyme arc maximal. Part of

the activity probably originated from protoplasts lysed duriiig

analytical column of Scphadex G-200 (see “Experimental Procedure”).

The procedure described below first separates the internal invertase from the external enzyme by repeated precipitat#ion with ammonium sulfate. The internal enzyme is prccipitat,ed at 70% saturation, whereas most of the external form remains in the suyernat,ant. The ammonium sulfate precipitate is dis- solved, treated with protamine sulfate, batch-adsorbed on DE%E- Sephadex A-50, and passed through a column of the same material. The active fractions are dialyzed and applied to a column of sulfoethyl Sephadex C-50. When examined by col- umn chromatography and analytical ultracent,rifugation, the active peak is free of detectable contamination. The purification obtained is in the order of 2500.fold in terms of protein.

Ml the steps were carried out at O-5” except for the breaking of the yeast and centrifugation in the Sharples centrifuge, which were performed at room temperature.

Preparation oj Crude E&act-In a typical experiment 15.3 kg of yeast (wet weight) were suspended in 40 liters of cool 0.05 M

‘Iris-HCl buffer, pH 7.5, and passed once through a Laboratory Sub Micron Disperser (Manton-Gaulin Manufacturing Com- pany, Everet,t, Massachusetts) at maximum continuous operat- ing pressure (8000 psi). The homogenate was collected over ice. One pass with the single stage homogenizing valve as- sembly was usually sufficient to break 70 to 90% of the yeast.*

2 The proportion of broken yeast was determined by estimating the fraction of invertase solubilixed. Althortgh this is not a strict criterion of breakage, the assay was sufficiently rapid to be con

handliiig of the santple and assay of the eiixynie. The values foi internal invertasr (t,olnl minris esterllal) are therefore minimal.

c Part of the invcrlase was not solubilized, and remained at-

tached to the pr.otoplastj residues. After Ldtrasoiiic disruption of

the pellet, the solubilizcd ertxyme behaved as large invertase.

d mmonium Sulfate Precipitation and Protamine Xuljate Treat- ment-To the crude extract, 516 g of solid ammonium sulfate per liter were added (80% saturation), and the solution was stored overnight at 4”. The precipitate, which contained the small invertase, was collected in a Sharples centrifuge. The purifica- tion obtained in terms of protein was small, but t,he bulk of the carbohydrate remained in the supernatant and, what, is more significant,, 70 to 80y0 of the external invcrtase remained in the supernatant. The precipitate (approximately 3 kg, wet weight) was dissolved in 12 liters of cold, distilled water, and 436 g of ammonium sulfate were added per liter (70% saturation)? After 20 hours the solution was centrifuged at 10,000 X g for 15 min, and the precipitate was redissolved and again precipitated at 700/, ammonium sulfate saturation. The precipitate was dissolved in 4 liters of distilled water and dialyzed against distilled water until the conductivity of the solution was similar to that of 1 M NaCl. At this point in the purification, the ratio between external and internal invertase was about l:l, and for further steps of purification it was not necessary to assay each fraction for internal enzyme by filtration through Sephadex G-200.

The volume of the enzyme solution was brought to 12 liters,

verrient. Microscopic estirnatioil is dilficLtlt because of the ex- tensive clumping that occurs with this yeast.

3 11LLring ptmificatiotl, the concetLtratioLi of crude enzyme so- lutions was achieved by precipitatioii with ammonium sulfate to 707, saturation. Pure or highly petrified enzyme was concen- trated by dialysis against sodium salts of carboxymethyl cellulose (AqLLacide 11) or by rcnderilrg the enzyme insoluble at pH 5.0 by dialysis against distilled water, or both.

1570 Internal Invertase of Yeast Vol. 243, No. 7

and the pH was adjusted to 6.5. Three liters of a 2% solution of protamine sulfate in water were added wit’h vigorous stirring, and the solution was kept at 4” overnight. The inactive precipitate was collected at 10,000 x g for 15 min. The protamine sulfate treatment removed not only inactive protein from the extract, but also particulate material and non-protein contaminants.

DEAE-Xephadex Batch Adsorption and Chromatography-The supernatant from the protamine sulfate treat.ment was adjusted to pH 7.5 with 0.5 M Tris, and distilled water was added to make the conductivity. equivalent to 0.05 M Tris-HCl buffer, pH 7.5, containing 0.15 M KaCl. The volume was adjusted to 38 liters with this buffer. DEXE-Sephadex (20 g, dry weight, previously equilibrated with the same buffer) was added to the enzyme

800

i

z 600-

a

5 400-

2 &

z zoo- 0.2 2

0.1

” ,L ” ”

0.5 1.0 1.5 2.0 2.5

VOLUME (LITERS1

FIG. 2. Fractionation of the int,ernal invertase on DEAE- Sephadex A-50. Fraction 5 of Table 11 was applied t,o a column (4 X 35 cm) previously equilibrated with 0.05 M Tris-RCl buffer, pH 7.5, containing 0.15 M ?jaCl. The column was washed with 300 ml of this bluffer, and a linear gradient, was established to 0.5 M ?;aCl in the same buffer. The total volume of the gradient was 4000 ml. The first peak of invert&se, which appeared before the gradient, was started, corresponds to the external enzyme. O---O, invertase activity; A--A, protein; L--A, carbo- hydrate; - - -; molarity of the elrlent.

solut,ion. After 2 hours with frequent stirring, all the internal invertase was adsorbed. The supernatant was discarded, and the ion exchange agent was washed twice with 1 liter of fresh buffer. The internal enzyme was removed by cluting twice with l-liter aliquots of 0.05 M Tris-HCl buffer, pH 7.5, containing 0.5 M NaCl. The purification in this step was approximately 20-fold, and most of t,he external invertase was also removed.

The pooled eluates from the batch adsorption were dialyzed against 0.05 M Tris-HCl buffer, pH 7.5, containing 0.15 M NaCl, and applied to a column of DEAE-Sephadex with the character- istics given in Fig. 2, which also illustrates the elution procedure. The batch adsorption and the column chromatography afforded a purification of ZOO-fold in terms of protein content.

Dialysis at pll 4.0 and Xulfoethyl Xephadex Chromatography- The more active fractions from the DEAE-Sephadex column were pooled, concentrated, and dialyzed against 0.05 M acetate buffer, pH 4.0, containing 0.05 M NaCl. The inactive precipitate was removed by centrifugation at 30,000 x g for 20 min. The supernatant was dialyzed against distilled water and centrifuged at 30,000 x g for 20 min, and the precipitate, which contained the enzyme, was dissolved in 0.05 M acetate buffer, pH 4.0, con-

I ’ I I I I I

t

BLUE DEXTRAN PEAK 0.08

I A 1.4 2

1.2 z

1.0 B >

0.8 Y 2

0.6 $

0.4 9

0.2

-i ;; 0.06- t 5

% &I 0.04-

? z

0.02-

1 40

VOLUME (ml)

FIG. 1. Gel filtration in Sephadex G-200 of extracts containing large and small invert,ases, and calibration of the column. Char- acteristics of the analytical collunn, buffer used, and elution sequence are given in “Experimental Procedure.” III each case, 1.5 units of invertase activit,y were applied to the column. The samples were the same as those described in Table I. For analyt- ical purposes, cells were broken in a Braun MSK homogenizer. O-O, enzyme released during protoplast formation from FII4C cellsgrown on high glucose medium; n----a, supernatant fraction of lysed protoplasts from strain 303-67 cells grown on high glucose medium; O---O, crude extract from FH4C cells. This crude extract is similar to the one used for the preparation of the internal enzyme. The column w-as calibrated with 10 my of blue dextran and 2.5 mg of lactate dehydrogenase dissolved in 1 ml of the same buffer used for ellltion. Blue dextrun was detected by its ab- sorption at GO0 mp, and the lactate dehydrogennse (A--A), by its absorption at 230 mp

Purijkhon of i. nternal invertase o-f yeast struin FHQC

Fraction VOlUllK

Internal invertase Protein External invertase

zmits

14,000,000

12,000,000 185,000 108,000

2,000 0

Total

ml

54,000 56,000

G,OOO 15,000

m !

304 ) 000

120,000 54,700 27,400

zcnits

3G0,OOO

3OG ) 000 158,000

141,000

Specific activity

wzits/mg pvotein

1.18

2.55 2.89

5.15

,000 150

1,200 74.1

122) 000 75,000

102

1,010

174 3i.0 GG ,000 1,780

5 8.4 24,000 2,900 37 0.2 18,000 2,900

I- 1. Yeast suspension. ’ 2. Crude extract

3. Ammonium sulfate (O-0.7) 4. Prolamine supernntant 5. l)EAB-Sephadex batch adsorp

tion.

6. UEAE-Sephsdex column. 7. 11ialysi.s against pTI 4 buffer

(supernatant) 8. I>ialysis against distilled water

(precipitate) 9. Sulfoethyl Sephadex column.

Issue of April 10, 1968 8. Gas&n and J. 0. Lampen

taining 0.05 M NaCl. The solubilized enzyme was adsorbed on a sulfoethyl Sephadex C-50 column (1 x 10 cm) that had been equilibrated with a similar buffer. The column was eluted as shown in Fig. 3. The specific acitvity along the active peak was constant. The active fractions were pooled, dialyzed against 0.05 M Tris-HCl buffer, pH 7.5, and stored.

The ultraviolet absorption of the purified enzyme shows a maximum at 282 rnp and a ratio of absorbances at 280 and 260 rnp of 1.75, indicating the absence of nucleotides and nucleic acids.

Alternative Purification Proced’ures-The procedure for purifi- cation just outlined was developed for working with large batches of yeast. In preliminary studies, good purification of internal invertase was obtained on preparative columns of Sephadex G-200 (5 x 60 cm) with reversed flow. The bulk of the protein, carbohydrate, and external invertase was present, in the fractions corresponding to the exclusion volume, whereas the internal invertase has an elution volume to exclusion volume ratio of approximately 1.7. This procedure proved impractical for large scale preparation, and after the protamine sulfate treatment the purification obtained by this method is relatively small. The gel filtration step was therefore omitted in routine purifica- tions.

Rechromatography of the purified enzyme on DEAE-Sephadex at pH 7.5 and 8.6 gave no further purification.

Molecular Weight and Sedimentation Velocity Determkation

Gel filtration of the purified internal invertase on Sephadex G- 200 was carried out in order to estimate its molecular weight. The enzyme was dissolved in 0.05 M Tris-HCl buffer, pH 7.5, and added to an analytical column calibrated with lactate dehydrogenase and blue dextran, according to the method of Andrews (23). Fig. 1 shows the elution profile of the lactate dehydrogenase as well as that of a crude preparation containing predominantly internal invertase (A in Fig. 1). In the gel filtration profile of purified internal invertase, there was no material in the region of external or large invertase, and it gave a sharper profile than the one shown in Fig. 1 (See Fig. 5 in the accompanying paper (19)). The peak obtained u7it.h the purified

400

t

I I I I

025

F 3CO- 2- 0.20- o - 1.00

G t

% a

5 k 0.15- -0.75

5

w 200- 2.

g 2

& f 1’ B

Y i? O.lO-

/Ifi _-’

-0.50 k

z a

loo- $ A’ 2

0.05- //’ 0.25

0 1

I ,

VOLUME (ml)

FIG. 3. Fractionation of t,he internal invertase in sulfoethyl Seuhadex G-50. Fraction 8 of Table II (2.3 me of protein) was ~ Y

ap’plied to a column (1 X 10 cm) previously equilibrated with 0.05 M acetate buffer containing 0.05 M N&l. A linear gradient was established to 1.0 M PIjaCl in the same buffer. The total volume of the gradient was 200 ml. O-O, invertase activity; O--O, absorbance at 280 mp; - - -, molarity of the eluent.

FIG. 4. Ultracentrifugation pattern of plu-ified internal invert- ase. The sedimentation velocity was determined with 0.4 ml of a sample containing 3 mg of protein per ml in 0.01 M Tris-HCl buffer, pH 7.5, containing 0.2 M NaCl. The sedimentation meas- urements were made with a Spinco model E ultracentrifuge at 20”. The photograph was taken approximately 5 min after a speed of 59,780 rpm was obtained. Sedimentation is proceeding from right to left.

enzyme and that of the lactate dehydrogenase overlap almost exactly. The elution volume of the internal enzyme suggested a molecular weight of 130,000 to 140,000.

Sedimentation velocity experiments revealed only one peak (Fig. 4) with s:~,~ of 8.8.

DISCUSSION

Invertase was first isolated by Berthelot in 1860 (30). Since then, the enzyme has been extensively studied and purified from a variety of sources and with different extraction methods (15). The existance of invertase inside protoplasts has been detected recently (4, 5, 29), and Sut,ton and Lampen (5) established that the internal enzyme had the same kinetic properties as the exter- nal (see also Gascon and Ottolenghi (14)). It is not surprising, however, that its presence has not been detected in the several preparations of invertase, in view of the small proportion of the internal enzyme in fully induced cells and the heterogeneous nature of invertase extracted by the usual autolytic procedures. In addition, owing to the different characteristics of both en- zymes, it is highly probable that internal invertase will be lost in one of the preliminary steps of purification.

The distribution and characteristics of invertase isozymes of Neurospora are markedly different from our findings and those of Gas&n and Ottolenghi (14), with invertase isozymes of yeast.

1572 Inntemal Invedase of Yeast Vol. 243, No. 7

Rletzenberg (7) has shown that in Nezaospora the two isozymes can be interconverted, and has previously reported that the heavy form contains little if any carbohydrat’c (31). It is also known (8) that both isoenzymes can coexist outside the cytoplasmic membrane, although the heavy one predominatjes inside the prohoplasts. In cont#rast,, in yeast the heavy aud light invert,ases correspond to the external and internal enzymes, and the external enzyme contains carbohydrate (15) whereas the internal is devoid of it (19).

Hoshino, Kaya, and Sato (11) have reported three forms of inverbase in bakers’ yeast, distinguishable by chromatography on DE.%E-cellulose columns. Forms I and II were eluted from the column; the third form was retained and was not further studied because it only represented 2 to 60/O of t,he t’otal activity. Hos- hino et al. used fully induced cells, and in view of t’he fact t,hat Forms I and II contained mannan (10) and constituted more than 90 y0 of the total invert’ase, they may represent the external invertase. Form III has two characteristics in common with our internal enzyme; that is, it represents a small fraction of the total, and it. adsorbs very strongly to anionic exchangers; how- ever, insufficient data have been published on the characteristics of this fraction to warra,nt, further speculation on that possibility.

The purified internal invertase from strain FH4C is homo- geneous by column chromatography and ultracentrifugation, and its specific act,ivity is in the same order of magnitude as that reported for external enzyme (15). In a companion paper (19)) a comparative study of the properties of both enzymes is made with special emphasis on a possible precursor-product relationship of the internal and external invertases. Some additional evidence for purit,y of the isolated enzyme is presented.

ilcknowledgments-~~~e wish to thank Dr. X. P. Neumann for the runs on t,he analytical ultracentrifuge. We are indebted to Dr. L. McDaniel and Mr. E. Bailey for t’heir assistance in the production of the yeast and their help in some steps of purifica- tion, and to Dr. P. Ottolenghi for making some of his results available to us in advance of publication.

REFEREXCES

1. L.YMPEN, J. O., NEUSUXS, N. P., C:.YSC~S, S., .wo MOSTENE- COUILT, B. S., irl H. J. \v~~~:~~, J. 0. LAMPEN, AwD \'. Bnu-

sol (Editors), Organizational biosynthesis, Academic Press, New York, 1967, p. 363.

2. WILKES, B. J., .\ND P.\LMxri, E. T., J. Gen. Physiol., 16, 233 (1932).

3.

4.

5.

A.

7. 8.

9.

10. 11.

12.

13.

14.

15.

16. 17.

18.

19.

20.

21.

22.

23. 24.

25.

20.

27. 28.

29.

30.

31.

I~EMIS, 11. J., HO~PHSTEIN, 9., .\ND MEIER, Ii., rZrch. Biochem. Hiophys., 48, 55 (1954).

PHIIS, J., .\ND OTTOLENGHI, P., Compl. Rend. ?‘?a~. Lab. Curlsberg, 31, 259 (1959).

SUTTON, II. I)., .&ND L.\MPES, J. O., Aiochim. Biophys. Acta, 56, 303 (1962).

EILICRS, F. I., ALLEN, J., TTILL, E:. P., AR‘D Hussnun-, 9. S., J. His&hem. Cytochem., 12, 448 (1964).

METZENBERG, It. L., Biochim. Hioph!/s. A&, 89, 291 (1964). TI~EVITHICK, J. R., .ixD MET~EXHERC:, It. L., Biochem. Bio-

phg’“. Res. Commun., 16, 319 (1964). TREVI'I'HICK. J. IL.. AND METZESHERG. R. L., J. Uacleriol.. 92,

1010 (1966j. ’ K.zu.1, T., J. Agr. Chem. Sot. Japan, 38, 417 (1964). IIosr-IINO, J., K1~y.*, T., AND S.\.ro, T., Plant Cell Physio(.,

5, 495 (1964). HOSHINO, J., AND MOMOSE, A., J. Uioc~hcm. (7’okyo), 59, 192

(19GG). HosHrn-o, J., AND MOMUSE, A., .J. Gen. Appt. Microbial., 12,

163 (1966). (;ASCfiN, s., Ai'iD O'I"I'OLENGH1, P., CO??@. Rend. 'I','af'. T,Ub.

Cc&berg, 36, 85 (1967). NEUMSNN, N. P., AND L.\I\IPEN, J. O., Jliochemistry, 6, 468

(1967). EYNR, E. II., J. Theor. Riot., 10, 89 (1965). SOOO.~K, M., iII N. 0. K..~PL.w \R‘I) E. P. KEn-l\-snY (Editors),

Current aspects of biochemical energetic-s, Ac:tdemic Press, Sew York, 1966, p. 459.

LANII'ES, J. O., in M. II. POLLOCK .\A-D M. 11. RICHW~ND (Editors), Pun&on and stwrcture in mirroorgani.sms, Carnhridge Urriversity Press, Loudon, 1965, p. 115.

ci.\SdN, S., NEUMANN, N. I’. , ASU hMPEN, J. O., J. Bid. Chern., 243, 1573 (1968).

KESUIN, A. S., Abstracts of the Ameriran Chemical Society Meeting, Dallas, April 1956, p. 31 C.

I>uno~s,~~., C&Es; K. A. I~.~NIILTON, J. K., RERERS, P. A., .WD SMITH. F.. ,4nal. Chew.. 28. 350 11956).

Lowtu, 0. H:, &EIIHOUGH, 2. J., F.w, ,d.'L., .\xD RAXDALL, II. J., J. Hiol. Chem., 193, 265 (1951).

Ah-~~n~~\-s, I’., Hiochem. J., 96, 595 (1965). COHX, E. J., AND EDS.\LL, T. J., Protein,s, aminw acids, and

peptides as ions and rtipolar ions, Reinhold Publishiny Corporatiorr, New York, 1943, p. 370.

WINGE, O., .AND ROBEI~TS, C., Compt. Rend. Trav. Lab. Carls- berg, Ser. Phyniol., 25, 419 (1957).

SYYINCTON,E. B., AND LAMPEN, J. O., ill V.M. SLAKH.%NOVA (Editor), Proceedings of the )Yinth International Congress o.f Microbiology, Moscow, J7dy 1966, p. 147.

I~AVIES, K., AND ELVIX, P. A., Hiochem. .I., 93, 8~ (1964). WI~KERH~M. L. J.. Tech. Hull. I.. S. Dept. ilw. No. 1029.

(1951). ’ _ ”

IsL.~, M. F., AND L.YMPEN, J. O., Riochim. Biophys. Acta, 58, 294 (1962).

BERTHELOT. M., Compf. Rend. Hebcl. Seunces Acad. Sri. 50, 980 (1860). -

ME~YZEXBERG, R.. L., Arch. Biochem. Biophys., 100, 503 (1963).