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
,llaterials-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 diluted 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 \lol. 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, containing 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 strains 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.
Eormation 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 lris-WC1 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 than 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 the
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
obtained. The enzyme released into thc 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
activity 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.
IuriJicution 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 quite 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
Wickerhams 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 for-
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
152
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 with 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 Procedures-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
applied 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 carbohydratc (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 total activity. Hos-
hino et al. used fully induced cells, and in view of the fact t,hat
Forms I and II contained mannan (10) and constituted more
than 90 y0 of the total invertase, 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 their 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.
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