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An improved method for the preparation of yeast enzymes in situ
Vol. 65, No. 4, 1975
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
AN IMPROVED METHOD FOR THE PREPARATION OF YEAST ENZYMES IN SITU.
H.T.A. Jaspers, K. Cb_ristianse and J. van Steveninek
Sylvius Laboratories Laboratory for Medical Chemistz7 Wassenaarseweg 72 Leiden, The Netherlands
Received
June 1 8 , 1 9 7 5
SUMMARY
Semi-permeable, enzymatic active yeast cells were prepared with a modification of the procedure, described in recent literature. To abolish the membrane barrier, chitosan was used instead of basic proteins. It appeared that the effect of chitosan was less susceptible to cations than the effect of basic proteins. Moreover, with ehitosan all cells of Saccharomyces cerevisiae were transformed into enzymatic active ghosts, whereas with protamine about 40% of the cells was unaffected.
INTRODUCTION
Schlenk et al. have described a method for the preparation of semi-permeable yeast cells, that retain full enzymatic activity (1-4). The method is based on the observation that basic proteins, like protamine, cytochrome c and pancreatic ribonuclease, abolish the memhrane barrier for small molecules. When yeast cells are incubated with a basic protein, cellular constituents of low molecular weight are released and the cells become permeable for substrates and products, whereas the enzymes are retained inside the semi-permeable cells. These yeast cell "ghosts" are very suitable for enzymatic studies (3). The enzymes are made accessible by a very simple, sparing method and can thus be studied under more or less physiological conditions (5,6).
Copy~ght© 19~byAcademicPress,~c. AHrigh~ofreproductioninanyform rese~ed.
1434
Vol. 65, No. 4, 1975
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
A disadvantage of the method is, that only about half of the yeast cells is made semi-permeable by the protein treatment (5,6). Further, the protein effect is inhibited strongly by the presence of cations (I). In experiments designed to improve the method on these points, the non-protein basic macromolecule chitosan was studied. The results of these studies are described in the present communication.
METHODS
The experiments were carried out with commercial baker's yeast, obtained from the Gist- en Spiritusfabriek,
Delft, and with Sacoharomyces fragilis, grown
on the medium described previously (7). A stock solution of chitosan was prepared by dissolving 500 mg deacetylated chitin (Mann Research Laboratories) in about 90 ml dilute acetic acid. After extensive dialysis against distilled water, the volume was adjusted to i00 ml. Semi-permeable ghosts were prepared by incubating a yeast cell suspension (2% wet weight/volume) with either protamine or chitosan at 30°C. The disappearance of the membrane barrier for small molecules was followed by measuring the release of cytoplasmic constituents, absorbing at 260 nm (2) and by selective staining of semi-permeable cells with uranyl nitrate and Ponceau Red
(8). Hexokinase activity was measured in triethano!amine, pH 7.0, 0.I M;
MgCI 2, 5 mM; ATP, 2.5 mM; glucose, 2.5 mM and ghosts, 5 mg/ml. The glucose concentration was measured at intervals, according to Washko and Rice (9). Glucose-6-phosphate dehydrogenase activity was measured as described by Schlenk and Zydek-Cwick (3).
~S~TS
During incubation of commercial bakerts yeast with protamine or chitosan, an increasing percentage of cells became stainable with uranyl nitrate and Ponceau Red. With protamine the maximal effect (about 65% of the cells stainable after 30 minutes) was reached at a final concentration o2 0.5 mg/ml; higher concentrations didn't increase the number of stainable cells. The optimal chitosan concentration appeared to be 0.05 mg/ml; with this concentration all
1435
Vol. 65, No. 4, 1975
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
cells became stainable within 15 minutes (fig. i). In all experiments there was a close parallel between the percentage of stainable cells and the release of cellular constituents, absorbing at 260 nm. The hexokinase and glucose-6-phosphate dehydrogenase activities of the ghost preparations are shown ih figs. 2 and 3. Both activities were considerably higher in chitosan-prepared ghosts, as compared to protamine-prepared ghosts. Increasing the protamine concentration to 1.0 or 2.0 mg/ml had no effect on the final enzyme activity. In control experiments it appeared that no enzyme activity was detectable in the supernatant after spinning down the ghosts. Apparently the enzyme molecules remained tightly bound to the ghosts, both after protamine and ehitosan treatment. As shown by Schlenk eta!.
cations strongly inhibit the effect of basic
proteins. This was confirmed in the present investigations.
The number of
stainable cells, the release of cell constituents absorbing at 260 nm and the measured enzyme activities decreased progressively, with increasing concentrations
,~ 1oo
D"--0----
O--
--
-0--
--
---0--
--
-0--
/
lu
=" "3
80
I
I
/
v
60
/Y 20
i/ I
I
I
20
I /,0
I
I 60 minutes
Fig. i. Staining of Saecharomyces cerevisiae cells with uranyl nitrate and Ponceau Red, after incubation with 0.Smg/ml protamine (@ --@ ) or with 0.05 mg/ml chitosan ( O - - O ).
1436
Vol. 65, No. 4, 1975
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
E
<~ 2.0
1.0
lio
210
30
410 minutes
Fig. 2. Hexokinase activity, as measured from the disappearance of glucose, of protamine-prepared ghosts ( O - - 0 )
and chitosan-prepared ghosts ( O - - O ) .
1.6 / /
1.2
J
i
/ /
/
o
/ /
0.8 i 0.4
i~iil/ I
I
I0
20
I
30 minutes
Fig. 3. Glucose-6-phosphate dehydrogenase activity, as measured from the formation of NADPH (A 340 nm), of protamine-prepared ghosts ( O - - O ) chitosan-prepared ghosts ( O - - ~ ) .
and
The ghost concentration was 5 mg/ml.
of cations. Although the effect of chitosanwas also susceptible to the presence of cations, the inhibition was much less pronounced. In fig. 4 the effect of cations on the release of cell constituents absorbing at 260 nm is depicted.
1437
Vol. 65, No. 4, 1975
BIOCHEMICALAND BIOPHYSICAL RESEARCH COMMUNICATIONS
=E 0./,
'
._o .
.
.
.
o - - - -
-.o--
----
o.--------
o-
\ \
0.3
\
\\
\\ k
0.2
h\
0.1
--
I
f
10
L I
A I
15 20 cation concentration mH
Fig. 4. Release of cell constituents~ absorbing at 260 nm~ after incubation during 30 minutes with 0.5 mg/ml protamine or 0.05 mg/ml chitosan, at varying cation concentrations in the medium. O--O - CaCI2; O - - O : chitosan - K C I ; ~ - - A
: protamine - K C I ; ~ - A
: protamine
: chitosan - CaCI 2.
Cell staining and enzyme activities were inhibited to the same extent. Similar results wore obtained with Saceharomyoes fragilis~ with one notable exception. Saceharomyces fragilis appeared to be more susceptible both to protamine and to chitosan. With 0.5 mg/ml protamine all cells were stainable with uranyl nitrate and Poneeau Red after an incubation time of 30 minutes, whereas optimal results with chitosan were obtained with 0.025 mg/ml. In accordanc% enzyme activities were equal in protamine- and ehitosan-treated cells.
DISCUSSION
Schlenk et al. suggested that the effect of basic proteins on the yeast cell membrane may be attributed to a binding of positively charged groups of the proteins to negative charges on the membrane surface (I)° The exact mechanism at a molecular level is still nnknown~ however. The present investigations show~ that the protein nature of the agens is not essential;
1438
Vol. 65, No. 4, 1975
BIOCHEMICALAND BIOPHYSICAL RESEARCH COMMUNICATIONS
similar effects were found with the cationic polysaccharide chitosan. These ghosts can be used successfully in enzymatic studies (5,6). Especially in studies on the relationship between phase of growth and enzyme activity this method is very useful (6). It should be realized, however, that utilizing Protamine or other basic proteins, a varying percentage of cells will not be attacked. In this respect there are differences between different yeast strains, as shown in the present communication. It is conceivable that similar differences would exist e.g. between cells, harvested during the log phase of growth and cells, harvested in the stationary phase. Further, even low concentrations of cations interfere strongly with the effect of basic proteins. The present results indicate that these problems may be solved by utilizing chitosan. Provisionally, q~antitative enzyme studies with this method should include control measurements of the number of stainable cells.
ACKNOWLEDGEMENT
These investigations were carried out under auspices of the Netherland Foundation for Biophysics and with financial aid from the Netherlands Organization for the Advancement of Pure Research (ZWO).
REFERENCES
i. Yphantis, D.A., Dainko, J.L. and Seh!enk, F. (1967) J. Bacteriol. 9~, 1509 - 1515. 2. Svihla, G., Dainko, J.L. and Sehlenk, F. (1969) J. Bacteriol. 100, 498 - 504. 3. Schlenk, F. and Zydek - Cwick, C.R. (1970) Arch. Biochem. Biophys. 138, 220 - 225. 4. Sohlenk, F. and Dainko, J.L. (1965) J. Baeteriol. 89, 428 - 436. 5. Kosow, D.P. and Rose, I.A. (1971) J. Biol. Chem. 246 , 2618 - 2625. 6. Reitzer, L.J. and Neet, K.E. (1974) Biochim. Biophys. Acta ~41, 201 - 212. 7. Van Steveninck, ft. (1972) Biochim. Biophys. Acta 274, 575 - 583. 8. Maas, M. and Van Steveninek, J. (1967) Experientia2_~, 405 - 406. 9. Washko, M.E. and Rice, E.W. (1961) Clin. Chem. ~, 542 - 545.
1439
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
AN IMPROVED METHOD FOR THE PREPARATION OF YEAST ENZYMES IN SITU.
H.T.A. Jaspers, K. Cb_ristianse and J. van Steveninek
Sylvius Laboratories Laboratory for Medical Chemistz7 Wassenaarseweg 72 Leiden, The Netherlands
Received
June 1 8 , 1 9 7 5
SUMMARY
Semi-permeable, enzymatic active yeast cells were prepared with a modification of the procedure, described in recent literature. To abolish the membrane barrier, chitosan was used instead of basic proteins. It appeared that the effect of chitosan was less susceptible to cations than the effect of basic proteins. Moreover, with ehitosan all cells of Saccharomyces cerevisiae were transformed into enzymatic active ghosts, whereas with protamine about 40% of the cells was unaffected.
INTRODUCTION
Schlenk et al. have described a method for the preparation of semi-permeable yeast cells, that retain full enzymatic activity (1-4). The method is based on the observation that basic proteins, like protamine, cytochrome c and pancreatic ribonuclease, abolish the memhrane barrier for small molecules. When yeast cells are incubated with a basic protein, cellular constituents of low molecular weight are released and the cells become permeable for substrates and products, whereas the enzymes are retained inside the semi-permeable cells. These yeast cell "ghosts" are very suitable for enzymatic studies (3). The enzymes are made accessible by a very simple, sparing method and can thus be studied under more or less physiological conditions (5,6).
Copy~ght© 19~byAcademicPress,~c. AHrigh~ofreproductioninanyform rese~ed.
1434
Vol. 65, No. 4, 1975
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
A disadvantage of the method is, that only about half of the yeast cells is made semi-permeable by the protein treatment (5,6). Further, the protein effect is inhibited strongly by the presence of cations (I). In experiments designed to improve the method on these points, the non-protein basic macromolecule chitosan was studied. The results of these studies are described in the present communication.
METHODS
The experiments were carried out with commercial baker's yeast, obtained from the Gist- en Spiritusfabriek,
Delft, and with Sacoharomyces fragilis, grown
on the medium described previously (7). A stock solution of chitosan was prepared by dissolving 500 mg deacetylated chitin (Mann Research Laboratories) in about 90 ml dilute acetic acid. After extensive dialysis against distilled water, the volume was adjusted to i00 ml. Semi-permeable ghosts were prepared by incubating a yeast cell suspension (2% wet weight/volume) with either protamine or chitosan at 30°C. The disappearance of the membrane barrier for small molecules was followed by measuring the release of cytoplasmic constituents, absorbing at 260 nm (2) and by selective staining of semi-permeable cells with uranyl nitrate and Ponceau Red
(8). Hexokinase activity was measured in triethano!amine, pH 7.0, 0.I M;
MgCI 2, 5 mM; ATP, 2.5 mM; glucose, 2.5 mM and ghosts, 5 mg/ml. The glucose concentration was measured at intervals, according to Washko and Rice (9). Glucose-6-phosphate dehydrogenase activity was measured as described by Schlenk and Zydek-Cwick (3).
~S~TS
During incubation of commercial bakerts yeast with protamine or chitosan, an increasing percentage of cells became stainable with uranyl nitrate and Ponceau Red. With protamine the maximal effect (about 65% of the cells stainable after 30 minutes) was reached at a final concentration o2 0.5 mg/ml; higher concentrations didn't increase the number of stainable cells. The optimal chitosan concentration appeared to be 0.05 mg/ml; with this concentration all
1435
Vol. 65, No. 4, 1975
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
cells became stainable within 15 minutes (fig. i). In all experiments there was a close parallel between the percentage of stainable cells and the release of cellular constituents, absorbing at 260 nm. The hexokinase and glucose-6-phosphate dehydrogenase activities of the ghost preparations are shown ih figs. 2 and 3. Both activities were considerably higher in chitosan-prepared ghosts, as compared to protamine-prepared ghosts. Increasing the protamine concentration to 1.0 or 2.0 mg/ml had no effect on the final enzyme activity. In control experiments it appeared that no enzyme activity was detectable in the supernatant after spinning down the ghosts. Apparently the enzyme molecules remained tightly bound to the ghosts, both after protamine and ehitosan treatment. As shown by Schlenk eta!.
cations strongly inhibit the effect of basic
proteins. This was confirmed in the present investigations.
The number of
stainable cells, the release of cell constituents absorbing at 260 nm and the measured enzyme activities decreased progressively, with increasing concentrations
,~ 1oo
D"--0----
O--
--
-0--
--
---0--
--
-0--
/
lu
=" "3
80
I
I
/
v
60
/Y 20
i/ I
I
I
20
I /,0
I
I 60 minutes
Fig. i. Staining of Saecharomyces cerevisiae cells with uranyl nitrate and Ponceau Red, after incubation with 0.Smg/ml protamine (@ --@ ) or with 0.05 mg/ml chitosan ( O - - O ).
1436
Vol. 65, No. 4, 1975
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
E
<~ 2.0
1.0
lio
210
30
410 minutes
Fig. 2. Hexokinase activity, as measured from the disappearance of glucose, of protamine-prepared ghosts ( O - - 0 )
and chitosan-prepared ghosts ( O - - O ) .
1.6 / /
1.2
J
i
/ /
/
o
/ /
0.8 i 0.4
i~iil/ I
I
I0
20
I
30 minutes
Fig. 3. Glucose-6-phosphate dehydrogenase activity, as measured from the formation of NADPH (A 340 nm), of protamine-prepared ghosts ( O - - O ) chitosan-prepared ghosts ( O - - ~ ) .
and
The ghost concentration was 5 mg/ml.
of cations. Although the effect of chitosanwas also susceptible to the presence of cations, the inhibition was much less pronounced. In fig. 4 the effect of cations on the release of cell constituents absorbing at 260 nm is depicted.
1437
Vol. 65, No. 4, 1975
BIOCHEMICALAND BIOPHYSICAL RESEARCH COMMUNICATIONS
=E 0./,
'
._o .
.
.
.
o - - - -
-.o--
----
o.--------
o-
\ \
0.3
\
\\
\\ k
0.2
h\
0.1
--
I
f
10
L I
A I
15 20 cation concentration mH
Fig. 4. Release of cell constituents~ absorbing at 260 nm~ after incubation during 30 minutes with 0.5 mg/ml protamine or 0.05 mg/ml chitosan, at varying cation concentrations in the medium. O--O - CaCI2; O - - O : chitosan - K C I ; ~ - - A
: protamine - K C I ; ~ - A
: protamine
: chitosan - CaCI 2.
Cell staining and enzyme activities were inhibited to the same extent. Similar results wore obtained with Saceharomyoes fragilis~ with one notable exception. Saceharomyces fragilis appeared to be more susceptible both to protamine and to chitosan. With 0.5 mg/ml protamine all cells were stainable with uranyl nitrate and Poneeau Red after an incubation time of 30 minutes, whereas optimal results with chitosan were obtained with 0.025 mg/ml. In accordanc% enzyme activities were equal in protamine- and ehitosan-treated cells.
DISCUSSION
Schlenk et al. suggested that the effect of basic proteins on the yeast cell membrane may be attributed to a binding of positively charged groups of the proteins to negative charges on the membrane surface (I)° The exact mechanism at a molecular level is still nnknown~ however. The present investigations show~ that the protein nature of the agens is not essential;
1438
Vol. 65, No. 4, 1975
BIOCHEMICALAND BIOPHYSICAL RESEARCH COMMUNICATIONS
similar effects were found with the cationic polysaccharide chitosan. These ghosts can be used successfully in enzymatic studies (5,6). Especially in studies on the relationship between phase of growth and enzyme activity this method is very useful (6). It should be realized, however, that utilizing Protamine or other basic proteins, a varying percentage of cells will not be attacked. In this respect there are differences between different yeast strains, as shown in the present communication. It is conceivable that similar differences would exist e.g. between cells, harvested during the log phase of growth and cells, harvested in the stationary phase. Further, even low concentrations of cations interfere strongly with the effect of basic proteins. The present results indicate that these problems may be solved by utilizing chitosan. Provisionally, q~antitative enzyme studies with this method should include control measurements of the number of stainable cells.
ACKNOWLEDGEMENT
These investigations were carried out under auspices of the Netherland Foundation for Biophysics and with financial aid from the Netherlands Organization for the Advancement of Pure Research (ZWO).
REFERENCES
i. Yphantis, D.A., Dainko, J.L. and Seh!enk, F. (1967) J. Bacteriol. 9~, 1509 - 1515. 2. Svihla, G., Dainko, J.L. and Sehlenk, F. (1969) J. Bacteriol. 100, 498 - 504. 3. Schlenk, F. and Zydek - Cwick, C.R. (1970) Arch. Biochem. Biophys. 138, 220 - 225. 4. Sohlenk, F. and Dainko, J.L. (1965) J. Baeteriol. 89, 428 - 436. 5. Kosow, D.P. and Rose, I.A. (1971) J. Biol. Chem. 246 , 2618 - 2625. 6. Reitzer, L.J. and Neet, K.E. (1974) Biochim. Biophys. Acta ~41, 201 - 212. 7. Van Steveninck, ft. (1972) Biochim. Biophys. Acta 274, 575 - 583. 8. Maas, M. and Van Steveninek, J. (1967) Experientia2_~, 405 - 406. 9. Washko, M.E. and Rice, E.W. (1961) Clin. Chem. ~, 542 - 545.
1439