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
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
