Tech Bull Fac Agr Kagawa Univ , Vol 41, No 1, 85-88, 1989
AUTOPROTEOLYTIC ACTIVITY IN SACCHAROMYCES CEREVISIAE CELLS
Masayuki SATO, Shunsuke UEMATU,
Haruki
SENOO
and Chitoshi NARIMATU
Saccharornyces cerevisiaeHB
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The CBB method for determination of autoproteolytic activity in Euglena gractlts by Krauspe et a1 could be applied to cell free extract of Saccharomyces cerevtszae The autoproteolytic activity of the yeast by the CBB method was nearly proportional to initial protein concentration in the cell free extract for 60 min at 30eC, pH 3 5 When a temperature sensitive mutant of S cerevzszae was incubated at the restrictive temperature (37"C), autoproteolytic activity by CBB method increased somewhat after 48 hr but decreased significantly by addition of cycloheximide in the incubation medium
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Oohashi et a1 (1) developed a new hypothesis, namely, "Programed Self-Decomposition Model," for living organisms In order to observe biological phenomena supporting the hypothesis, we have been studing on self-decomposition of cellular proteins in yeasts In our previous papers intracellular proteinase activities were shown to increase during the autolytic process of Saccharomyces cerevzszae cells (2,3) In the proteinase assay hemoglobin or azocoll has been generally used as the substrate We thought that endogenous proteins in the cells must be more suitable as the substrate for determination of autoproteolysis than hemoglobin or azocoll
Coomassie brilliant blue (CBB) dye-binding technique was developed for determination of autolyrtic protein breakdown in Euglena graczlts by Krauspe et al (4) They used the CBB method for determination of autolysis rates of Euglena cell free extracts In the present study it is shown that the CBB method can be applied to measurement of autoproteolytic activity in yeast cell free extracts And the fact is also reported that increase of autoproteolytic activity during death phase of yeast is repressed by cycloheximide
Materials and Methods
Organtsm and culttvatton Saccharomyces cerevzszae M-88, a temperature sensitive mutant, was used
OLIVE 香川大学学術情報リポジトリ
86 Tech Bull Fac Agr Kagawa Univ, Vol 41, No 1, 1989
in this study This organism was obtained by EMS (ethylmethansulfonate) treatment (5) of S cerevtstae I F 0 0847 and isolated by the methods according to Cold Spring Harbor Laboratory Manual (6) T h e mutant, M-88, can grow at 30°C but can't at 37'C In a nutrient medium The culture medium contained 40g of glucose, 3 5g of polypeptone, 3g of yeast extract, 2g of KHzPOa, l g of MgSO, 7Hz0 in one liter of deionized water The pH value of the medium was adjusted to 5 5 The organism was grown in the m e d ~ u m on a rotaly shaker for 3 days at 27°C
Incubatton o f cell suspenszon The cells were harvested by centrifugation and then washed with 0 1 M citrate-sodium phosphate buffer (pH 5 0) The cell suspension in the same buffer was incubated on a shaker at 37°C or 30°C
Preparatton o f cell free extract After the incubation, the cells were washed with 0 05 M sodium phosphate buffer (pH 7 5) containing 0 7 M sorbitol and then treated with Zymolyase according to Yamamura et a1 (7) The resulting protoplasts were washed with cold water and disruped by ultrasonic radiation at 0°C in water The sonicate was ultracentrifuged at 40,000 rpm(100,200Xg, 2") for 60 min T h e supernatant was used as Cell free extract
Assay methods Azocaseinolytic activity was assayed by the method of Krauspe et a1 (4) modified a s follows ; 0 25 ml of azocasein-buffer (pH4 0) and 0 25 ml of cell free extract were mixed and incubated for 30 min at 30°C and precipitated by 0 7 ml of 10% trichloroacetic acid and centrifuged at about 1,500Xg One unit of the activity was defined as the amount of enzyme which increase 0 01 of optical dencity at 366 nm per min under the above conditions Autoproteolytic activity was measured as breakdown rate of endogenous proteins in the cell free extract according to the CBB method of Krauspe et a1 (4) modified a s follows T h e appropriate amount of cell free extract was diluted with 0 1 M citrate-sodium phosphate buffer (pH3 5) preincubated at 3 0 C , and immediately aliquots (0 1 ml) of the dilution were mixed with 4 ml of the dye reagent and after 10 min absorbance of the mixture was measured at 595 nm This value is the zero-time value (Ass5, t=O min) Usually the cell free extract was diluted with the buffer to A5s5, t=O of 0 8-0 9 After different incubation periods at 30C, 0 1 ml of aliquots of the dilution were m e a s u ~ e d a s described (AsQ5. t = x min) Autoproteolysis rates were determined a s AAsss, the difference of Asss, t=O and AsQs, t= x and are expressed as AAsss per hour or A Ass5 per hour per mg of initial protein Proteins and their hydrolyzates were assayed by the method of Lowry et a1 (8) using bovine serum albumin a s a standard The number of living cells was counted by the colony plate method
Reagents Coomassie brilliant blue G (75%), bovine serum albumin and azocasein were purchased from Sigma Chemical Co Zymolyase-20000 was from Seikagaku Kogyo Co
Results
1 Autoproteolysis determination by CBB method
The difference, AAss5, of Asss, t=O and Asss, t = x by CBB method were plotted against incubation time in Fig 1 The approximately linear relationships were shown for 60 min with different dilution of the cell free extract At higher dilutions the linear relationship holds for 90 or 120 min And it was also
M SAIO, S UEMATU, H SENOO, C NARIMAIU : Autoproteolytic activity of yeast
0 15 30 45 60 2 4 6 8
Incubation tlme (mln) pH
Flg 2 Effect of pH on Autoproteolys~s F I ~ 1 Tlme dependence of autoproteolys~s measurement by CBB method
measurement by CBB method at pH (@), 0 1 M sodlum acetate-HCI buffer. 3 5 In~tlal Am, ( t = 0 mln), were
(0)
, 0 1 M cltrate-sodlum phosphate buffer, lnd~cated on the rlght of each curve (A) , 0 1~ glyc~ne-NaOH bufferdemonstrated that A Asg5 after the incubation time of 60 min is proportional to the initially used protein
concentration The effect of pH on AAsg5 per hour is shown in Fig 2 T h e major and minor peaks of AAss5 per hour appear at pH 3 5 and 8 0, respectively From these results, it was demonstrated that autoproteolysis in yeast cell free extracts can be assayed by the CBB method Acid proteinase may play main role in the autoproteolysis of yeast near at pH 3 5
2 Effect of cycloheximide on autoproteolysis
Table I shows extinction ratio of cells, residual intracellular Lowry positive compounds and proteolytic activities in cells after the incubation for 48 hr at 30°C In this experiments cells were preincubated at 30°C for 6 hr with or without cycloheximide and then incubated at 30 or 37°C for 48 hr A t 37"C, over 90% of M-88 cells died and about half of intracellular Lowry positive compounds was released outside the cells
Table 1 Effect of cycloheximide on proteolytic activities in Saccharomyces cerevtstae M-88 cells
Incubation* Extinction Residual intra- Autoproteolytic Azocaseinolytic cellular Lowry activity activity
addition Temp Time ratio*** positive by CBB
compound method ( ) (hr)
0
0
( A AssJhr/mg) (unit/mg) None - o** 0 100 30 48 3 1 9 1 37 48 9 1 52 Cyclo- - 0** 14 100 heximide 37 48 93 63*Cells were preincubated with or without cycloheximide ( 1 0 0 , ~ g/ml) in 0 1 M citrate-sodium phosphate buffer (pH5 0) at 30'C for 6 hr and followed by incubation at 30°C or 37°C for 48 hr
**Data at zero time were obtained from assay in cells after the preincubation for 6 hr Initial living cells - Residual living cells after incubation
***Extinction ratio = X 1 0 0 0
Initial living cells
88 Tech Bull Fac Agr Kagawa Univ , Vol 41, No 1, 1989
after incubation for 48 hr At 30"C, extinction ratio and proteolytic activities were considerably lower than at 37°C Death and autoproteolysis in this organism seemed to be induced by incubation at the restrictive temperature Autoproteolytic activity by CBB method increased somewhat after 48 hr at 37C, but decreased significantly by addition of cycloheximide in the incubation medium under the same conditions Azocaseinolysis activity at 37'C was also lower with cycloheximide than without it
Discussion
In this study it was shown that assay of autoproteolysis by CBB method could be applied to yeast cells This method appear to be more suitable for studing natural self-decomposition of yeast cells but is necessary to considerably careful procedure for obtaing reproducible values
Activation of yeast proteinases had been discussed by many investigations relating to specific proteinase inhibitors and protein modification But there were little reports which suggested de novo synthesis of proteinase in autolysis process of yeasts The decrease of autoproteolytic activity by cycloheximide in Table I suggest that biosynthesis of proteinases playing autoproteolysis is stimulated in the death phase yeast cells Oohashi et a1 (4) also observed similar results on Tetrahymena cells Subsequently biosynthesis of lytic enzymes during autolytic process must be proved by further investigations such as incorporation of 'T-amino acids into intracellular proteins
A temperature sensitive mutant of yeast was used in this study Because we thought that proteinases might be more stable in dead cells at 37°C than in dead cells by heating at 60'C for 5 min a s using in the previous study (3)
Acknowledgments We wish to thank Professor Shojiro Iwahara of Kagawa University, and Dr Tutomu Oohashi of the University of Tsukuba for their valualbe discussions and encouragement
References
( 1 ) OOHASHI, T , D NAKATA, T KIKUTA and K MURAKAMI, J Jpn Assoc Phtlo Sct , 18,
79 (1987)
( 2 ) SATO, M and Y OHTA, Tech Bull Fac Agr Kagawa Untv , 37, 11 (1985)
( 3 ) SATO, M , H MORMOTO and T OOHASHI, Agrtc Btol Chem , 51, 2609 (1987)
( 4 ) KRAUSPE, R and A SCHEER, Anal Btoc hem , 153, 242 (1986)
( 5 ) LINDEGREN, G , Y L HAUNG, Y OSHIMA and C C LINDEGREN, Can J Genet C y t o l , 7, 491 (1965)
( 6 ) SHERMAN,
F
, G R FINK and T D PETES, "Laboratory Manual for a Course Methods in Yeast Genetics, " Cold Spring Harbor Labor- atory for Quantitative Biology, Cold Spring Harbor, 1977, pp 4-8( 7 ) YAMAMURA, M , Y TERANISHI, A TANAK- A and S FUKUI, Agrtc Btol Chem , 39, 13 (1975)
( 8 ) LOWRY, 0 H , h' J ROSEBROUGH, A L FARR and R J RANDALL, J Btol Chem,
193, 265 (1951)
(Received October 31, 1988)
