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Destruction of cocarboxylase in active dry yeast
ARCHIVES
OF
BIOCHEMISTRY
AND
BIOPHYSICS
62,
29%-3i)d
(1956)
Destruction of Cocarboxylase in Active Dry Yeast S. L. Chen and H. J. Peppler From the Research Department, Biochemistry Laboratory, Red Star Yeast and Products Company, Milwaukee, Wisconsin
Received November 10, 1955
INTRODUCTION It is a common experience that the activity of baker’s yeast decreases during storage. Such decline in activity can be accelerated by exposure to moderately high temperatures for short periods. Thus, under controlled conditions, factors affecting yeast stability and metabolic changes in active dry yeast can be systematically investigated. Reported in this communication are some changes in the pyruvate decarboxylation system of yeast cells during accelerated exposure. METHODS Both fermentation and pyruvete decarboxylation by yeast cells were measured under an atmosphere of nitrogen by the conventional techniques of Warburg manometry (1). For anaerobic fermentations, each Warburg flask contained 1.0 ml. of phosphate buffer (pH 6.0, 0.1 M), 0.1 ml. MgSOd (0.1 M), 0.9 ml. water, 0.5 ml. of yeast suspension (2.76 mg. yeast solids) in the vessel, and 0.5 ml. of glucose solution (0.1 M) in the side arm. Pyruvate decarboxylation was measured after the introduction of 0.5 ml. citrate buffer (pH 6.0), 0.1 ml. MgSO, (0.1 M), 1.4 ml. water, 0.5 ml. yeast suspension (5.5 mg. yeast solids) toeachvessel, and0.5 ml. of pyruvate (0.5 M, pH 5.9) to the side arm. All measurements were made at 30”. Crude yeast carboxylase was prepared by washing 1 g. of active dry yeast (ADY) three times with 400 ml. of NasHPOl (0.1 M) and once with distilled water. The alkali-washed yeast was resuspended in phosphate buffer (pH 6.0, 0.1 M). This preparation contained only apocarboxylase, and, without thiamine pyrophosphate and MgS04, it showed very little pyruvate decarboxylation activity. For the determination of carboxylase activity, the following composition was used for each Warburg vessel: 1.0 ml. of phosphate buffer (pH 6.0, 0.1 M), 0.1 ml. of MgSOa (O.l1M), 1.0 ml. of crystalline thiamine pyrophosphate solution (100 pg.), and 0.4 ml. of alkali-washed yeast (18 mg. yeast solids) ; 0.5 ml. of pyruvate (0.5 M, pH 5.9) was placed in the side arm. Cocarboxylase content in the yeast extracts was estimated under similar con299
300
S. L. CHEN AND H. J. PEPPLER
ditions, i.e., 1.0 ml. of phosphate buffer (pH 6.0, 0.1 M), 0.1 ml. MgSOa (0.1 M), 0.4 ml. water, 0.5 ml. of the extract containing cocarboxylase, 0.5 ml. of alksliwashed yeast (36 mg. yeast solids) in the vessel, and 0.5 ml. of pyruvate in the side arm.
EXPERIMENTAL
RESULTS
Deterioration of Active Dry Yeast During Accelerated Exposure Samples of Red Star active dry yeast (ADY) were exposed under oxygen at 48” for periods of l-8 days. The activity of these exposed samples was determined by measuring their anaerobic fermentation and pyruvate decarboxylation rates, as well as their baking performance. The results of fermentation and pyruvate decarboxylation measurements are shown in Table I; they are compared on a relative basis with the bake test (2) in Fig. 1. A parallelism between all values is apparent. The pyruvate decarboxylation rate of the alkali-washed yeast was measured in the presence of 100 pg. of crystalline thiamine pyrophosphate to determine whether the deterioration is due in part to the denaturation of apocarboxylase. The results of such measurements (Table II) show no progressive decline of carboxylase activity with continued exposure of the yeast, indicating slight or no damage to the apoensyme. Destruction of Cocarboxylase During Exposure Changes in cocarboxylase content of ADY during exposure were determined from the activity of the cocarboxylase extracted. The optimum conditions of extraction were determined by rehydrating 0.5 g. of ADY TABLE Anaerobic
I
Fermentation and Pyruvate Decarboxylation Yeast (ADY) Exposed at 48°C. Time of exposure dlZYS
Anaerobic fermentation rl. COP
0
185 163 149 125 119 106 93 89
1 2 3 4 6 7 8
Q In 25 min. per 2.7 mg. yeast solids. ) In 45 min. per 5.5 mg. yeast solids.
Pyrllvate . deca$$$on
136 118 116 95 91 89 81 77
of Active
Dry
DESTRUCTION
OF
301
COCARBOXY5ASE
2
4
a
6
EXPOSURE TIME, DAY FIG. 1. A comparison and pyruvate
of relative baking decarboxylation
activity
TABLE Carboxylase
Activity
of Alkali-Washed Time of exposure days
0 2 3 4 6 7 8
(X),
anaerobic
fermentation
(0)
(0) of exposed active dry yeast. II Yeast after Exposure
at &‘C.
(hboxylase activity pl. COP
83 73 78 76 103 95 100
= In 25 min. per 10 mg. yeast solids.
in 6 ml. of water at 45” for 5 min., then immediately transferring the suspensions to water baths held at O”, 25”, 45”, 55”, 65’, 75”, 85”, and 95”. Then 4.0 ml. of NazHPOd solution (0.5 M) was added to each sample. After extraction for 10 min., the samples were cooled in ice and the cells were removed by centrifugation. All supernatant liquids were made up to 9 ml. in volume with distilled water. The cocarboxylase activity of these extracts was determined with a fresh preparation of alkali-washed carboxylase. The effect of temperature on cocarboxylase extraction (Table III) shows that the optimum temperature for extraction was between 65” and 75”. Extraction of cocarboxylase in this temperature range seemed to be complete since a sixfold increase of the Na2HP04 to yeast ratio during rehydration and extraction produced no change in extraction efficiency. Accelerated exposure conditions progressively decrease the cocarboxylase activity of the yeast cells (Table IV). These results together with
302
S.
L.
CHEN
AND
H.
TABLE E$ect of Temperature
J.
PEPPLER
III
on Cocarboxylase
Extraction
from ADY
TemE?ture
Cocarb~~y~~~~activity
2 25 45 55 65 75 85 95
0 0 1 11 104 100 76 57
0 In 25 min. per 0.5 ml. extract. TABLE Cocarboxylase
IV
Content of ADY
Time of exposure days
0 2 3 4 6 8
co,
after Exposure
Coca~boz+w p$llctm
129 93 91 53 44 37
at .&PC.
actiyity Relatwe )W
activity
cent 100.0 72.1 70.6 41.1 34.2 28.7
S In 25 min. per 0.5 ml. extract.
those in Table II show that this decline of pyruvate decarboxylation activity in yeast is due to the deterioration of cocarboxylase but not to apocarboxylase. Restoration of Pyruvate Decarboxylation Activity in Exposed ADY Since the decline of pyruvate decarboxylation activity in exposed samples is due to the destruction of cocarboxylase in the cells, it is conceivable that the lost activity may be partly restored with externally supplied thiamine pyrophosphate (TPP). The results in Table V show that crystalline thiamine pyrophosphate increased the pyruvate decarboxylation activity of the exposed samples to the level of the control in the presence of MgSOd and ATP. Obviously, the response to added TPP increases with extended exposure. For example, a 73 % increase was observed in a-day-exposed ADY as compared with a 6% increase in the control.
DESTRUCTION
Pyruvate
OF
303
COCARBOXYLASE
TABLE V Decarboxylation Activity of Exposed ADY in the Presence of Thiamine Pyrophosphate, ATP and Mg* Pyruvate decarboxylation activity’ ADY samples No thiamine With thiamine (Exposjre$me) pyr;ThF Jmte pyr;fhF *hate a .B .B
fictivity mcrease ger cent
lllb 103 75 68
5.4 13.6 70.7 73.5
0
2 6 8
117b 117 128 118
a Composition of medium: 0.5 ml. citrate-phosphate buffer (pH 6.0), 0.1 mg. MgSO( (0.1 M), 0.4 ml. ATP (10 mg./ml.), 1.0 ml. thiamine pyrophosphate (100 mg.), 0.5 ml. yeast suspension (5.5 mg. yeast solids), plus 0.5 ml. pyruvate (0.5 M, p.EI5.8) in the side arm. For those with no thiamine pyrophosphate, 1.5 ml. of water was added to each vessel to replace MgSOa, ATP, and TPP. b Microliters COJ30 minJ5.5 mg. yeast solids. DISCUSSION
The parallelism between pyruvate decarboxylation activity and anaerobic fermentation as well as baking performance of exposed yeast samples is apparent (Fig. 1). Since the CO2 produced during baking is derived directly from pyruvate decarboxylation, it is conceivable that any deterioration in the latter system may account for the general decline of yeast activity. During accelerated exposure at 48” under oxygen, the deterioration of the pyruvate decarboxylation system was due to the destruction of cocarboxylase. This was proved by the fact that (a) no systematic and significant denaturation of apocarboxylase was detected, (5) less cocarboxylase was extracted from samples subjected to progressive exposure, and (c) a greater response of exposed samples occurred toward externally supplied thiamine pyrophosphate. While the exact nature of cocarboxylase deterioration was not apparent, it was thought that cocarboxylase might have been converted into an inactive form by oxidation. Myrbiick et al. (3) reported that a synthetic thiamine pyrophosphate could be oxidized in alkaline solution to cocarboxylase disulfide (thiamine disulfide pyrophosphate) . Karrer and Visconti (4) synthesized this compound and found it was completely inactive. This was confirmed by Wachtmeister and MyrbZick (5). Although the pyruvate decarboxylation activity of the exposed active dry yeast can be increased in a buffered system containing ATP, Mg++,
304
S. L. CHEN AND H. J. PEPPLER
and thiamine pyrophosphate, the complete reactivation of the lost activity in the cells could not be demonstrated. Leijnse and Terpstra (6) enhanced the cocarboxylase content of fresh yeast by incubating resting cells (under nitrogen) with thiamine and (NHJzS04. Exposed dried yeast subjected to similar treatment, however, exhibited no recovery of pyruvate decarboxylation activity. Westenbrink et al. (7) believed that the cocarboxylase synthesized under such conditions was not free TPP but probably a TPP-protein symplex not identical with carboxylase, and therefore, played no part in the dissimilation of glucose or pyruvic acid. SUbXMARY
A parallelism between pyruvate decarboxylation, anaerobic fermentation, and baking activity was obtained in ADY samples exposed from 1 to 8 days at 48” under oxygen. The deterioration of the pyruvate decarboxylation system in the exposed ADY samples was not due to denaturation of carboxylase (apoenzyme) but to destruction of cocarboxylase in yeast cells. The pyruvate decarboxylation activity lost during accelerated exposure of ADY could be restored to the control level by externally supplied thiamine pyrophosphate, Mg++, and ATP. REFERENCES 1. UMBREIT, W. W., BURRIS, R. H., AND STAUFFER, J. F., “Manometric Techniques,” 2nd ed. Burgess Publishing Company, Minneapolis, 1949. pp. 385-9. Siebel Publishing 2. PYLER, E. J., “Baking Science and Technology,” Company, 1952. 3. MYRBXCK, K., VALLIN, I., AND MAGNELL, I., Svensk Kern. Tidskr. 67,124 (1945). 4. KARRER, P., AND VISCONTI, M., Helv. Chim. Acta 29, 711 (1946). 5. WACHTMEISTER, C. A., AND MYRBXCK, K., Arkiv Kemi, Mineral. Geol. 24A, No. 8 (1946). 6. LEIJNSE, B., AND TERPSTRA, W., Biochim. et Biophys. Acta 7, 574 (1951). 7. WESTENBRINK, H. G. K., STEYN-PARVE, E. P., AND VELDMAN, H., Biochim. et Biophys. Acta 1, 154 (1947).
OF
BIOCHEMISTRY
AND
BIOPHYSICS
62,
29%-3i)d
(1956)
Destruction of Cocarboxylase in Active Dry Yeast S. L. Chen and H. J. Peppler From the Research Department, Biochemistry Laboratory, Red Star Yeast and Products Company, Milwaukee, Wisconsin
Received November 10, 1955
INTRODUCTION It is a common experience that the activity of baker’s yeast decreases during storage. Such decline in activity can be accelerated by exposure to moderately high temperatures for short periods. Thus, under controlled conditions, factors affecting yeast stability and metabolic changes in active dry yeast can be systematically investigated. Reported in this communication are some changes in the pyruvate decarboxylation system of yeast cells during accelerated exposure. METHODS Both fermentation and pyruvete decarboxylation by yeast cells were measured under an atmosphere of nitrogen by the conventional techniques of Warburg manometry (1). For anaerobic fermentations, each Warburg flask contained 1.0 ml. of phosphate buffer (pH 6.0, 0.1 M), 0.1 ml. MgSOd (0.1 M), 0.9 ml. water, 0.5 ml. of yeast suspension (2.76 mg. yeast solids) in the vessel, and 0.5 ml. of glucose solution (0.1 M) in the side arm. Pyruvate decarboxylation was measured after the introduction of 0.5 ml. citrate buffer (pH 6.0), 0.1 ml. MgSO, (0.1 M), 1.4 ml. water, 0.5 ml. yeast suspension (5.5 mg. yeast solids) toeachvessel, and0.5 ml. of pyruvate (0.5 M, pH 5.9) to the side arm. All measurements were made at 30”. Crude yeast carboxylase was prepared by washing 1 g. of active dry yeast (ADY) three times with 400 ml. of NasHPOl (0.1 M) and once with distilled water. The alkali-washed yeast was resuspended in phosphate buffer (pH 6.0, 0.1 M). This preparation contained only apocarboxylase, and, without thiamine pyrophosphate and MgS04, it showed very little pyruvate decarboxylation activity. For the determination of carboxylase activity, the following composition was used for each Warburg vessel: 1.0 ml. of phosphate buffer (pH 6.0, 0.1 M), 0.1 ml. of MgSOa (O.l1M), 1.0 ml. of crystalline thiamine pyrophosphate solution (100 pg.), and 0.4 ml. of alkali-washed yeast (18 mg. yeast solids) ; 0.5 ml. of pyruvate (0.5 M, pH 5.9) was placed in the side arm. Cocarboxylase content in the yeast extracts was estimated under similar con299
300
S. L. CHEN AND H. J. PEPPLER
ditions, i.e., 1.0 ml. of phosphate buffer (pH 6.0, 0.1 M), 0.1 ml. MgSOa (0.1 M), 0.4 ml. water, 0.5 ml. of the extract containing cocarboxylase, 0.5 ml. of alksliwashed yeast (36 mg. yeast solids) in the vessel, and 0.5 ml. of pyruvate in the side arm.
EXPERIMENTAL
RESULTS
Deterioration of Active Dry Yeast During Accelerated Exposure Samples of Red Star active dry yeast (ADY) were exposed under oxygen at 48” for periods of l-8 days. The activity of these exposed samples was determined by measuring their anaerobic fermentation and pyruvate decarboxylation rates, as well as their baking performance. The results of fermentation and pyruvate decarboxylation measurements are shown in Table I; they are compared on a relative basis with the bake test (2) in Fig. 1. A parallelism between all values is apparent. The pyruvate decarboxylation rate of the alkali-washed yeast was measured in the presence of 100 pg. of crystalline thiamine pyrophosphate to determine whether the deterioration is due in part to the denaturation of apocarboxylase. The results of such measurements (Table II) show no progressive decline of carboxylase activity with continued exposure of the yeast, indicating slight or no damage to the apoensyme. Destruction of Cocarboxylase During Exposure Changes in cocarboxylase content of ADY during exposure were determined from the activity of the cocarboxylase extracted. The optimum conditions of extraction were determined by rehydrating 0.5 g. of ADY TABLE Anaerobic
I
Fermentation and Pyruvate Decarboxylation Yeast (ADY) Exposed at 48°C. Time of exposure dlZYS
Anaerobic fermentation rl. COP
0
185 163 149 125 119 106 93 89
1 2 3 4 6 7 8
Q In 25 min. per 2.7 mg. yeast solids. ) In 45 min. per 5.5 mg. yeast solids.
Pyrllvate . deca$$$on
136 118 116 95 91 89 81 77
of Active
Dry
DESTRUCTION
OF
301
COCARBOXY5ASE
2
4
a
6
EXPOSURE TIME, DAY FIG. 1. A comparison and pyruvate
of relative baking decarboxylation
activity
TABLE Carboxylase
Activity
of Alkali-Washed Time of exposure days
0 2 3 4 6 7 8
(X),
anaerobic
fermentation
(0)
(0) of exposed active dry yeast. II Yeast after Exposure
at &‘C.
(hboxylase activity pl. COP
83 73 78 76 103 95 100
= In 25 min. per 10 mg. yeast solids.
in 6 ml. of water at 45” for 5 min., then immediately transferring the suspensions to water baths held at O”, 25”, 45”, 55”, 65’, 75”, 85”, and 95”. Then 4.0 ml. of NazHPOd solution (0.5 M) was added to each sample. After extraction for 10 min., the samples were cooled in ice and the cells were removed by centrifugation. All supernatant liquids were made up to 9 ml. in volume with distilled water. The cocarboxylase activity of these extracts was determined with a fresh preparation of alkali-washed carboxylase. The effect of temperature on cocarboxylase extraction (Table III) shows that the optimum temperature for extraction was between 65” and 75”. Extraction of cocarboxylase in this temperature range seemed to be complete since a sixfold increase of the Na2HP04 to yeast ratio during rehydration and extraction produced no change in extraction efficiency. Accelerated exposure conditions progressively decrease the cocarboxylase activity of the yeast cells (Table IV). These results together with
302
S.
L.
CHEN
AND
H.
TABLE E$ect of Temperature
J.
PEPPLER
III
on Cocarboxylase
Extraction
from ADY
TemE?ture
Cocarb~~y~~~~activity
2 25 45 55 65 75 85 95
0 0 1 11 104 100 76 57
0 In 25 min. per 0.5 ml. extract. TABLE Cocarboxylase
IV
Content of ADY
Time of exposure days
0 2 3 4 6 8
co,
after Exposure
Coca~boz+w p$llctm
129 93 91 53 44 37
at .&PC.
actiyity Relatwe )W
activity
cent 100.0 72.1 70.6 41.1 34.2 28.7
S In 25 min. per 0.5 ml. extract.
those in Table II show that this decline of pyruvate decarboxylation activity in yeast is due to the deterioration of cocarboxylase but not to apocarboxylase. Restoration of Pyruvate Decarboxylation Activity in Exposed ADY Since the decline of pyruvate decarboxylation activity in exposed samples is due to the destruction of cocarboxylase in the cells, it is conceivable that the lost activity may be partly restored with externally supplied thiamine pyrophosphate (TPP). The results in Table V show that crystalline thiamine pyrophosphate increased the pyruvate decarboxylation activity of the exposed samples to the level of the control in the presence of MgSOd and ATP. Obviously, the response to added TPP increases with extended exposure. For example, a 73 % increase was observed in a-day-exposed ADY as compared with a 6% increase in the control.
DESTRUCTION
Pyruvate
OF
303
COCARBOXYLASE
TABLE V Decarboxylation Activity of Exposed ADY in the Presence of Thiamine Pyrophosphate, ATP and Mg* Pyruvate decarboxylation activity’ ADY samples No thiamine With thiamine (Exposjre$me) pyr;ThF Jmte pyr;fhF *hate a .B .B
fictivity mcrease ger cent
lllb 103 75 68
5.4 13.6 70.7 73.5
0
2 6 8
117b 117 128 118
a Composition of medium: 0.5 ml. citrate-phosphate buffer (pH 6.0), 0.1 mg. MgSO( (0.1 M), 0.4 ml. ATP (10 mg./ml.), 1.0 ml. thiamine pyrophosphate (100 mg.), 0.5 ml. yeast suspension (5.5 mg. yeast solids), plus 0.5 ml. pyruvate (0.5 M, p.EI5.8) in the side arm. For those with no thiamine pyrophosphate, 1.5 ml. of water was added to each vessel to replace MgSOa, ATP, and TPP. b Microliters COJ30 minJ5.5 mg. yeast solids. DISCUSSION
The parallelism between pyruvate decarboxylation activity and anaerobic fermentation as well as baking performance of exposed yeast samples is apparent (Fig. 1). Since the CO2 produced during baking is derived directly from pyruvate decarboxylation, it is conceivable that any deterioration in the latter system may account for the general decline of yeast activity. During accelerated exposure at 48” under oxygen, the deterioration of the pyruvate decarboxylation system was due to the destruction of cocarboxylase. This was proved by the fact that (a) no systematic and significant denaturation of apocarboxylase was detected, (5) less cocarboxylase was extracted from samples subjected to progressive exposure, and (c) a greater response of exposed samples occurred toward externally supplied thiamine pyrophosphate. While the exact nature of cocarboxylase deterioration was not apparent, it was thought that cocarboxylase might have been converted into an inactive form by oxidation. Myrbiick et al. (3) reported that a synthetic thiamine pyrophosphate could be oxidized in alkaline solution to cocarboxylase disulfide (thiamine disulfide pyrophosphate) . Karrer and Visconti (4) synthesized this compound and found it was completely inactive. This was confirmed by Wachtmeister and MyrbZick (5). Although the pyruvate decarboxylation activity of the exposed active dry yeast can be increased in a buffered system containing ATP, Mg++,
304
S. L. CHEN AND H. J. PEPPLER
and thiamine pyrophosphate, the complete reactivation of the lost activity in the cells could not be demonstrated. Leijnse and Terpstra (6) enhanced the cocarboxylase content of fresh yeast by incubating resting cells (under nitrogen) with thiamine and (NHJzS04. Exposed dried yeast subjected to similar treatment, however, exhibited no recovery of pyruvate decarboxylation activity. Westenbrink et al. (7) believed that the cocarboxylase synthesized under such conditions was not free TPP but probably a TPP-protein symplex not identical with carboxylase, and therefore, played no part in the dissimilation of glucose or pyruvic acid. SUbXMARY
A parallelism between pyruvate decarboxylation, anaerobic fermentation, and baking activity was obtained in ADY samples exposed from 1 to 8 days at 48” under oxygen. The deterioration of the pyruvate decarboxylation system in the exposed ADY samples was not due to denaturation of carboxylase (apoenzyme) but to destruction of cocarboxylase in yeast cells. The pyruvate decarboxylation activity lost during accelerated exposure of ADY could be restored to the control level by externally supplied thiamine pyrophosphate, Mg++, and ATP. REFERENCES 1. UMBREIT, W. W., BURRIS, R. H., AND STAUFFER, J. F., “Manometric Techniques,” 2nd ed. Burgess Publishing Company, Minneapolis, 1949. pp. 385-9. Siebel Publishing 2. PYLER, E. J., “Baking Science and Technology,” Company, 1952. 3. MYRBXCK, K., VALLIN, I., AND MAGNELL, I., Svensk Kern. Tidskr. 67,124 (1945). 4. KARRER, P., AND VISCONTI, M., Helv. Chim. Acta 29, 711 (1946). 5. WACHTMEISTER, C. A., AND MYRBXCK, K., Arkiv Kemi, Mineral. Geol. 24A, No. 8 (1946). 6. LEIJNSE, B., AND TERPSTRA, W., Biochim. et Biophys. Acta 7, 574 (1951). 7. WESTENBRINK, H. G. K., STEYN-PARVE, E. P., AND VELDMAN, H., Biochim. et Biophys. Acta 1, 154 (1947).