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Reversal by L-cysteine of the inactivation of urease by L-usnic acid
Plant Science Letters, 15 (1979) 165--168 © ]~lsevier]North-HoUand Scienhfic Publishers Ltd.
165
REVERSAL BY L.CYSTEINE OF THE INACTIVATION OF UREASE BY L-USNIC ACID
C. VICENTE and BLANCA CIFUENTES Cdtedra de Fisiologia Vegetal, Facultad de Biologia, Universidad Complutense, Madrid 3 (Spain)
(Received December 22nd, 1977) (Revision received December 12th, 1978) (Accepted December 12th, 1978)
SUMMARY
L-usnic acid inactivates urease by inducing the formation of high molecular weight aggregates. L-cysteine reverses this process not by enzyme protection but by stimulating the appearance of active high molecular weight polymers which are inactive under normal conditions.
INTRODUC~ON
After Marshak and Fager [1] demonstrated that L-usnic acid inactivates DNAase in the presence of cobalt usnic acids have frequently been shown to be powerful enzyme inactivators. While studying the effect of sodium L-usnate on a NADP(H) linked glutamate dehydrogenase from Proteus mirabilis, Vicente et al. [2] noticed a kinetic conversion from the allosteric to michaelian that was related to the formation of molecular aggregates among different enzyme sub-units. In further studies with crystalline urease, Vicente et al. [3] showed that the inactivation of urease by sodium L-usnate was related to the formation of high molecular weight aggregates. They also noticed that the presence of urea in the incubation mixture accelerated the process, while the addition of both L-alanine and L-proline totally prevented enzyme inactivation. Later, Vicente et al. [4] showed that the inactivation process is dependent upon the inactivator concentration and that the degree of inactivation was determined by the molecular size of the aggregates formed. They also showed that urea alone causes aggregation and these aggregates had a molecular weight higher than those obtained by treating with L-usnic acid. The polymers formed by the action of urea were not necessary inactive. The results presented in this paper report the effect of L-cysteine on the inactivation process.
166 MATERIALS AND METHODS
Solutions of crystalline urease (type III, Sigma Chemical Company) preincubated with L-cysteine and L-usnic acid were prepared as previously described [ 3 ]. Specific activities were determined using Conway's microdiffusion method [ 5]. Protein was measured using the method of Warburg and Christian [6]. Molecular weights of the different aggregates were determined by passing the sample through a Sepharose 6B column, (21 cm × 3 cm) equilibrated with 75 mM phosphate buffer (pH 6.9) using as markers tyroglobulin, glutamJne synthetase, glutamate dehydrogenase, a-urease, catalase or myosin. Molecular weights were ~!~o determined by electrophoresis in 7.5% acrylamide gels using Fishbein's method [7] and staining with amido-black 10-B. RESULTS
The results presented in Fig. I show that urease, at a concentration of 0.1 mg in a final volume of 3 ml is inactivated in the presence of L-usnic acid. When 50 pmol of L-cysteine are present in the incubation mixture, 12.25% of the activity was recovered. Separation of different aggregates was achieved by Sepharose 6B filtration. The elution profile of an active urease is shown in Fig. 2. The enzyme is
3.0 20J
25
100
29
80
/-.
01
E 1.5
N
60
c o
~oo
¢D
3m t-
o ~
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o 1,0
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05
20
Q.
01
&
I
233
I
I
I
I
/66 699 932 11.65 (L-usnic acidl mM
I
20
J
60
I00 1,40 filtrate (ml)
Fig. 1. (left) Effect o f L-usnic acid on urease activity. Urease (0.1 mE) and L-usnic acid were pre-incubated for 5 min at 37°C in 3 ml of 75 mM phosphate buffer (pH 6.9) (e-----e). 50 pmol/ml of L~ysteine added at zero time (o-----o). Each point is the average of 4 experiments. Vertical bars give S.E.M. Fig. 2. (right) Diagram of filtration of a sample of urease (10 mg protein/10 ml of buffer) on Sepharose 6B. (e----~)protein; (o----o) enzyme activity.
167
f O1
E
1.0
B
A
O
.
N
/
20
r-
e-
o Q,.
!
,
I
e
!
..-i
30 60 g0 120150 30 60 90 120 IN) filtrate (ml)
Fig. 3. Diagram of filtration on Sepharose 6B of ureas ~ samples incubated for 5 min at 37°C with (A) 35 pmoi/ml L-usnic acid or (B) 35 pmol]ml L-usnic acid and 50 pmol/m] L-cysteine added at zero time. ( e - - e ) protein; (o . . . . . o) enzyme activity.
eluted in a main peak at 130 ml which retains the maximum enzyme activity. This peak corresponds to a - u r e a s e with a molecular weight of 480 000 [8]. The elution and separation of urease treated with 35 mM L-usnic acid or urease treated with both L-usnic acid (35 mM) and L-cysteine (50 mM) are reported in Fig. 3. The enzyme incubated with L-usnic acid shows two peaks of inactive protein, eluted at 20 ml and 40 ml. The main peak of active enzyme is eluted at 130 ml filtration (Fig. 3A).
TABLE I AC'FIVE POLYMERS OF UREASE FORMED BY ACTION OF BOTH L4~YSTEINE AND L-USNIC ACID 50 mM L-cysteine and 11.65 mM L-usnic acid in the incubation mixture. Fraction from Sepharose 6B (ml) 10 0.14 0.24 Electrophoretie mobility 880 000 820 000 Mol. wt estimated from electrophoretic mobility 55 51 Number of monomers 0.40 Enzyme units Percentage of remaining activity 3.54 Observed 3.44 Expected 0.58 Protein (mg) Percentage of total protein 5.98 Observed 5.8 Expected
30
80
100
0.28 780 000
0.44 670 000
0.51 630 000
49 1.13
42 0.60
39 3.00
10.02 9.73 0.48
5.32 5.16 0.50
26.~1 25.83 0.46
5.04 4.8
5.25 5.0
4.83 4.6
168
Remarkable changes in this behaviour appear when L.cysteine is included in the incubation mixture (Fig. 3B). The most characteristic peaks of protein have been used for determination of the molecular weight by acrylamide gel electrophoresis. The different aggregates formed retain a certain proportion of enzyme activity. Any inactive aggregates do not appear in this case. The polymeric form retaining the highest enzyme activity is restricted to a molecular weight of 630 000 without any correspondence with the a-urease form previously described (Fig. 2). A summary of these results is shown in Table L DISCUSSION
Two active polymers of urease are formed by the action of L-usnic acid in the presence of L-cysteine. The molecular weights of these polymers, determined by acrylamide gel electzophoresis, are 880 000 and 820 000 respectively. Table I shows an analysis of these polymers as structures formed by 55 and 51 monomers respectively. These numbers have been calculated according to Sekita and Mamiya [9] assuming a monomer molecular weight of 16 000 instead of 83 300 as defined by Reithel et al. [10]. Several degrees of polymerization have been determined. Aggregates formed with 49,42 and 39 monomers retain a certain amount of enzyme activity, the most active being a polymer composed of 39 monomers and with a molecular weight of 630 000. Therefore, L-cysteine seems to reverse the inactivation of higher molecular weight forms of urease. Inactivation of urease by L.usnic acid has been described as a process independent of the --SH groups of the enzyme [ 3]. However since active aggregates of molecular weight higher than 800 000 have now been found the hypothesis that the aggregation state is the only cause of enzyme inactivation appears to be untenable. ACKNOWLEDGEMENT
We are indebted to Prof. F. Bustinza for a generous supply of L-usnic acid. REFERENCES 1 2 3 4
A. Marshak and J. Fager, J. Cell. Comp. Physiol., 35 (1950) 317. C. Vicente, H. Guerra and M.T. Valle, Rev. Esp. Fisiol., 29 (1973) 293. C. Vicente, H. Guerra and M.T. Valle, Rev. Esp. Fisiol., 30 (1974) 1. C. Vicente, A. Azpiroz, M.P. Est~vez and M.L. Gonzalez, Plant Cell Environment, 1 (1978) 29. 5 E.J. Conway, Microdiffusion Analysis and Volumetric Error, Crosby Lockwood, London, 1957. 6 0 . Warburg and W. Christian, Biochem. Z., 310 (1941) 384. 7 W.N. Fishbein, Anal. Biochem., 46 (1972) 388. 8 W.N. Fishbein, Ann. N.Y. Acad. Sc., 147 (1969) 857. 9 K. Sekita and G. Mamiya, Proc. 7th Intern. Congr. Biochem., Abstracts, IV (1967) 761. •10 F.J. Reithel, J.E. Robbins and G. Gorin, Arch. Biochem .... ~phys., 108 (1964) 409.
165
REVERSAL BY L.CYSTEINE OF THE INACTIVATION OF UREASE BY L-USNIC ACID
C. VICENTE and BLANCA CIFUENTES Cdtedra de Fisiologia Vegetal, Facultad de Biologia, Universidad Complutense, Madrid 3 (Spain)
(Received December 22nd, 1977) (Revision received December 12th, 1978) (Accepted December 12th, 1978)
SUMMARY
L-usnic acid inactivates urease by inducing the formation of high molecular weight aggregates. L-cysteine reverses this process not by enzyme protection but by stimulating the appearance of active high molecular weight polymers which are inactive under normal conditions.
INTRODUC~ON
After Marshak and Fager [1] demonstrated that L-usnic acid inactivates DNAase in the presence of cobalt usnic acids have frequently been shown to be powerful enzyme inactivators. While studying the effect of sodium L-usnate on a NADP(H) linked glutamate dehydrogenase from Proteus mirabilis, Vicente et al. [2] noticed a kinetic conversion from the allosteric to michaelian that was related to the formation of molecular aggregates among different enzyme sub-units. In further studies with crystalline urease, Vicente et al. [3] showed that the inactivation of urease by sodium L-usnate was related to the formation of high molecular weight aggregates. They also noticed that the presence of urea in the incubation mixture accelerated the process, while the addition of both L-alanine and L-proline totally prevented enzyme inactivation. Later, Vicente et al. [4] showed that the inactivation process is dependent upon the inactivator concentration and that the degree of inactivation was determined by the molecular size of the aggregates formed. They also showed that urea alone causes aggregation and these aggregates had a molecular weight higher than those obtained by treating with L-usnic acid. The polymers formed by the action of urea were not necessary inactive. The results presented in this paper report the effect of L-cysteine on the inactivation process.
166 MATERIALS AND METHODS
Solutions of crystalline urease (type III, Sigma Chemical Company) preincubated with L-cysteine and L-usnic acid were prepared as previously described [ 3 ]. Specific activities were determined using Conway's microdiffusion method [ 5]. Protein was measured using the method of Warburg and Christian [6]. Molecular weights of the different aggregates were determined by passing the sample through a Sepharose 6B column, (21 cm × 3 cm) equilibrated with 75 mM phosphate buffer (pH 6.9) using as markers tyroglobulin, glutamJne synthetase, glutamate dehydrogenase, a-urease, catalase or myosin. Molecular weights were ~!~o determined by electrophoresis in 7.5% acrylamide gels using Fishbein's method [7] and staining with amido-black 10-B. RESULTS
The results presented in Fig. I show that urease, at a concentration of 0.1 mg in a final volume of 3 ml is inactivated in the presence of L-usnic acid. When 50 pmol of L-cysteine are present in the incubation mixture, 12.25% of the activity was recovered. Separation of different aggregates was achieved by Sepharose 6B filtration. The elution profile of an active urease is shown in Fig. 2. The enzyme is
3.0 20J
25
100
29
80
/-.
01
E 1.5
N
60
c o
~oo
¢D
3m t-
o ~
P~
o 1,0
`40
05
20
Q.
01
&
I
233
I
I
I
I
/66 699 932 11.65 (L-usnic acidl mM
I
20
J
60
I00 1,40 filtrate (ml)
Fig. 1. (left) Effect o f L-usnic acid on urease activity. Urease (0.1 mE) and L-usnic acid were pre-incubated for 5 min at 37°C in 3 ml of 75 mM phosphate buffer (pH 6.9) (e-----e). 50 pmol/ml of L~ysteine added at zero time (o-----o). Each point is the average of 4 experiments. Vertical bars give S.E.M. Fig. 2. (right) Diagram of filtration of a sample of urease (10 mg protein/10 ml of buffer) on Sepharose 6B. (e----~)protein; (o----o) enzyme activity.
167
f O1
E
1.0
B
A
O
.
N
/
20
r-
e-
o Q,.
!
,
I
e
!
..-i
30 60 g0 120150 30 60 90 120 IN) filtrate (ml)
Fig. 3. Diagram of filtration on Sepharose 6B of ureas ~ samples incubated for 5 min at 37°C with (A) 35 pmoi/ml L-usnic acid or (B) 35 pmol]ml L-usnic acid and 50 pmol/m] L-cysteine added at zero time. ( e - - e ) protein; (o . . . . . o) enzyme activity.
eluted in a main peak at 130 ml which retains the maximum enzyme activity. This peak corresponds to a - u r e a s e with a molecular weight of 480 000 [8]. The elution and separation of urease treated with 35 mM L-usnic acid or urease treated with both L-usnic acid (35 mM) and L-cysteine (50 mM) are reported in Fig. 3. The enzyme incubated with L-usnic acid shows two peaks of inactive protein, eluted at 20 ml and 40 ml. The main peak of active enzyme is eluted at 130 ml filtration (Fig. 3A).
TABLE I AC'FIVE POLYMERS OF UREASE FORMED BY ACTION OF BOTH L4~YSTEINE AND L-USNIC ACID 50 mM L-cysteine and 11.65 mM L-usnic acid in the incubation mixture. Fraction from Sepharose 6B (ml) 10 0.14 0.24 Electrophoretie mobility 880 000 820 000 Mol. wt estimated from electrophoretic mobility 55 51 Number of monomers 0.40 Enzyme units Percentage of remaining activity 3.54 Observed 3.44 Expected 0.58 Protein (mg) Percentage of total protein 5.98 Observed 5.8 Expected
30
80
100
0.28 780 000
0.44 670 000
0.51 630 000
49 1.13
42 0.60
39 3.00
10.02 9.73 0.48
5.32 5.16 0.50
26.~1 25.83 0.46
5.04 4.8
5.25 5.0
4.83 4.6
168
Remarkable changes in this behaviour appear when L.cysteine is included in the incubation mixture (Fig. 3B). The most characteristic peaks of protein have been used for determination of the molecular weight by acrylamide gel electrophoresis. The different aggregates formed retain a certain proportion of enzyme activity. Any inactive aggregates do not appear in this case. The polymeric form retaining the highest enzyme activity is restricted to a molecular weight of 630 000 without any correspondence with the a-urease form previously described (Fig. 2). A summary of these results is shown in Table L DISCUSSION
Two active polymers of urease are formed by the action of L-usnic acid in the presence of L-cysteine. The molecular weights of these polymers, determined by acrylamide gel electzophoresis, are 880 000 and 820 000 respectively. Table I shows an analysis of these polymers as structures formed by 55 and 51 monomers respectively. These numbers have been calculated according to Sekita and Mamiya [9] assuming a monomer molecular weight of 16 000 instead of 83 300 as defined by Reithel et al. [10]. Several degrees of polymerization have been determined. Aggregates formed with 49,42 and 39 monomers retain a certain amount of enzyme activity, the most active being a polymer composed of 39 monomers and with a molecular weight of 630 000. Therefore, L-cysteine seems to reverse the inactivation of higher molecular weight forms of urease. Inactivation of urease by L.usnic acid has been described as a process independent of the --SH groups of the enzyme [ 3]. However since active aggregates of molecular weight higher than 800 000 have now been found the hypothesis that the aggregation state is the only cause of enzyme inactivation appears to be untenable. ACKNOWLEDGEMENT
We are indebted to Prof. F. Bustinza for a generous supply of L-usnic acid. REFERENCES 1 2 3 4
A. Marshak and J. Fager, J. Cell. Comp. Physiol., 35 (1950) 317. C. Vicente, H. Guerra and M.T. Valle, Rev. Esp. Fisiol., 29 (1973) 293. C. Vicente, H. Guerra and M.T. Valle, Rev. Esp. Fisiol., 30 (1974) 1. C. Vicente, A. Azpiroz, M.P. Est~vez and M.L. Gonzalez, Plant Cell Environment, 1 (1978) 29. 5 E.J. Conway, Microdiffusion Analysis and Volumetric Error, Crosby Lockwood, London, 1957. 6 0 . Warburg and W. Christian, Biochem. Z., 310 (1941) 384. 7 W.N. Fishbein, Anal. Biochem., 46 (1972) 388. 8 W.N. Fishbein, Ann. N.Y. Acad. Sc., 147 (1969) 857. 9 K. Sekita and G. Mamiya, Proc. 7th Intern. Congr. Biochem., Abstracts, IV (1967) 761. •10 F.J. Reithel, J.E. Robbins and G. Gorin, Arch. Biochem .... ~phys., 108 (1964) 409.