Planta
Planta 141, 145-153 (1978)
9 by Springer-Verlag 1978
Proteinase Activity in Potato Plants K. Santarius and H.-D. Belitz Institut fiir Lebensmittelchemie, Technische Universitfit Mfinchen, D-8046 Garching, Federal Republic of Germany
Abstract. Several vegetative tissues of potato plants were screened for proteinase activity. Both endopeptidase and exopeptidase activities were investigated using gelatin and L-amino acid-4-nitroanilides (benzoylL-arginine-4-nitroanilide/BAPA, glutaryl-L-phenylalanine-4-nitroanilide/GLUPHEPA, alanine-4-nitroanilide/APA, leucine-4-nitroanilide/LPA, and benzoylL-tyrosine-4-nitroanilide/BTPA) as substrates. Leaves and rootes were found to contain the highest levels of endopeptidase activity; lesser activities were detected in flower petals, sprouts, and tubers. Three different types of proteinases, L-BAPAase (serine proteinase), APAase (thiol proteinase), and BTPAase (sensitive to reducing agents), were characterized in various physical and chemical properties. Their temperature optima were determined to be 25 ~ (L-BAPAase) and 40 ~ (BTPAase, APAase) respectively; their p H o p t i m u m was between 8.6 and 9.0, their isoelectric points were between p H 4.25 and 6.0, and their molecular weight was estimated 70,000 (L-BAPAase, APAase) and between 150,000-250,000 (BTPAase). The trypsin-like activity against L-BAPA was inhibited by diisopropylfluorophosphate and by tosyllysine-chloromethyl ketone, but not by trypsin inhibitors from potato and legume.
of proteinases (i.e., serine, metal, and sulfhydryl) have been identified and in most instances several different inhibitor or isoinhibitor forms have been found. Most of the inhibitors have specificities against the serine proteinases of animals and microorganisms and are thought to be agents to help protect the tissues from pest attack. Although proteolytic enzymes have been identified in both leaves and tubers of potato plants (Tracey, 1948; Niemann, 1956; Hojima et al., 1971; N o w a k and Skwiercz, 1975), no systematic efforts have been made to determine whether the inhibitors present in tubers could inhibit endogenous enzymes of the plants. In this report we have examined proteinase activities present in various vegetative tissues of potato plants. Tissues with the highest levels of proteolytic activity were leaves and roots. Proteolytic activities with various substrates were identified and the influence of purified potato and legume proteinase inhibitors on their activities was determined.
Key words: Proteinase S o & n u m .
Chemicals
Introduction Potato tubers are one of the richest sources of proteinase inhibitors known. Inhibitors of all three classes Abbreviations: APA=alanine-4-nitroanilide; BAPA=benzoyl-L-
arginine-4-nitroanilide; BTPA = benzoyl-L-tyrosine-4-nitroanilide; DFP=diisopropylfluorophosphate; DMF=dimethyl formamide; EDTA=ethylenedinitrilotetraacetic acid; GLUPHEPA=glutarylL-phenylalanine-4-nitroanilide ; LPA = Ieucine-4-nitroanilide; PHMB=p-hydroxy-mercuribenzoate; Pl-I=potato chymotrypsin inhibitor I; PPl=potato proteinase leaf; PPr=potato proteinase root; PPt=potato proteinase tuber; PVP=polyvinylpyrrolidone; TLCK tosyl-L-lysinechloromethyl ketone; TPCK tosyl-Lphenylalanyl chloromethane
Materials and Methods
Polyvinylpyrrolidone (PVP), mol.wt. 40,000, and myoglobin, sperm whale, cryst, salt-free from Mann Research Lab. ; chloramphenicol, ponceau S, imidazole (grade I), bovine ,/-globulin (fraction lI), p-hydroxymercuribenzoate(PHMB), dimethyl formamide (DMF), glutaryl-L-phenylalanine-4-nitroanilide(GLUPHEPA), Lleucine-4-nitroanilide (LPA), N-benzoyl-L-tryrosine-4-nitroanilide (BTPA), N~-p-tosyl-L-Iysine-chloromethylketone (TLCK) from Sigma Chemical Co. ; lima bean trypsin inhibitor from Worthington Biochem. Corp.; benzoyl-L-arginine-4-nitroanilide(L-BAPA), L-alanine-4-nitroanilide (APA), sodium cyanide, cysteine, 2-iodacetamide, 2,2-bipyridin, 2-mercaptoethanol, 1.10-phenantroliniumchloride, acrylamide, and iodacetic acid from Merck and Co.; light green SF, ovalbumin, soybean trypsin inhibitor and bovine serum albumin from Serva; 5-sulfosalicylicacid from Pierce; chymotrypsinogen A, aldolase, and catalase from Boehringer; Sephacryi S 200 sf from Pharmacia; ampholine carrier arnpholytes from LKB-Producter; pepstatin from Banyu Pharmaceutical Co. Ud./ Tokyo; N-tosyl-L-phenylalanylchloromethane (TPCK) from Cal-
O032-0935/78/0141/0145/$01.80
146 biochem; diisopropylfluorophosphate (DFP) from Serva; ethylenedinitrilotetraacetic acid disodium salt (EDTA) from Mallinckrodt; dialysis membranes from Union Carbide Corp. Potato material was obtained from tuberizing potato plants (variety Russet Burbank) on fields and in the greenhouse.
Extraction Methods' 1. Tubers: The tubers employed in the study were either freshly harvested in late summer or were stored one year and allowed to sprout four to five weeks in pots in a greenhouse. All procedures were carried out at 4 ~ C. Three hundred grams of washed tubers were cut into small pieces and blended with 100 ml 1-M Tris/HC1 buffer, pH 7.2, 4• 1 0 - 3 M EDTA and 0.25 x 10 3 M PVP, mol. wt. 40,000. The juice was extruded through cheesecloth and clarified by centrifugation at 12,000 g for 30 rain. Solid ammonium sulfate was added to give an 80% saturated solution. After standing 30 min, the precipitate was collected by centrifugation, the supernatant discarded, and the precipitate was dissolved in about 30 ml 0.1-M Tris/HC1 buffer. Insoluble debris were removed by centrifugation at 15,000 g 15 min and the clear supernatant was dialyzed against 0.05 M NHcHCO~ for 8 h and against distilled water for 3 h and lyophilized. Yields of lyophilized material were 405 mg from fresh tubers (PPtl) and 460 mg from year-old, sprouted tubers (PPt2). 2. Roots: Thin roots (not the thick underground sprouts!) from potato plants after four to five weeks growing in pots in a greenhouse were used. From one hundred grams of washed and air-dried roots, extract was prepared by macerating the tissue with a ceramic mortar and pestle and expressing the juice by squeezing the tissue between the mortar and pestle. To about 50 ml juice (pH 6.2) was added an equal volume of 1 M Tris/HC1 buffer, pH 7.2, 4 x 10 -a M EDTA. After centrifugation for 15 rain at 15,000g the clear supernatant was adjusted to p H 7.0 and ammonium sulfate was added to give 80% saturation. The precipiatate obtained after centrifugation at 15,000 g was dissolved in about 20 ml of the above buffer diluted 1 to I0 and dialyzed 2 • h against NH4HCO3 and 3 h against distilled water. All operations were carried out at 4 ~ C. The clear solution was lyophilized and 105 mg crude potato proteinase PPr were obtained.
3. Leaves: This preparation was obtained as described previously (Santarius and Ryan, 1977). The main proteinase activity was extracted after the first extraction with 20 m M EDTA solution (crude potato proteinase PPll). With a second extraction of the cell debris with 1 M KC1 solution, further endogenous proteinase activity was recovered (crude potato proteinase PP12). From 200 g potato leaves, 520mg PPll and l l 2 m g PP12, respectively, were obtained.
K. Santarius and H.-D. Belitz: Proteinase Activity in Potato Plants then stained for 30 rain with a solution of equal parts of 0.2% light green SF in 0.5% sulfosalicylic acid, 0.2% ponceau red in 5 % trichloroacetic acid, and 0.1% amido black 10 B in 5% trichloroacetic acid (this mixture was prepared four days before using). Destaining was performed in both cases in 7.5% acetic acid with an ,,Ames" Quick-destainer. Enzyme activity against L-amino acid-4-nitroanilides was demonstrated by incubation of the electrophoresis gels for (a) 15 min in 50 m M immidazole/HC1 buffer, pH 7.6, at 4 ~ C, (b) 15 min in same buffer but containing 2.5 m M L-amino acid-4-nitroanilide at 4 ~ C and (c) 15 min or longer (depending on enzyme activity present) in same solution at 37~ (Hennrich et al., 1973). Due to its low solubility in aqueous solution, BTPA was dissolved in a mixture of D M F and 50 m M imidazole/HC1 buffer 1 to 2.
Measurement o f Enzyme and Inhibitor Activities To 0.05-0.5 ml enzyme solution was added 0.2 M Tris/HC1 buffer, pH 8.6, at BTPAase and L-BAPAase (pH 8.8 at APAase and LPAase), 4 x 1 0 - 4 M EDTA, to a final volume of 1.4ml. After 5 min preincubation at 35~ the reaction was initiated by the addition of 0.1 ml 3 m M L-amino acid-4-nitroanilide in DMF. The reaction was normally terminated after 2 h with 0.5 ml of 30% acetic acid. The yellow color that resulted from the liberation of p-nitroanilin was measured in 1-cm cuvettes in a Beckman DB-G spectrophotometer at 405 nm. A unit of activity is defined as an absorbance change of 0.01 per rain at 405 nm under the conditions described. The inhibition stock solutions of TLCK, TPCK, and 2,2'bipyridin were dissolved in methanol; the proteinase inhibitors of potato and beans were in 10-3 N HCI. All other inhibitors were dissolved and added in 0.1 M Tris/HC1 buffer, pH 7.4. The endopeptidase activity against gelatin was measured by radial diffusion assay as described previously (Santarius and Ryan, 1977). The measurement for content of potato chymotrypsin inhibitor I = P I - I (Melville and Ryan, 1972) was carried out by immunological test (Ryan, 1967).
Isoelectric Focusing Isoelectric focusing was carried out according to Haglund (1967) using the small column (8100-10, gradient volume 110 ml) of LKBProducter AB. Concentration of the ampholines in the density gradient was 1%. A voltage of 1600 V was employed at a temperature of 4 ~ C for 16-18 h. The column was emptied by pumping with a flow rate of 100 ml/h. One-ml fractions were collected and the absorbance at 280 nm (protein) and the pH were continuously measured. The hydrolyzing L-amino acid-4-nitroanilide activity of the fractions was determined as described above.
Estimation of Molecular Weight Disc Electrophoresis Polyacrylamide gel disc electrophoresis was carried out at pH 9.5 in system 1 according to Maurer (1971). Running time was 3 h at 150V (24mA at 12 tubes). The protein patterns were stained with a solution of 0.05% amido black 10 B in 7.5% acetic acid. To render the endopeptidase activity visible, parallel runs were carried out using polyacrylamide gel containing 0.1% gelatin (Stegemann, 1968) followed by incubation of the gel columns for 5 h at 37 ~ C in 0,05 M phosphate buffer, pH 7.4. The gels were
A Sephacryl S 200 sf column ( 2 x 8 5 cm) was equilibrated and eluted with 0.1 M Tris/HCl buffer, pH 8.0, 4 x 10 4 M EDTA, at 10~ C. The absorbance of the fiactions was measured at 280 nm (protein) and at 405 nm after reaction with a solution of L-amino acid-4-nitroanilides. Standard proteins (0.5-7 mg in 0.5 ml buffer) were myoglobin (mol.wt. 17,800), chymotrypsinogen A (25,000), aldolase (140,000-150,000), bovine 7-globuline (150,000-171,000), and catalase (225,000-250,000). The void volume (V0) was determined by dextran blue.
147
K. Santarius and H.-D. Belitz: Proteinase Activity in Potato Plants Table 1. Endopeptidase activity and inhibitor PI-I in different tissues at different growth stages of the potato plant Tissue a
Growth stage
Inhibitor PI-I (gg/ml)
Petals
About 21/2 months after sprouting in greenhouse
102
5
49
53
21
396
172
33
192
Old complete yellow leaves Green leaves
Four to five weeks after sprouting in greenhouse
Endopept. activity in trypsin equival. (~g/ml) x 10- 2
Ratio endopept./inhibitor (x 10 5)
Upper green sprouts
61
3
49
Lower green sprouts
53
3
57
121
1
8
White sprouts (already with green leaves)
About 3 weeks after field planting
White sprouts (without green leaves) White underground sprouts
102 Four to five weeks after sprouting in greenhouse
0.4
4
190
17
89
69
40
579
New tuber (less 1 cm in ,~)
510
30
59
New tuber (1 to 3 cm in ~ )
478
2
4
388
2
5
186
I
5
+
1
(1000)
1
23
Roots
Tuber apical end
Stored for one year
Tuber stem end Tuber apical end
Four to five weeks after sprouting in greenhouse
Tuber stem end
44
Plant extracts were made according to Santarius and Ryan (1977) and diluted 1:1 with 1 M Tris/HCl buffer, pH 7.4, containing 2x 10 -3 M chloramphenicol and 0.5x I0 -3 M PVP
Results
Distribution of Proteinase Activity in the Potato Plant Table 1 shows the distribution of proteinase activity a g a i n s t g e l a t i n in c o m p a r i s o n to the c o n t e n t o f t h e potato chymotrypsin inhibitor PI-I (Melville and R y a n , 1972). O v e r a l l the c o n t e n t o f p r o t e i n a s e a c t i v i t y is c o m p a r a t i v e l y low. R e l a t i v e l y h i g h p r o t e i n a s e a c t i v ity a n d l o w c o n t e n t o f i n h i b i t o r was f o u n d in t h e roots of the potato plant. In contrast, leaves exhibited a high proteinase activity with a stronger inhibitor a c t i v i t y . A v e r y l o w level o f p r o t e i n a s e a c t i v i t y was d e t e c t e d in t u b e r s . T h e h i g h a m o u n t o f i n h i b i t o r a c t i v ity in t u b e r s d e c l i n e s r a p i d l y d u r i n g the first w e e k s of sprouting. Crude proteinase preparations of potato tubers, r o o t s (PPr), a n d l e a v e s w e r e p r e p a r e d a n d t e s t e d f o r proteinase activity. All crude proteinase preparations s h o w e d a c t i v i t y a g a i n s t g e l a t i n m e a s u r e d by r a d i a l d i f f u s i o n assay. A c t i v i t y a g a i n s t several L - a m i n o acid4 - n i t r o a n i l i d e s is i n d i c a t e d in T a b l e 2. T h e c r u d e preparation of PPtl was made from tubers harvested
Table 2. Survey of enzyme activities in the crude proteinase preparations of potato tuber, root, and leaf against several L-amino acid-4-nitroanilides. Each 150 gg of the samples, dissolved in 0.15ml 0.1 M Tris/HC1 buffer, pH7.4, containing 4 x l 0 - ~ M EDTA, was assayed as described in Materials and Methods for 2 h at 35~ Crude proteinase Activity (units/mg crude proteinase) against L-amino acid-4-nitroanilides BAPA Potato Potato Potato Potato Potato
tuber PPtl _+ tuber PPt2 _+ root PPr 0.87 leaf PPI1 leaf PPlz .
.
GLUPHEPA
APA
LPA
BTPA
-
2.55 0.69 4.33 0.96 .
0.65 0.18 1.16 0.31
1.08 2.01 -
.
.
Note: = No detectable activity; + = Weak activity, only detectable at the attempts of isoelectric focusing
in late s u m m e r , a n d PPt2 f r o m o n e - y e a r s t o r e d t u b e r s a f t e r f o u r to five w e e k s s p r o u t i n g in p o t s in a g r e e n h o u s e . P P l l o f the p o t a t o l e a v e s was the E D T A ext r a c t ; PP12, the KC1 e x t r a c t , f o l l o w e d . T h e l e a f e x t r a c t PP12 d i d n o t s h o w a n y a c t i v i t y a g a i n s t t h e s e s y n t h e t i c
148
K. Santarius and H.-D. Belitz: Proteinase Activity in Potato Plants
PP I
! a
b
PP%
PPr
PPi 0.2
BTPA
APA/LPA
APA/LPA
d
tZ...'J~
,[•BTPA
9 ",~
ul
/
,~
01 4PAILPA ~ BAPA
~ 4PAILPA
[~
1 c
a
b c
a
b
c
I a
Z, b
c !+)
Fig. 1. Polyacrylamide gel electrophoresis of crude proteinase preparations of potato tuber (PPt~, PPt2), root (PPr), and leaf (PP10. The attempt was carried out as described in the method part. 300 gg (1000 gg PPr at L-BAPAase) of the samples were dissolved in 25 gl of spacer gel buffer. The gels show: (a) protein patterns after staining with amido black, (b) endopeptidase activity againts gelatin as substrate (clearzones),and (c) proteinase activity against several L-amino acid-4-nitroanilides as substrate
x.
X //~ ' ~ " x
~//~e"".e... ~
2'0
Jo
io
6
7
INCUBATION
8
9
1o
pH
Fig. 3. PH optima of BTPAase (-o--o-), APAase @a a-), LPAase ( - + - + =), and L-BAPAase (. ~ - 9 .). 0.15-0.8 mg crude proteinase PPr-according to the activity of the tested enzyme-dissolved in 0.15 ml 0.1 M Tris/ HC1 buffer, pH 7.4, containing 4 x 10 4 M EDTA, were assayed as described in Materials and Methods with buffer solutions of different pH (0.1 M Nacitrate/HC1, pH 2.0-3.5; 0.2 M acetate/NaOH, pH 4.0 5.5; 0.1 M phosphate/S6rensen, pH6.0 7.0; 0.2M Tris/HC1, pH7.2-9.0; 0.2 M borate buffer, pH9.2-10.0). The incubation time was 120 min at 35~ C
Polyacrylamide Gel Electrophoresis of the Crude Proteinase Preparations of Potato Tuber, Root and Leaf
/~',~
..o' ".../,'"
5
s'o
INCUBAtiON TEMPERA TURE (C)
Fig. 2. Temperature optima of BTPAase @o--o-), APAase ( A - - A - ) , and L-BAPAase (- e- 9 9 .). 0.15 0.5 mg crude preparation PPt2 (BTPAase), PPtl (APAase), and PPr (L-BAPAase) respectively-according to the activity of the enzymes-dissolved in 0.15ml 0.1 M Tris/HC1 buffer, pH7.40 containing 4x10 4M EDTA, were assayed as described in Materials and Methods at different temperatures. The incubation time was 120 min (APAase, 60 min)
substrates. A c t i v i t y a g a i n s t L - B A P A was f o u n d m a i n l y in the r o o t s o f the p o t a t o p l a n t ; o n l y a very low c o n c e n t r a t i o n has been d e t e c t e d in b o t h t u b e r p r e p a r a t i o n s . It is r e m a r k a b l e t h a t e n z y m e activity h y d r o l y z i n g B T P A in m e a s u r a b l e a m o u n t s in tissues o t h e r t h a n the r o o t s occurs only in t u b e r s after s p r o u t i n g in the g r e e n h o u s e . P r o t e i n a s e activity a g a i n s t A P A a n d L P A was f o u n d in leaves, tubers, a n d r o o t s o f the p o t a t o plant. F u r t h e r e x p e r i m e n t s were c a r r i e d o u t o n l y with the p r o t e i n a s e activities a g a i n s t the s u b s t r a t e s B T P A , A P A , L P A , a n d L - B A P A . The enzymes were n a m e d B T P A a s e , A P A a s e ( L P A a s e ) , a n d L - B A P A a s e , respectively.
The m i g r a t i o n o f the b a n d s o f A P A a s e a n d L P A a s e (Fig. 1) indicates t h a t these enzymes are p r e s e n t in all f o u r c r u d e p r o t e i n a s e p r e p a r a t i o n s . T h e wide activity r a n g e o b t a i n e d by e m p l o y i n g gelatin a n d benzoylL - t y r o s i n e - 4 - n i t r o a n i l i d e as s u b s t r a t e s for the crude p r e p a r a t i o n s o f r o o t a n d leaf is striking. A l s o , m u c h lower p r o t e i n a s e c o n c e n t r a t i o n s d i d n o t l e a d to smaller activity b a n d s . O n the s t r e n g t h o f low activity o f L - B A P A a s e in b o t h t u b e r p r e p a r a t i o n s , their activity b a n d s were here n o t detectable.
Temperature and pH Optimum of BTPAase, APAase, and L-BAPAase T h e t e m p e r a t u r e o p t i m a for the three e n z y m e activities were d e t e r m i n e d to be 2 5 ~ for L - B A P A a s e a n d a b o u t 40 ~ C for the o t h e r two e n z y m e activities (Fig. 2). T h e p H o p t i m u m for all activities was in the a l k a l i n e region. B T P A a s e a n d A P A a s e ( L P A a s e ) e x h i b i t e d an o p t i m u m at p H 8.6 a n d p H 9.0, respectively, a n d L - B A P A a s e e x h i b i t e d an o p t i m a l p H r a n g e o f 8.6 to 9.0 (Fig. 3).
Isoelectric Focusing of the Crude Proteinases To c o m p a r e the e n z y m e activities in the different vegetative p a r t s o f the p o t a t o plant, isoelectric focusing
K. Santarius and H.-D. Belitz: Proteinase Activity in Potato Plants
149
OG
2,0, t8
o3 ,,
Planta 141, 145-153 (1978)
9 by Springer-Verlag 1978
Proteinase Activity in Potato Plants K. Santarius and H.-D. Belitz Institut fiir Lebensmittelchemie, Technische Universitfit Mfinchen, D-8046 Garching, Federal Republic of Germany
Abstract. Several vegetative tissues of potato plants were screened for proteinase activity. Both endopeptidase and exopeptidase activities were investigated using gelatin and L-amino acid-4-nitroanilides (benzoylL-arginine-4-nitroanilide/BAPA, glutaryl-L-phenylalanine-4-nitroanilide/GLUPHEPA, alanine-4-nitroanilide/APA, leucine-4-nitroanilide/LPA, and benzoylL-tyrosine-4-nitroanilide/BTPA) as substrates. Leaves and rootes were found to contain the highest levels of endopeptidase activity; lesser activities were detected in flower petals, sprouts, and tubers. Three different types of proteinases, L-BAPAase (serine proteinase), APAase (thiol proteinase), and BTPAase (sensitive to reducing agents), were characterized in various physical and chemical properties. Their temperature optima were determined to be 25 ~ (L-BAPAase) and 40 ~ (BTPAase, APAase) respectively; their p H o p t i m u m was between 8.6 and 9.0, their isoelectric points were between p H 4.25 and 6.0, and their molecular weight was estimated 70,000 (L-BAPAase, APAase) and between 150,000-250,000 (BTPAase). The trypsin-like activity against L-BAPA was inhibited by diisopropylfluorophosphate and by tosyllysine-chloromethyl ketone, but not by trypsin inhibitors from potato and legume.
of proteinases (i.e., serine, metal, and sulfhydryl) have been identified and in most instances several different inhibitor or isoinhibitor forms have been found. Most of the inhibitors have specificities against the serine proteinases of animals and microorganisms and are thought to be agents to help protect the tissues from pest attack. Although proteolytic enzymes have been identified in both leaves and tubers of potato plants (Tracey, 1948; Niemann, 1956; Hojima et al., 1971; N o w a k and Skwiercz, 1975), no systematic efforts have been made to determine whether the inhibitors present in tubers could inhibit endogenous enzymes of the plants. In this report we have examined proteinase activities present in various vegetative tissues of potato plants. Tissues with the highest levels of proteolytic activity were leaves and roots. Proteolytic activities with various substrates were identified and the influence of purified potato and legume proteinase inhibitors on their activities was determined.
Key words: Proteinase S o & n u m .
Chemicals
Introduction Potato tubers are one of the richest sources of proteinase inhibitors known. Inhibitors of all three classes Abbreviations: APA=alanine-4-nitroanilide; BAPA=benzoyl-L-
arginine-4-nitroanilide; BTPA = benzoyl-L-tyrosine-4-nitroanilide; DFP=diisopropylfluorophosphate; DMF=dimethyl formamide; EDTA=ethylenedinitrilotetraacetic acid; GLUPHEPA=glutarylL-phenylalanine-4-nitroanilide ; LPA = Ieucine-4-nitroanilide; PHMB=p-hydroxy-mercuribenzoate; Pl-I=potato chymotrypsin inhibitor I; PPl=potato proteinase leaf; PPr=potato proteinase root; PPt=potato proteinase tuber; PVP=polyvinylpyrrolidone; TLCK tosyl-L-lysinechloromethyl ketone; TPCK tosyl-Lphenylalanyl chloromethane
Materials and Methods
Polyvinylpyrrolidone (PVP), mol.wt. 40,000, and myoglobin, sperm whale, cryst, salt-free from Mann Research Lab. ; chloramphenicol, ponceau S, imidazole (grade I), bovine ,/-globulin (fraction lI), p-hydroxymercuribenzoate(PHMB), dimethyl formamide (DMF), glutaryl-L-phenylalanine-4-nitroanilide(GLUPHEPA), Lleucine-4-nitroanilide (LPA), N-benzoyl-L-tryrosine-4-nitroanilide (BTPA), N~-p-tosyl-L-Iysine-chloromethylketone (TLCK) from Sigma Chemical Co. ; lima bean trypsin inhibitor from Worthington Biochem. Corp.; benzoyl-L-arginine-4-nitroanilide(L-BAPA), L-alanine-4-nitroanilide (APA), sodium cyanide, cysteine, 2-iodacetamide, 2,2-bipyridin, 2-mercaptoethanol, 1.10-phenantroliniumchloride, acrylamide, and iodacetic acid from Merck and Co.; light green SF, ovalbumin, soybean trypsin inhibitor and bovine serum albumin from Serva; 5-sulfosalicylicacid from Pierce; chymotrypsinogen A, aldolase, and catalase from Boehringer; Sephacryi S 200 sf from Pharmacia; ampholine carrier arnpholytes from LKB-Producter; pepstatin from Banyu Pharmaceutical Co. Ud./ Tokyo; N-tosyl-L-phenylalanylchloromethane (TPCK) from Cal-
O032-0935/78/0141/0145/$01.80
146 biochem; diisopropylfluorophosphate (DFP) from Serva; ethylenedinitrilotetraacetic acid disodium salt (EDTA) from Mallinckrodt; dialysis membranes from Union Carbide Corp. Potato material was obtained from tuberizing potato plants (variety Russet Burbank) on fields and in the greenhouse.
Extraction Methods' 1. Tubers: The tubers employed in the study were either freshly harvested in late summer or were stored one year and allowed to sprout four to five weeks in pots in a greenhouse. All procedures were carried out at 4 ~ C. Three hundred grams of washed tubers were cut into small pieces and blended with 100 ml 1-M Tris/HC1 buffer, pH 7.2, 4• 1 0 - 3 M EDTA and 0.25 x 10 3 M PVP, mol. wt. 40,000. The juice was extruded through cheesecloth and clarified by centrifugation at 12,000 g for 30 rain. Solid ammonium sulfate was added to give an 80% saturated solution. After standing 30 min, the precipitate was collected by centrifugation, the supernatant discarded, and the precipitate was dissolved in about 30 ml 0.1-M Tris/HC1 buffer. Insoluble debris were removed by centrifugation at 15,000 g 15 min and the clear supernatant was dialyzed against 0.05 M NHcHCO~ for 8 h and against distilled water for 3 h and lyophilized. Yields of lyophilized material were 405 mg from fresh tubers (PPtl) and 460 mg from year-old, sprouted tubers (PPt2). 2. Roots: Thin roots (not the thick underground sprouts!) from potato plants after four to five weeks growing in pots in a greenhouse were used. From one hundred grams of washed and air-dried roots, extract was prepared by macerating the tissue with a ceramic mortar and pestle and expressing the juice by squeezing the tissue between the mortar and pestle. To about 50 ml juice (pH 6.2) was added an equal volume of 1 M Tris/HC1 buffer, pH 7.2, 4 x 10 -a M EDTA. After centrifugation for 15 rain at 15,000g the clear supernatant was adjusted to p H 7.0 and ammonium sulfate was added to give 80% saturation. The precipiatate obtained after centrifugation at 15,000 g was dissolved in about 20 ml of the above buffer diluted 1 to I0 and dialyzed 2 • h against NH4HCO3 and 3 h against distilled water. All operations were carried out at 4 ~ C. The clear solution was lyophilized and 105 mg crude potato proteinase PPr were obtained.
3. Leaves: This preparation was obtained as described previously (Santarius and Ryan, 1977). The main proteinase activity was extracted after the first extraction with 20 m M EDTA solution (crude potato proteinase PPll). With a second extraction of the cell debris with 1 M KC1 solution, further endogenous proteinase activity was recovered (crude potato proteinase PP12). From 200 g potato leaves, 520mg PPll and l l 2 m g PP12, respectively, were obtained.
K. Santarius and H.-D. Belitz: Proteinase Activity in Potato Plants then stained for 30 rain with a solution of equal parts of 0.2% light green SF in 0.5% sulfosalicylic acid, 0.2% ponceau red in 5 % trichloroacetic acid, and 0.1% amido black 10 B in 5% trichloroacetic acid (this mixture was prepared four days before using). Destaining was performed in both cases in 7.5% acetic acid with an ,,Ames" Quick-destainer. Enzyme activity against L-amino acid-4-nitroanilides was demonstrated by incubation of the electrophoresis gels for (a) 15 min in 50 m M immidazole/HC1 buffer, pH 7.6, at 4 ~ C, (b) 15 min in same buffer but containing 2.5 m M L-amino acid-4-nitroanilide at 4 ~ C and (c) 15 min or longer (depending on enzyme activity present) in same solution at 37~ (Hennrich et al., 1973). Due to its low solubility in aqueous solution, BTPA was dissolved in a mixture of D M F and 50 m M imidazole/HC1 buffer 1 to 2.
Measurement o f Enzyme and Inhibitor Activities To 0.05-0.5 ml enzyme solution was added 0.2 M Tris/HC1 buffer, pH 8.6, at BTPAase and L-BAPAase (pH 8.8 at APAase and LPAase), 4 x 1 0 - 4 M EDTA, to a final volume of 1.4ml. After 5 min preincubation at 35~ the reaction was initiated by the addition of 0.1 ml 3 m M L-amino acid-4-nitroanilide in DMF. The reaction was normally terminated after 2 h with 0.5 ml of 30% acetic acid. The yellow color that resulted from the liberation of p-nitroanilin was measured in 1-cm cuvettes in a Beckman DB-G spectrophotometer at 405 nm. A unit of activity is defined as an absorbance change of 0.01 per rain at 405 nm under the conditions described. The inhibition stock solutions of TLCK, TPCK, and 2,2'bipyridin were dissolved in methanol; the proteinase inhibitors of potato and beans were in 10-3 N HCI. All other inhibitors were dissolved and added in 0.1 M Tris/HC1 buffer, pH 7.4. The endopeptidase activity against gelatin was measured by radial diffusion assay as described previously (Santarius and Ryan, 1977). The measurement for content of potato chymotrypsin inhibitor I = P I - I (Melville and Ryan, 1972) was carried out by immunological test (Ryan, 1967).
Isoelectric Focusing Isoelectric focusing was carried out according to Haglund (1967) using the small column (8100-10, gradient volume 110 ml) of LKBProducter AB. Concentration of the ampholines in the density gradient was 1%. A voltage of 1600 V was employed at a temperature of 4 ~ C for 16-18 h. The column was emptied by pumping with a flow rate of 100 ml/h. One-ml fractions were collected and the absorbance at 280 nm (protein) and the pH were continuously measured. The hydrolyzing L-amino acid-4-nitroanilide activity of the fractions was determined as described above.
Estimation of Molecular Weight Disc Electrophoresis Polyacrylamide gel disc electrophoresis was carried out at pH 9.5 in system 1 according to Maurer (1971). Running time was 3 h at 150V (24mA at 12 tubes). The protein patterns were stained with a solution of 0.05% amido black 10 B in 7.5% acetic acid. To render the endopeptidase activity visible, parallel runs were carried out using polyacrylamide gel containing 0.1% gelatin (Stegemann, 1968) followed by incubation of the gel columns for 5 h at 37 ~ C in 0,05 M phosphate buffer, pH 7.4. The gels were
A Sephacryl S 200 sf column ( 2 x 8 5 cm) was equilibrated and eluted with 0.1 M Tris/HCl buffer, pH 8.0, 4 x 10 4 M EDTA, at 10~ C. The absorbance of the fiactions was measured at 280 nm (protein) and at 405 nm after reaction with a solution of L-amino acid-4-nitroanilides. Standard proteins (0.5-7 mg in 0.5 ml buffer) were myoglobin (mol.wt. 17,800), chymotrypsinogen A (25,000), aldolase (140,000-150,000), bovine 7-globuline (150,000-171,000), and catalase (225,000-250,000). The void volume (V0) was determined by dextran blue.
147
K. Santarius and H.-D. Belitz: Proteinase Activity in Potato Plants Table 1. Endopeptidase activity and inhibitor PI-I in different tissues at different growth stages of the potato plant Tissue a
Growth stage
Inhibitor PI-I (gg/ml)
Petals
About 21/2 months after sprouting in greenhouse
102
5
49
53
21
396
172
33
192
Old complete yellow leaves Green leaves
Four to five weeks after sprouting in greenhouse
Endopept. activity in trypsin equival. (~g/ml) x 10- 2
Ratio endopept./inhibitor (x 10 5)
Upper green sprouts
61
3
49
Lower green sprouts
53
3
57
121
1
8
White sprouts (already with green leaves)
About 3 weeks after field planting
White sprouts (without green leaves) White underground sprouts
102 Four to five weeks after sprouting in greenhouse
0.4
4
190
17
89
69
40
579
New tuber (less 1 cm in ,~)
510
30
59
New tuber (1 to 3 cm in ~ )
478
2
4
388
2
5
186
I
5
+
1
(1000)
1
23
Roots
Tuber apical end
Stored for one year
Tuber stem end Tuber apical end
Four to five weeks after sprouting in greenhouse
Tuber stem end
44
Plant extracts were made according to Santarius and Ryan (1977) and diluted 1:1 with 1 M Tris/HCl buffer, pH 7.4, containing 2x 10 -3 M chloramphenicol and 0.5x I0 -3 M PVP
Results
Distribution of Proteinase Activity in the Potato Plant Table 1 shows the distribution of proteinase activity a g a i n s t g e l a t i n in c o m p a r i s o n to the c o n t e n t o f t h e potato chymotrypsin inhibitor PI-I (Melville and R y a n , 1972). O v e r a l l the c o n t e n t o f p r o t e i n a s e a c t i v i t y is c o m p a r a t i v e l y low. R e l a t i v e l y h i g h p r o t e i n a s e a c t i v ity a n d l o w c o n t e n t o f i n h i b i t o r was f o u n d in t h e roots of the potato plant. In contrast, leaves exhibited a high proteinase activity with a stronger inhibitor a c t i v i t y . A v e r y l o w level o f p r o t e i n a s e a c t i v i t y was d e t e c t e d in t u b e r s . T h e h i g h a m o u n t o f i n h i b i t o r a c t i v ity in t u b e r s d e c l i n e s r a p i d l y d u r i n g the first w e e k s of sprouting. Crude proteinase preparations of potato tubers, r o o t s (PPr), a n d l e a v e s w e r e p r e p a r e d a n d t e s t e d f o r proteinase activity. All crude proteinase preparations s h o w e d a c t i v i t y a g a i n s t g e l a t i n m e a s u r e d by r a d i a l d i f f u s i o n assay. A c t i v i t y a g a i n s t several L - a m i n o acid4 - n i t r o a n i l i d e s is i n d i c a t e d in T a b l e 2. T h e c r u d e preparation of PPtl was made from tubers harvested
Table 2. Survey of enzyme activities in the crude proteinase preparations of potato tuber, root, and leaf against several L-amino acid-4-nitroanilides. Each 150 gg of the samples, dissolved in 0.15ml 0.1 M Tris/HC1 buffer, pH7.4, containing 4 x l 0 - ~ M EDTA, was assayed as described in Materials and Methods for 2 h at 35~ Crude proteinase Activity (units/mg crude proteinase) against L-amino acid-4-nitroanilides BAPA Potato Potato Potato Potato Potato
tuber PPtl _+ tuber PPt2 _+ root PPr 0.87 leaf PPI1 leaf PPlz .
.
GLUPHEPA
APA
LPA
BTPA
-
2.55 0.69 4.33 0.96 .
0.65 0.18 1.16 0.31
1.08 2.01 -
.
.
Note: = No detectable activity; + = Weak activity, only detectable at the attempts of isoelectric focusing
in late s u m m e r , a n d PPt2 f r o m o n e - y e a r s t o r e d t u b e r s a f t e r f o u r to five w e e k s s p r o u t i n g in p o t s in a g r e e n h o u s e . P P l l o f the p o t a t o l e a v e s was the E D T A ext r a c t ; PP12, the KC1 e x t r a c t , f o l l o w e d . T h e l e a f e x t r a c t PP12 d i d n o t s h o w a n y a c t i v i t y a g a i n s t t h e s e s y n t h e t i c
148
K. Santarius and H.-D. Belitz: Proteinase Activity in Potato Plants
PP I
! a
b
PP%
PPr
PPi 0.2
BTPA
APA/LPA
APA/LPA
d
tZ...'J~
,[•BTPA
9 ",~
ul
/
,~
01 4PAILPA ~ BAPA
~ 4PAILPA
[~
1 c
a
b c
a
b
c
I a
Z, b
c !+)
Fig. 1. Polyacrylamide gel electrophoresis of crude proteinase preparations of potato tuber (PPt~, PPt2), root (PPr), and leaf (PP10. The attempt was carried out as described in the method part. 300 gg (1000 gg PPr at L-BAPAase) of the samples were dissolved in 25 gl of spacer gel buffer. The gels show: (a) protein patterns after staining with amido black, (b) endopeptidase activity againts gelatin as substrate (clearzones),and (c) proteinase activity against several L-amino acid-4-nitroanilides as substrate
x.
X //~ ' ~ " x
~//~e"".e... ~
2'0
Jo
io
6
7
INCUBATION
8
9
1o
pH
Fig. 3. PH optima of BTPAase (-o--o-), APAase @a a-), LPAase ( - + - + =), and L-BAPAase (. ~ - 9 .). 0.15-0.8 mg crude proteinase PPr-according to the activity of the tested enzyme-dissolved in 0.15 ml 0.1 M Tris/ HC1 buffer, pH 7.4, containing 4 x 10 4 M EDTA, were assayed as described in Materials and Methods with buffer solutions of different pH (0.1 M Nacitrate/HC1, pH 2.0-3.5; 0.2 M acetate/NaOH, pH 4.0 5.5; 0.1 M phosphate/S6rensen, pH6.0 7.0; 0.2M Tris/HC1, pH7.2-9.0; 0.2 M borate buffer, pH9.2-10.0). The incubation time was 120 min at 35~ C
Polyacrylamide Gel Electrophoresis of the Crude Proteinase Preparations of Potato Tuber, Root and Leaf
/~',~
..o' ".../,'"
5
s'o
INCUBAtiON TEMPERA TURE (C)
Fig. 2. Temperature optima of BTPAase @o--o-), APAase ( A - - A - ) , and L-BAPAase (- e- 9 9 .). 0.15 0.5 mg crude preparation PPt2 (BTPAase), PPtl (APAase), and PPr (L-BAPAase) respectively-according to the activity of the enzymes-dissolved in 0.15ml 0.1 M Tris/HC1 buffer, pH7.40 containing 4x10 4M EDTA, were assayed as described in Materials and Methods at different temperatures. The incubation time was 120 min (APAase, 60 min)
substrates. A c t i v i t y a g a i n s t L - B A P A was f o u n d m a i n l y in the r o o t s o f the p o t a t o p l a n t ; o n l y a very low c o n c e n t r a t i o n has been d e t e c t e d in b o t h t u b e r p r e p a r a t i o n s . It is r e m a r k a b l e t h a t e n z y m e activity h y d r o l y z i n g B T P A in m e a s u r a b l e a m o u n t s in tissues o t h e r t h a n the r o o t s occurs only in t u b e r s after s p r o u t i n g in the g r e e n h o u s e . P r o t e i n a s e activity a g a i n s t A P A a n d L P A was f o u n d in leaves, tubers, a n d r o o t s o f the p o t a t o plant. F u r t h e r e x p e r i m e n t s were c a r r i e d o u t o n l y with the p r o t e i n a s e activities a g a i n s t the s u b s t r a t e s B T P A , A P A , L P A , a n d L - B A P A . The enzymes were n a m e d B T P A a s e , A P A a s e ( L P A a s e ) , a n d L - B A P A a s e , respectively.
The m i g r a t i o n o f the b a n d s o f A P A a s e a n d L P A a s e (Fig. 1) indicates t h a t these enzymes are p r e s e n t in all f o u r c r u d e p r o t e i n a s e p r e p a r a t i o n s . T h e wide activity r a n g e o b t a i n e d by e m p l o y i n g gelatin a n d benzoylL - t y r o s i n e - 4 - n i t r o a n i l i d e as s u b s t r a t e s for the crude p r e p a r a t i o n s o f r o o t a n d leaf is striking. A l s o , m u c h lower p r o t e i n a s e c o n c e n t r a t i o n s d i d n o t l e a d to smaller activity b a n d s . O n the s t r e n g t h o f low activity o f L - B A P A a s e in b o t h t u b e r p r e p a r a t i o n s , their activity b a n d s were here n o t detectable.
Temperature and pH Optimum of BTPAase, APAase, and L-BAPAase T h e t e m p e r a t u r e o p t i m a for the three e n z y m e activities were d e t e r m i n e d to be 2 5 ~ for L - B A P A a s e a n d a b o u t 40 ~ C for the o t h e r two e n z y m e activities (Fig. 2). T h e p H o p t i m u m for all activities was in the a l k a l i n e region. B T P A a s e a n d A P A a s e ( L P A a s e ) e x h i b i t e d an o p t i m u m at p H 8.6 a n d p H 9.0, respectively, a n d L - B A P A a s e e x h i b i t e d an o p t i m a l p H r a n g e o f 8.6 to 9.0 (Fig. 3).
Isoelectric Focusing of the Crude Proteinases To c o m p a r e the e n z y m e activities in the different vegetative p a r t s o f the p o t a t o plant, isoelectric focusing
K. Santarius and H.-D. Belitz: Proteinase Activity in Potato Plants
149
OG
2,0, t8
o3 ,,
