Download Proteases of Senescing Oat Leaves

Plant Physiol. (1978) 61, 501-505
Proteases of Senescing Oat Leaves
II. REACTION TO SUBSTRATES AND INHIBITORS'
Received for publication March 23, 1977 and in revised form November 2,
1977
ROLF H. DRIVDAHL2 AND KENNETH V. THIMANN3
Thimann Laboratories, University of California, Santa Cruz, California 95064
ABSTRACT
MATERIALS AND METHODS
Two proteases isolated from senescent oat (Avens saiva) leaves have
Plant Material. Seeds of A. sativa cv. Victory, obtained from
been subjected to further study. One of these, an acid protease active at the USDA, were husked, soaked, and grown in Vermiculite as
pH 4.2, is inhibited by phenylmethylsulfonyl fluoride (PMSF) but not by described previously (23). After 8 days of growth in light the intact
iodoacetamide (lAc). The other, active at pH 6.6, is inhibited by both plants were left in darkness for 2 to 3 days, in order to maximize
PMSF and IAc. These results, together with previously reported evidence protease yields.
that mercaptoethanol stimulates the activity of only the neutral protease,
Protease Extraction and Purification. Procedures for homogeare taken to indicate that the acid protease is probably of the serine type, nization of the tissue and purification of the extracts have been
whereas the neutral enzyme is of the sulfhydryl type. Both enzymes are described (9). For the experiments presented here, the final step in
Inhibited by irradiation in the presence of rose bengal, a selective histidine purification, involving chromatography on DEAE-Sephadex, was
modification reagent. The acid protease was completely unaffected by omitted. Material eluted from a hemoglobin-Sepharose affinity
chelators, but data on the neutral protease were equivocal.
column was dialyzed against 10 mm sodium phosphate buffer (pH
All protein substrates tested were attacked by both enzymes, though at 6) at 0 C, and either used directly or concentrated by ultrafiltration
strkingly different rates. Characterization of the digestion products, with in an Amicon cell equipped with a PM-10 membrane. As shown
denatured hemogiobin as substrate, indicated that the acidic enzyme is an in the preceding paper (9), the enzymes are highly purified at this
endoprotease, while the neutral one is an exoprotease. Evidence is pre- point, and omission of the DEAE step avoids the large loss of
sented that these proteases undergo autolysis in vitro.
activity normally encountered at the final stage; the primary
disadvantage is that they are not separated from each other, since
this was accomplished by the ion exchanger. However, their effects
were easily differentiated by working at the respective pH optima.
Protease Assay. Except where indicated, both proteases were
assayed with denatured hemoglobin as substrate; details of the
Several studies in recent years have indicated a major role for incubation procedure and analysis of the reaction products were
proteases in the senescence of leaves (1, 13, 17, 25). In at least one described in the previous report (9). One unit is defined as that
case the observed increases in proteolytic activity have correlated amount of enzyme which liberates I ,umol of leucine equivalents
with the liberation of a-amino nitrogen which is such a character- in 1 hr at 50 C. Use of this high temperature, which was shown
istic concomitant of the senescence process. In senescing leaves of previously (9) to be the optimum for short incubations, gave
Avena sativa, previous work from this laboratory has shown the results qualitatively similar to those obtained at lower temperapresence of at least two proteases (17). One of these, active at acid tures, while permitting the use of smaller enzyme quantities and
pH values, showed marked increases in activity during the brief shorter reaction times. The optimum assay temperature for the
experimental senescence period, while the other, active in the acid protease of wheat seedling leaves has been reported as 52 C;
neutral range, increased more slowly. Extensive purification and the reaction rate at 50 C was linear for at least 90 min (11).
a preliminary characterization of these two proteases have now
Inhibitor Studies. pCMB' was dissolved in 20 mm sodium
been reported (9). They have virtually identical mol wt, as deter- acetate buffer (pH 5), and equal volumes of inhibitor and enzyme
mined by gel filtration, show unexpectedly high temperature stock solution were incubated for 30 min at 30 C. The exact
optima, close to 50 C, and both exert their activity over broad pH concentration of the pCMB stock solution was determined specranges. The neutral enzyme required the presence of a sulfhydryl trophotometrically, using a molar extinction coefficient of 1.69 x
compound for maximal activity.
104 M-1 cm-' (6). Protease activity remaining after these and all
The present work extends the biochemical study of these two other preincubations with inhibitors was estimated by the standard
proteases. Evidence is adduced that the acidic enzyme is probably hemoglobin assay.
an endoprotease, having seine at the active site, while the neutral
PMSF was dissolved in 100%o ethanol and diluted to 0.1 M with
enzyme appears to be an exoprotease and has a sulfhydryl group 20 mm sodium phosphate buffer (pH 7). Concentrations of 10 mm
at its active site. Both probably undergo autolysis in vitro just as PMSF in 10%o ethanol were obtainable in this fashion. Equal
they probably do in vivo as the leaf senescence progresses (17). volumes of inhibitor and enzyme were mixed and the reaction
Given the apparently generalized nature of the protein degrada- allowed to proceed at 30 C for 30 min. The final concentration of
tion that occurs during senescence, the presence of proteases of ethanol in all preincubations was thus 5%; controls in 5% ethanol
varying activities and specificities in the leaf is not unexpected. without inhibitor indicated negligible effects of this level of
ethanol on protease activity.
'Supported in part by National Science Foundation Grant GB 35238
to K. V. T.
2 Present address: 1521 Pine Street, Olympia, Washington 98502.
4 Abbreviations used in this paper: lAc: iodoacetamide; pCMB: parachloromercuribenzoic acid; PMSF: phenylmethylsulfonyl fluoride.
'To whom requests for reprints should be sent.
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502
DRIVDAHL AND THIMANN
Reaction with lAc was carried out at pH 7 in 20 mm sodium
phosphate buffer; the incubation times were varied, and these are
reported with the results.
Solutions of rose bengal in phosphate-citrate buffer were mixed
with equal volumes of enzyme and irradiated at room temperature
with a 450-w spotlight for 5 min. The light was placed 5 cm from
the tubes, and a distilled H20 screen was positioned between the
tubes and the light to minimize heating. Controls contained rose
bengal but were protected from the light by wrapping in aluminum
foil; the dye was removed from the mixture by chromatography
on Sephadex G-25 (coarse) prior to enzyme assay.
Metal ions and metal chelators were dissolved in the appropriate
assay buffers, and the enzymes were added in the standard assay
proportions previously described (9). The mixtures were then
incubated for 30 min at 30 C prior to addition of the substrate.
Substrate Specifcity Studies. Gelatin, casein, and BSA were
dissolved or suspended in water and simply substituted for hemoglobin in the standard assays. Azocoli (Calbiochem), a powdered insoluble cowhide derivative covalently bound to a red dye,
was added in 10-mg amounts to small flasks containing 1 ml of
enzyme solution and 4 ml ofthe appropriate assay buffer. Samples
were incubated at the optimum temperature (50 C) for both
enzymes with sufficiently vigorous shaking to keep the substrate
particles in an even suspension. The reaction was similar at 37 C,
though naturally slower. Filtration of the mixture through lens
paper removed the residual substrate and thus terminated the
reaction; the A of the filtrate was read at 520 nm.
Chromatography of Reaction Products. To compare the products of proteolysis at pH 4.2 and 6.6, the reactions were run as
described above, but with the volumes scaled up by a factor of 10.
The reactions were terminated by immersing the tubes in a boiling
water bath for 5 min. At pH 4.2 the hemoglobin was not precipitated by boiling, and this necessitated the addition of sufficient
0.4 M Na2HP04 to raise the pH above 6 before boiling. The
mixtures were cooled at 0 C for 1 hr and then centrifuged; the
resultant clear supernatants were concentrated in an air stream.
The concentrated solutions were then applied to a column (1.5 x
95 cm) of Sephadex G-50 (medium) equilibrated with 50 mm
sodium phosphate (pH 7.2). From this column 2-ml fractions were
collected and the soluble a-amino nitrogen determined by the
ninhydrin reaction.
Substrate Specificity-Synthetic Substrates. Several synthetic
protease substrates were tested with both the acidic and neutral
proteases, in hopes of learning more about their peptide bond
specificity and of finding a more convenient substrate for kinetic
Plant Physiol. Vol. 61, 1978
work. All compounds were obtained from Sigma Chemical Corp.
All were assayed spectrophotometrically, making use of the shift
in the absorption maximum between the substrate and the product
of hydrolysis. Difference spectra maxima employed were as follows: p-tosyl-L-arginine methyl ester, 247 nm; benzoyl-L-arginine
ethyl ester, 253 nm; benzoyl-L-tyrosine ethyl ester, 256 nm; benzoyl-L-arginine p-nitroanilide, 410 nm. The substrates were dissolved in dimethylsulfoxide and diluted into appropriate assay
buffer, and 1.5 ml of this solution was mixed with 0.5 ml of
suitably diluted enzyme stock and incubated at 35 C for I hr.
Results were expressed as the change in A of the reaction mix in
the 1-hr period. Soybean trypsin inhibitor was from Sigma.
Tests for Autolysis in Solution. To study the possible reappearance of small mol wt peaks, samples of the partially purified
protease from the affinity chromatography step were applied to a
column (1.5 x 90 cm) of Sephadex G-100 in phosphate-citrate
buffer (pH 6). Because of their closely similar mol wt, both
proteases eluted in a single symmetrical peak. The active fractions
were pooled, concentrated, stored for 72 hr at 6 C, and then
rechromatographed on the same column. The appearance of new
peaks indicated proteolysis.
RESULTS
Kinetics. In the particular case of proteolytic enzymes, Lineweaver-Burk plots and determinations of Km values can be complicated, since if some protein substrate is introduced as an impurity, then even in the absence of hemoglobin or other added
substrate some proteolysis occurs. This is a more serious problem
at low substrate concentrations than at saturating levels, for in the
latter the relatively enormous amounts of hemoglobin in the assay
mixture render any contribution of substrate by the enzyme
preparation negligible. This difficulty was overcome by measuring
the small amount of hydrolysis occurring in the absence of added
hemoglobin and subtracting this from all other values. The resulting linear relationship between 1/S and l/V, obtained for both
Avena proteases, is shown in Figure 1, a and b. The approximate
Km values from the figure are 0.11% hemoglobin for the acidic
protease and 0.067% for the neutral one. Although the Km for the
neutral enzyme is lower, the Vmaz is also lower. This effect is more
specifically a feature of the substrate than of the enzyme, since
experiments reported below, using other protein substrates, indicate a higher V.. for the neutral protease. The acidic protease of
wheat leaves yields a value of 0.026% (I 1).
It was somewhat surprising to discover that the acidic protease,
2.
I1.9
I
1.0
1.01
0.8[
0.8~
0Vo.
Km -O.115%
0.6k
Heoglobin
Km a Q067 %
Hemogloin
0.4
0.4
0.2
021
a
I
5
I-
lS
'S
20
I~~~~~~~~~~~~~~~
25
30
b
F
I
I
5
10
I
15
1
I
20
25
30
FIG. 1. Lineweaver-Burk plots depicting the relationship between substrate (hemoglobin) concentration and rate of hydrolysis by the A vena proteases
at 50 C. a: Neutral protease; b: acidDownloaded
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Plant Physiol. Vol. 61, 1978
PROTEASES OF SENESCING OAT LEAVES
Table I
Hydrolysis of different proteins by Avena leaf proteases
Azocoll was used as a suspension at 10 mg/ml; all other
substrates at a concentration of 4%. Assay was performed
at 50 C for 90 min. A unit is defined as the amount of
enzyme that liberates one imol of amino nitrogen per hr. for all
substrates except Azocoll, for which one unit is the amount
of enzyme which produces a AOD520 of 0.1 in 1 hr.
Substrate
503
The photosensitive dye rose bengal has been shown to be a
highly selective reagent for modification ofhistidine residues when
the reaction is carried out at pH near neutral (4, 28). Its effect on
the A vena proteases is illustrated in Figure 3. Strong inhibition of
both is evident, indicating the presence of histidine at the active
sites. Although tyrosine, tryptophan, and methionine are also
attacked by rose bengal, the latter two react at appreciable rates
Units
pH 6.6
pH 4.2
Table II
Inhibition of Avena leaf proteases by IAc and pOIB
Hemoglobin
Bovine serum albumin
Gelatin
Casein (insoluble)
Azocoll
4.53
1.12
0.95
0.40
1.02
3.17
1.58
2.92
3.38
6.40
originally thought to be the more potent enzyme by virtue of its
Preincubations at 30 C for indicated times prior
to standard hemoglobin assay.
Inhibition at
Preincubation
pH 4.2
pH 6.6
10 mM IAc, 30 min
10 mM IAc, 150 min
0.04 mM pCMB, 30 min
0.4 mM pCMB, 30 min
1.4
7.2
0
2.1
18.3
48.0
0
4.0
reactivity against hemoglobin, is actually less active than the
neutral enzyme against other proteins (Table I). The most pronounced differences are apparent with gelatin, casein, and Azocoll,
with which substrates the neutral activity is three to eight times as
great. It is suggestive that the BSA and casein samples used were
native proteins, whereas the hemoglobin was acid-denatured.
Although the limited number of proteins tested makes generalizations only tentative, the apparent differences in susceptibility of
native and denatured proteins raise interesting possibilities with
regard to the in vivo significance of these proteases in senescence.
This will be considered below.
2
Action of Inhibitors. Since previous experiments have indicated
z
that only the neutral protease requires a reduced sulfhydryl group
z
for its functioning (9), the effects of pCMB and lAc on enzyme
A'
activities were tested. Table II shows that the activity at pH 4.2 is
only slightly affected by IAc, but the activity at pH 6.6, following
a 3-hr preincubation, shows a 50% inhibition. Complete reaction
of sulfhydryl groups with lAc often requires long incubation
periods and conditions that involve protein denaturation (3), so
the incompleteness of the inhibition is not surprising. Histidine
residues are also susceptible to attack by lAc, but the relative rate
of this reaction between pH 6 and 8 is extremely slow.
.001
.01
.1
1.0
The lack of any significant inhibition by pCMB is an apparently
PMSF CONCENTRATION (MxalO)
contradictory result, but since this and other organic mercurials
FIG. 2. Effect of PMSF on the activities of Avena proteases. Enzyme
show very limited solubility in water, their approach to relatively was preincubated with the inhibitor in 5% ethanol for 30 min at 30 C.
hydrophilic centers of proteins can be limited also. Furthermore, Controls contained 5% ethanol without PMSF. Hemoglobin assay, 50 C.
recent experiments have shown that inhibition of the neutral
protease by HgCl2 is partially reversible by the P-mercaptoethanol
in the assay buffer, and this could be occurring in the case of
pCMB as well. The reagent N-bromo-succinimide inhibits both
enzymes, 1 mM preincubated with the enzyme for 30 min at 30 C
pH 4.
sufficing for 95% inhibition. However, the specificity of this re2
/
10ISO
agent is not sufficiently narrow to allow for definite deductions.
The same is true for the actions of mercury and silver salts, both
of which were found to cause 60 to 100% inhibition at 10 mm.
Both trypsin and chymotrypsin are powerfully inhibited by
20
PMSF, through its reactivity with the active serine hydroxyl; it
z 6
also attacks the hydroxyl of acetylcholinesterase, though more
slowly (5, 10). In addition, it retards the senescence of detached
oat leaves (25). Figure 2 demonstrates effective inhibition by
PMSF of both Avena proteases, with a slightly greater effect on
the neutral enzyme. Whitaker and Perez-Villasenor (29) have
clearly demonstrated inhibition of papain (a sulfhydryl protease)
by PMSF, and this ability to attack free sulfhydryls probably
explains the inhibition of the neutral protease. Since the neutral
protease from Avena is stimulated by mercaptoethanol and inI
I
rt 4 0
hibited by IAc, while the acid protease is unaffected by these
".001
.1
1.0
.01
PMSF
indicate
that
the
obtained
with
the
results
compounds,
ROSE BENGAL CONCENTRATION (%)
neutral enzyme is sulfhydryl-dependent, and the acid enzyme is
FIG. 3. Effect of rose bengal on the activities of A vena proteases. The
hydroxyl-dependent. In contrast, the acid protease from germi- enzyme was irradiated under a spotlight in the presence of the dye for 5
nating sorghum seeds shows no sensitivity to either -SH or -OH min prior to assay. Controls contained the dye but were protected from
Hemoglobin
assay, 50 C.
reagents (12).
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Plant Physiol. Vol. 61, 1978
DRIVDAHL AND THIMANN
504
Table III
Effects of metal ion chelators on activity of Avena leaf proteases
The enzymes were incubated in chelator solutions for 30 min at
30 C prior to standard hemoglobin assay.
Chelator
Inhibition at:
pH 6.6
pH 4.2
0
Na2EDTA, 1.0 mN
2.4
Na2EDTA, 10.0 mM
a,a-Dipyridyl, 1.0 mM 0
a,a-Dipyridyl, 10.0 mM 0
41.1
45.0
15.3
52.4
only below pH 4, while tyrosine reacts only above pH 8 (18).
The involvement of histidine may explain the slight inhibition
of the acid protease by lAc (Table II). The evidence indicates that
this enzyme contains no reactive sulfhydryl, and in the absence of
this group lAc will attack histidine (5). However, the reactivity of
the imidazole group toward lAc is low (14), and thus only a very
small inhibition is produced.
Table III shows the effects of two different metal ion chelators
on protease activities. No inhibition of the acid protease was seen,
and hence a metal ion of the type removable by these reagents is
probably not involved in its function. Both EDTA and dipyridyl
exerted a moderate inhibition of the neutral protease, but the
concentrations required were higher than would be expected. In
contrast, Vallee and Neurath (26) reported complete inhibition of
bovine pancreatic carboxypeptidase by I mM EDTA or 5 mM
dipyridyl. Since the effect of EDTA shown here is nearly maximal
at I mm and is increased only slightly by a 10-fold increase in
concentration, there may be a metal atom present which is not
absolutely essential for activity. Alternatively, a small amount of
contaminating metallo-protease may be present. A similar explanation for partial EDTA inhibition of a-amylase has been proposed by Jacobsen et aL (15).
Evidence on Mechanism of Action. As seen in Figure 4, the
nitrogenous products produced upon digestion of hemoglobin by
the acid protease are of heterogeneous sizes; in contrast, digestion
by the neutral enzyme produces essentially one size class, the
elution volume of which closely approximates that of an amino
acid standard. It is thus reasonable to designate the acidic enzyme
as an endoprotease and the neutral one as an exoprotease. The
acidic enzyme of mung bean cotyledons is also apparently an
endoprotease (8).
Another difference between the two proteases was seen in their
attack on synthetic substrates. Table IV shows that the three
arginine derivatives were more sensitive to the neutral than to the
acid enzyme, while the tyrosine derivative was much more rapidly
attacked by the acid enzyme. Such differences, as weli as that
above, give some indication of how the rapid and extensive
proteolysis and liberation of free amino nitrogen are achieved in
leaf senescence.
Neither enzyme was sensitive to the soybean trypsin inhibitor,
even at a concentration of 500 mg/I.
Evidence for Autllysis. It has been suggested that proteases
present unique problems in protein purification, not only because
of their tendency to aggregate with other proteins but more
particularly because they may undergo autolysis in solution. That
autolysis may occur in the enzyme preparations discussed here is
clearly shown by Figure 5. In this experiment, a partially purified
sample was chromatographed on Sephadex G- 100, and the active
fractions were pooled and stored at 6 C for 3 days. They were
then rechromatographed on the same column. Even though the
final peak of material absorbing at 280 nm had been discarded
after the first column run, a very similar peak appeared on the
second run (upper figure). Since this peak consists of low mol wt
peptides and amino acids, the results indicate proteolytic digestion
of proteins in the enzyme preparation. Gel electrophoresis has
shown that the proteases are almost the only components of the
preparation at this stage (see Fig. 3 of ref. 9), and since a significant
loss of activity is apparent, the renewed second peak most probably
represents autolytically generated fragments. The occurrence of
such autolysis supports the suggestion of Martin and Thimann
(17) that the rapid decrease in protease activity toward the end of
the senescence period in oat leaves resulted from self-degradation.
DISCUSSION
In any consideration of the regulation of proteolytic activity the
problem of substrate susceptibility is crucial. Several proteins were
degraded by the preparations from oat leaves, but at strikingly
different rates. It has been suggested (19) that such differences in
rate are due to the existence in some proteins of susceptible peptide
bonds in exposed regions of the molecule which are characterized
by a high degree of local flexibility, thus facilitating "fit" into the
active center of the protease. Such susceptibility can be increased
by the breakage of certain bonds which cause the polypeptide
chain to unfold (20). It seems likely, therefore, that in vivo, the
activity of one of the A vena proteases renders some of the leaf
substrates more prone to attack by the other. Since the data of
Figure 4 have shown that only the acidic enzyme is an endoprotease, its activity could produce fragments which are more readily
reduced to amino acids by the exoprotease. Correspondingly, in
the senescing leaf it is the acidic protease whose activity increases
first, while that of the neutral protease develops more slowly (17).
The A vena leaf proteases evinced rather broad specificities, in
that ali proteins tested were degraded to some extent. Given the
multiplicity of proteins to be degraded in a senescing leaf, this
breadth of specificity would be an appropriate feature.
In the previous report (9) we noted that mercaptoethanol stim-
FRACTION NUIMER
FIG. 4. Sephadex G-50 chromatography of acidic and neutral protease
reaction products (hemoglobin assay). Column dimensions were 1.5 x 95
cm, eluting buffer was 0.02 M sodium phosphate (pH 7).
Table IV
Hydrolysis of synthetic substrates by Avena leaf proteases
All substrates were used at 1.0 mM. Assay conditions as
described in Methods. All data from linear proportionality ranges.
Substrate
HTydrolysis change
pH 6.6
pH 4.2
&OD/hr
Benzoyl-L-arginine ethyl ester
Benzoyl-L-arginine p-nitroanilide
Tosyl-L-arginine methyl ester
Benzoyl-L-tyrosine ethyl ester
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Copyright © 1978 American Society of Plant Biologists. All rights reserved.
0.325
0.285
0.080
0.575
0.610
0.642
0.430
0.340
Plant Physiol. Vol. 61, 1978
PROTEASES OF SENESCING OAT LEAVES
1.2
3.0
0.6
2.0
ENZYME
PROTEIN
1.0
0O4-A
PROTEIN
ol.2
Z
~ENZYME
'hi0
04
0.6
1.
505
from oat seedlings with a mol wt of about 60,000, but the preparations also contained a number of minor active species, having
mol wt as low as 10,000; it was suggested that these were autolytically generated fragments. Similarly, at least some of the multiple
forms of a protease from Rhizopus oligosporus are produced by
autolysis (27). However, in the case of ficin (16) and a barley
endopeptidase (7), autolysis was ruled out as an explanation for
multiple forms; the existence of several distinct gene products was
the preferred explanation.
The continued generation of autolytic fragments in solution can
create serious problems in enzyme purification and characterization. Yet, if such fragmentation occurs in vivo, with minimal loss
of proteolytic activity, it would lend a useful diversity and stability
to the process of protein degradation during senescence.
LITERATURE CITED
20
40
60
30
FRACTION NUMBER
FIG. 5. Test for autolysis of protease preparation by repeated G-100
chromatography of active fractions. Lower figure shows the chromatography of the extract at the G-5O stage. Right hand peak ("protein") was
then discarded. Upper figure shows the rechromatography of the active
fractions ("4enzyme"9) from the first column after 3 days of storage at 6 C
at pH 6. Column dimensions were 1.5 x 90 cm, eluting buffer was 0.05
M sodium phosphate (pH 6), 3-mi fractions. Protease assay at pH 4.2 and
SOC.
ulated the activity of the neutral protease, whereas it had no effect
on the acid one, except for a small inhibition at high concentrations. This stimulation, together with the inhibition of the neutral
protease by both IAc and PMSF, strongly suggests that it is a
sulfliydryl enzyme. Since the acidic enzyme is not affected by IAc,
the inhibitory effect of PMSF on it is probably due to the acylation
of an active site hydroxyl group.
Attempts to implicate histidine residues in protease activity
have frequently employed as specific labels the chloromethyl
ketones of phenylalanine and lysine. In trypsin and chymotrypsin
these compounds act by alkylating histidine residues at the active
site (22). However, tosyl-lysylchloromethyl ketone alkylates cysteine in the active site of papain (2, 29), and hence the more
specific reagent, rose bengal, was used in this study. Means and
Feeney (18) considered photooxidation to be the best general
procedure for selective histidine modification. The results shown
here indicate a critical role for histidine in the functioning of both
Avena proteases.
No requirement for metal ions in catalysis by the acid protease
is apparent, but the partial inhibition of neutral enzyme activity
by EDTA and dipyridyl remains unexplained. It could be the
result of the presence of a metal atom which accelerates hydrolysis
but is not essential for it, or of the presence of a contaminating
metallo-protease. However, no stimulation of activity was observed upon addition of various metal ions (Cu2+, Zn2+, Mg2+,
Mn2e) to assay buffers at concentrations up to 10mt (results not
shown). In view of this, it is unlikely that the previously reported
inhibition of leaf senescence by chelators (24) is related to any
effect on protease activity.
The phenomenon of autolysis probably explains the previously
reported shoulders in the pH curve for the acid proteases of oats
(9) and of wheat (11). Pike and Briggs (21) purified a protease
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