Yakov E. Dunaevsky and Mikhail A. Belozersky Protease ofLimited ...

PROTEASE OF LIMITED SPECrnCITY IN BUCKWHEAT SEEDS
Yakov E. DUNAEVSKYand Mikhail A. BELOZERSKY
A. N. Belozersky Institute of Physico-Chemical Biology,
Moscow State University, Moscow 119899
Abstract
Two proteinases with limited specificity have been isolated and studied from the seeds
of buckwheat Fagopyrum esculentum Moench. It was demonstrated that one of them was a
serine proteinase, which hydrolyzed well p-nitroanilid of N-benzoyl-DL-arginine (BAPA),
protamins, to a lesser extent - histones and did not hydrolyse buckwheat seed globulins and
albumins as well as a number of protein substrates commonly used for determination of
proteolytic activity. This proteinase was a single chain enzyme with molecular mass 70 kD,
isoelectric point 4.5 and pH-optimum 9.0. Its substrate specificity was similar to that of
trypsin but in contrast to trypsin it was not inhibited by trasilol and soybean trypsin inhibitor.
The second enzyme studied belonged to the class of metalloproteinases and performed limited
proteolysis of the 13S globulin - the major storage protein in buckwheat seeds. In the course
of this proteolysis only high molecular mass subunits of 13S globulin were hydrolyzed. The
molecular mass of metalloproteinases was 34 kD, isoelectric protein inhibitor with molecular
mass 10 kD. The possible functional role of the studied buckwheat seed proteinases with
limited substrate specificity is discussed.
Introduction
Proteins of buckwheat seeds may be divided into two groups according to their
physiological role. The first group involves storage proteins, comprising the main part of seed
protein and includes a relatively small number of proteins. The second group consists of
proteins, maintaining the normal flow of cell metabolism, present in the seed in relatively
small amount, and includes a large number of proteins. For the past years much attention was
paid to the study of individual representatives of both groups of proteins as well as to revealing
interrelations between proteins of different groups. Proteases occupy a key position among he
functionally active proteins of the second group since they not only perform proteolysis of the
seed storage proteins, which are the main source of nitrogen for the growing embryo, but play
an important role in transpon, secretion, morphogenesis, differentiation and apoptosis
(Vierstra 1993; Pennell and Lamb 1997). Proteolytic enzymes with limited substrate
specificity are the most interesting for investigation from the point of participation in
regulatory processes. Most often these proteinases by limited proteolysis provide
transformation of non-active preCursors into active enzymes as well as modification and
inactivation of macromolecules, participate in cell growth and division, plant seed maturation
and germination (Lokshina 1994; Dalbey et al. 1997).
The purpose of our work was the study of proteolytic enzymes of buckwheat
(Fagopyrum esculentum Moench) seeds and elucidation of their possible role in seed
metabolism (identification of their natural substrates, mechanism of action and regulation of
activity. Among proteases, which we isolated from dry and germinating buckwheat seeds two
enzymes, which belonged to different classes but possessed limited substrate specificity, were
found. Their propenies and possible functional roles are described in the present paper.
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Materials and Methods
Buckwheat Fagopyrum escu[entum seeds cv. Shatilovskaya-5 were used. Purification
of BAPAase, proteinase hydrolyzing BAPA, was carried out by acetone fractionation, ionexchange
chromatography on DEAE-Sephadex A-50, gel-filtration on Sephadex G-150 and
preparative PAGE (pH 8.3). Metalloproteinase was isolated as described previously
(Belozersky et al. 1990), using gel-filtration on Sepharose 6B, ion-exchange chromatography
on DEAE-Toyopearl, gel-filtration on Ultra-gel AcA 54 and two FPLC procedures on Mono S
at pH 5.8 and 8.15 The metalloproteinase inhibitor was isolated by affinity chromatography
on trypsin-Sepharose 4B and ion-exchange chromatography on DEAE-cellulose
(Voskoboynikova et al. 1990).
Proteolytic activity towards BAPA and N-benzoyl-DL-Iysine p-nitroanilide (BLPA) was
determined spectrophotometrically at 410 om in 0.1 M phosphate, pH 8.0 at 37°C (Erlanger et
al. 1961). Activity on protein substrates was determined by trinitrophenylation (Habeeb 1966)
in 0.01 M phosphate, pH 8.0 at 37°C after incubation of the enzymes with substrates for 18 h.
Results and Discussion
Protease, hydrolyzing well synthetic substrate N-benzoyl-DL-arginine-p-nitroanilide
and named BAPAase, proved to be the most typical representative of enzymes with limited
specificity. It was purified to homogeneity from dry buckwheat seeds what was evidence by
PAGE analysis in the absence and presence of SDS, isoelectric focussing (pH gradient 4-6)
and analytical untracentrifugation. Main characteristics of the purified enzyme are presented
in Table 1.
Table 1.
Properties of proteases of limited specificity from buckwheat seeds
Enzyme Source M r (kD) pH-optimum pi S20,w
BAPAase dry seeds 65 (g-f) 9.0 4.5 4.3
70 (PAGE)
Metalloproteinase dry seeds 39 (g-f) 8.2 > 9.6 n.d.
34 (PAGE)
*g-f - gel-filtration
Molecular mass of the BAPAase was 65 kD according to gel-filtration on Sephadex G­
100 and 70 kD according to SOS-PAGE analysis. These data indicate that buckwheat seed
BAPAase is a single chain enzyme. The isoelectric point of BAPAase was 4.5 and its pHoptimum
was in the alkaline region of pH 9.0.
Investigation of the functional groups at the active centre of the enzyme revealed that
BAPAase is a serine proteinase with serine and histidine residues at the active centre (Table 2).
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Table 2.
Effect of inhibitors and activators on BAPAase and metalloproteinase activities
Residual activity (%)
Concentration
Metal10-
Reagent (nM) BAPAase proteinase
DiisopropyIfluorophosphate 0.1 0 100
Tosyl-L-lysinechloromethyl ketone 0.1 0 n.d.
Tosyl-L-phenylalaninechloromethyl ketone 0.1 100 n.d.
p-Chloromercuribenzoate 1.0 148 80
NaCI 170 100 n.d.
N-ethylmaleimide 1.0 72 n.d.
EDTA 3.0 100 0
Trasilol 0.01 98 n.d.
Soybean trypsin inhibitor 0.1% 100 n.d.
1.1O-phenantroline 1.0 n.d. 0
Zincon 1.0 n.d. 0
Pepstatin 0.16 100 100
It was surprising that BAPAase was activated with p-chloromercuribenzoate (to 50%)
and this activation was not caused by Cl- ions.
The study of the substrate specificity of the BAPAase showed that this enzyme
hydrolyzed peptide, amide and ester bonds, formed by carboxyl groups of arginine and lysine
when N- and C- terminal groups of amino acid residue in the used substrates were blocked.
BAPAase showed a higher affinity to arginine residues than to lysine. When the same
concentrations of BAPA and BLPA were used the initial rate of hydrolysis of the first substrate
was 2.5-fold higher than of the second. Buckwheat seed BAPAase did not hydrolyse albumins
and globulins from these seeds as well as a number of other proteins, usually used as
proteinase substrates, weakly hydrolyzeP histones and good - protarnins. Analysis of peptide
bonds, cleaved by the enzyme irr the oxidized insulin B-chain, carried out by dansil technique
indicated that only a single bond Arg-Gly was cleaved (Fig. 1).
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BAPAase
5 10 15 20 25 30
F-V-N-Q-H-L-C-G-S-H-L-V-E-A-L-Y-L-V-C-G-E-R-G-F-F-Y-T-P-K-A
I
S03H
Metalloproteinase 1 1
I
S03H
Fig. 1 Cleavage sites of insulin B-chain by buckwheat seed BAPAase and
metalloproteinase
In its action on synthetic substrates and insulin B-chain buckwheat seed BAPAase is
very similar to trypsin. However, in contrast to trypsin it was not inhibited by trasilol (trypsin
inhibitor from calf lungs) and soybean trypsin inhibitor (Table 2).
Thus, the obtained data on the effect of buckwheat seed BAPAase on protein substrates
indicate that this enzyme is a proteinase with limited substrate specificity and, possibly, may
have its specific substrate. As a possible substrate might serve an arginine-rich protein,
determined histochemically in seeds of several plants (Fulcher et al. 1972). On the other
hand, one cannot exclude that BAPAase may carry out limited proteolysis of few (one-two)
key peptide bonds in proteins what is rather difficulty to determine by usual techniques.
Another proteinase with narrow specificity and capable of limited proteolysis was also
found in dry buckwheat seeds but belonged to a different class of proteolytic enzymes. It was
purified 1000-fold, obtained homogeneous and was completely inhibited only by
metalloenzymes inhibitors such as EDTA and 1,10-phenantroline (Table 2). The isolated
enzyme was classified as metalloproteinase according to atomic-absorption spectroscopy which
showed that the studied proteinase contained one Zn 2 + ion per enzyme molecule. Inhibition of
the enzyme with EDTA did not result in removal of the zinc ion from the active centre.
Activity of the enzyme, inactivated with EDTA, was completely restored after addition of
Zn2+, eo2+ or Mg 2 + ions. At the same time treatment of the active enzyme preparation with
additional amount of these cations as well as Ca2+ ions did not activate it more. According to
these data one may suggest that Mg2+, Co2+ and Zn 2 + activate inactive metalloproteinase
competing with Zn2+ at the active centre of the enzyme for binding EDTA. The main
characteristics of the studied metalloproteinase are given in Table 1. Its molecular mass was
34 kD according to SDS PAGE-analysis and 39 kD according to gel-filtration on Sephadex G­
150. These data indicate that metalloproteinase is a single chain enzyme. The pH-optimum of
the enzyme towards buckwheat seed 13S globulin was 8.0-8.2 and isoelectric point was higher
9.6.
To study the effect of metalloproteinase on protein substrates typical seed storage
proteins (buckwheat seed 13S globulin, .soybean glycinin) were used as well as exogenous
proteins (BSA, casein, hemoglobin), which are often used as the substrates for determination
of proteolytic activity (Table 3).
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Substrate
BSA
Hemoglobin
Soybean lIS globulin
Buckwheat 13S globulin
Table 3.
Effect of metalloproteinase on protein substrates.
Amino groups released, Bonds hydrolyzed,
nmol.mg- 1 %.mol- 1
10
15
85
135
0.12
0.16
1.00
1.50
Measurement of the amino groups liberated after hydrolysis indicated that
metalloproteinase expressed weak activity towards the used substrates. Calculations showed
that degree of hydrolysis of 13S globulin and glycinin was approximately similar. The enzyme
split 30 peptide bonds (1 %) per glycinin molecule and 36 bonds (1.5% per 13S globulin
molecule. Detailed analysis of hydrolysis by metalloproteinase of endogenous buckwheat seed
13S globulin revealed that is predominantly cleaved high molecular mass subunits (57.5, 47.6
and 44.7 kD) of this protein. Data on the quantitative analysis of 13S globulin hydrolysis and
the study of electrophoretograms of the hydrolysis products indicate that the studied
metalloproteinase performs limited proteolysis of the storage protein. As it was demonstrated
in our further experiments only the storage protein, modified by metalloproteinase through
limited proteolysis, could be later subjected to profound proteolysis by other less specific
buckwheat seed proteases.
Hydrolysis of the oxidized insulin B-chain, carried out to reveal the peptide bonds
cleaved by metalloproteinase, and followed by separation of hydrolysis products by reverse·
phase HPLC and their amino acid analysis, indicated that three peptide bonds Ala I4 _Leu 1s ,
Leu lS _Tyr 16 and Phe 24·Phe25 (Fig. 1) were split. Comparison of the peptide bonds, hydrolyzed
by metalloproteinase in the oxidized insulin B-chain, with the products of its hydrolysis by
some bacterial and animal metalloproteinases showed that the studied buckwheat proteinase
possessed the most narrow substrate specificity. It hydrolyzed mainly only three peptide bonds
whereas other metalloproteinases hydrolyzed from 6 to 14 bonds.
We have found that activity of the investigated metalloproteinase was controlled by
endogenous protein inhibitor. Highly purified inhibitor had molecular mass 10-12 kD and
isoelectric point 4.8. It was acid and thermostable what may be, possibly, due to the tighmess
of its globular structure and is a characteristic property of almost all protein protease inhibitors
of plant origin. Complete inactivation of metalloproteinase was observed in its presence
expressed in the loss of ability of the enzyme to cleave high molecular mass subunits of 13S
globulin. The presence of the metalloproteinase inhibitor in dry seeds together with the
narrow specificity of metalloproteinase indicated that proteolysis of the main buckwheat seed
storage protein was a precisely regulated process, playing an important role in the effective
supply of the growing embryo with nitrogen compounds.
Thus, dry buckwheat seeds contain at least two highly specific proteinases, which
belong to different classes ofproteolytic enzymes and performing, apparently, different
functions. The existence of enzymes of this kind gives living organisms a possibility to
regulate precisely vitally imponant metabolic processes.
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Acknowledgements
The work was supponed by grants from the Russian Foundation for Basic Research and
State Program "International Projects".
References
1. Belozersky M.A., Dunaevsky Y.E., Voskoboynikova N.E. (1990). Biochern. J. 272,
677-682.
2. Dalbey R.E., Lively M.O., Bron S., Van Dijl J.M. (1997). Protein Science 6,1129­
1138.
3. Erlanger B.F., Kokowsky N., Cohen W. (1961). Arch. Biochem. Biophys. 95,271­
278.
4. Fulcher RG., O'Brien T.P., Simmonds D.H. (1972). Austral. J. BioI. Sci. 25,487­
492.
5. Habeeb T.S. (1966). Anal. Biochern. 14, 328-336.
6. Lokshina L.A. (1994). Bioorgan. Chern. 20, 134-142.
7. Pennell R 1., Lamb C. (1997). Plant Cell 9, 1157-1168.
8. Vierstra RD. (1993). Ann. Rev. Plant PhysioI. Plant Mol. BioI. 44, 385-410.
9. Voskoboynikova, N.E., Dunaevsky Y.E., Belozersky M.A. (1990). Biochemistry 55,
839-847.
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