Download Acid Carboxypeptidases: Their Occurrence in Plants, Intracellular

p-Hydroxy-DL-phenylglycin 16 , farblose Nadeln aus
n-Propanol/Wasser 3 : 1 , Schmp. 215 —216 °C,
Zers., 46 mg ( = 55% d. Theorie).
3.5-Dihydroxy-DL-phenylglycin, farblose Nadeln
aus n-Propanol/Wasser 3 : 1 , Sdimp. 232 — 234 °C,
Zers., 23 mg ( = 25,1% d. Theorie).
Elementaranalyse: C 8 H 9 N0 4
Gef. C 52,24 H 5,11
Ber. C 52,45 H 4,92
N 7,89,
N 7,65.
Herrn Prof. Dr. K. MOTHES danken wir für ständig
anregende Diskussionen.
Acid Carboxypeptidases: Their Occurrence in Plants, Intracellular Distribution
and Possible Function
H.
ZUBER
Department of Molecular Biology
and
PH.
MATILE
Department of General Botany
Swiss Federal Institute of Technology Zurich, Switzerland
(Z. Naturforsch. 23 b, 663—665 [1968] ; eingegangen am 17. November 1967)
Saure Carhoxypeptidase, welche Phenylalanin von CbO-Leu-Phe-OH bei pH 5,3 abspaltet, ist ein
in höheren Angiospermen weit verbreitetes Enzym. Es scheint dagegen in primitiven Angiospermen
(Fagus und Magnolia) sowie in Gymnospermen und Kryptogamen (Ausnahme: Equisetum) zu
fehlen. Bei Zitrone ist die Aktivität saurer Carboxypeptidase von Natur und Alter der extrahierten
Gewebe abhängig (Exocarp und Endocarp der Früchte, Blätter). In Zellen der Wurzelspitzen von
Maiskeimlingen ist das Enzym in meristematischen Vakuolen lokalisiert. Funktionell ist die saure
Carboxypeptidase als lysosomales Enzym, welches am intrazellulären Proteinabbau teilnimmt, anzusprechen.
1
has purified and characterized a carboxypeptidase extracted from the exocarp of citrus fruit.
A study of the occurrence of similar enzymes in
various organisms of the plant kingdom has now
yielded evidence that carboxypeptidases with a pH
optimum around 5 are common constituents of
many angiospermous plants (Table 1 ) . It is unlikely that the carboxypeptidases which occur in
different plant species are identical with respect to
structure, activity and specificity; rather would it
seem that they belong to a family of enzymes, subsequently termed acid carboxypeptidases,
which
hydrolyze typical substrates at a pH of 5.3.
With two exceptions all of the angiospermous
plants tested so far contain acid carboxypeptidase
activity: from the point of view of phylogenetic classification, it should be noted that Fagus and Magnolia, which lade this enzyme, belong to the primitive
orders of Fagales and Polycarpicae. CarboxypeptiZUBER
1
H. ZUBER, Nature [London] 201, 613 [1964].
2
F . FELIX and J . LABOUESSE-MERCOUROFF, Biochim. b i o p h y s i c a
Acta [Amsterdam] 21, 303 [1956]; F.FELIX and N.BRUILLET
122,
127
[1966];
139, 382 [1967],
K . MORIHARA,
T.OKA,
and
H . TSUZUKI
dase is also absent in extracts from needles of the
gymnosperm Picea. Of some Cryptogams tested the
horse-tail Equisetum contains acid carboxypeptidase;
considering the phylogenetic situation of the Equisetales, it appears that acid carboxypeptidases may
have originated independently at the evolutionary
levels of the Pteridophyta (class of Equisetales
which has not passed through further development)
and of the Angiosperms. Carboxypeptidase activity,
finally, is present in the thermophilic fungi Talaromyces duponti (Ascomycetes) Humicula lanuginosa
(Deuteromycetes); these enzymes, however, belong
to a different family of alkaline carboxypeptidases
since they are optimally active at pH 8 — 9. Similar
enzymes have recently been isolated from yeast and
Streptomyces
Fradiae 2 .
The total carboxypeptidase activity which is extractable from a certain mass of plant material
seems to be dependent both on the species of plant
and on the kind of tissue used. It appears from the
data listed in Table 1 that a very high yield was obtained by extracting cauliflower buds, a tissue which
consists almost exclusively of metabolically very
active meristematic cells.
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Plant
Tissue
Yield
Units per 100 g
of fresh weight
C r values
Acid carboxypeptidase
Artichoke (Cynara scolymus)
Tomato (Solanum lycopersicum)
Orange (Citrus aurantiacum)
Pea (Pisum sativum)
Cauliflower (Brassica oleracea)
Spinach (Spinacia oleracea)
Nettle (Urtica dioica)
Corn (Zea mais)
Beech (Fagus silvatica)
Magnolia spec.
Spruce (Picea excelsa)
Horse-tail (Equisetum arvense)
Fern (Athyrium filix-femia)
Talaromyces du ponti
Bracts
Fruit
Fruit, Exocarp
Seedling
Flowerbuds
Leaf
Leaf
Seedling
Leaf
Leaf
Needle
Shoot
Sporophyll
Mycelium
1 619
150
600
400
11 692
4 761
300
2 090
0
0
0
114
0
0
0.020
0.023
0.040
0.0004
0.042
0.016
0.005
0.002
0
0
0
0.012
0
0
Alkaline
carboxypeptidase
Talaromyces du ponti
Mycelium
17 300
0.143
Table 1. Occurrence of carboxypeptidases in plants.
The tissues were extracted with 2.3% NaCl as described previously Precipitation of the protein with (NH 4 ) 2 S0 4 (70%
saturation) yielded a crude enzyme product which was
dialyzed against water and lyophilized. The determination of
the carboxypeptidase activity present in 1 mg of crude product was based on the hydrolysis of CbO-Leucyl-phenyl-
alanine-OH (2 x 1 0 ~ 2 M substrate in 0.1 M Na-citrate-buffer
pH 5.3; production of phenylalanine in 10, 30, 60 and
120 min of incubation at 37 °C). Q-values (proteolytic
coefficient) represent the quotient of kx (velocity constant of
first order reaction) and E (concentration of protein in mg
protein-N). (1 u n i t = C t x /ug protein-N; 15% N.)
In lemon the exocarp of very young fruits contains twice as much carboxypeptidase than the same
tissue of mature fruits (Table 2). Thus the carboxypeptidase content seems to be related to the metabolic activity of cells. In citrus not only the fruit but
experiments had shown that approximately onethird of the total activity present in an extract from
corn seedlings (grown under sterile conditions at
28 °C for 40 hrs) is sedimentable. An even higher
proportion of the enzyme is structurally bound in
extracts from root tips (approx. 5 mm long), prepared by grinding the tissue in the presence of sand
and a medium containing 0.5 M sorbitol, 0.05 M
tris-HCl-buffer pH 7.6 and 1 mM EDTA. Differential
centrifugation of the low-speed supernatant of a root
tip extract yielded a mitochondrial (15 min
20,000 g ) , a microsomal (30 min 150,000 g) and
a soluble fraction. Carboxypeptidase activity is present in all of these subcellular fractions; thus, the
enzyme appears to be both soluble and structurally
bound (Table 3 ) . The highest specific activity is present in the mitochondrial fraction which, according
to a previous study 3 contains particles carrying a
considerable number of lysosomal enzymes. Several
types of lysosomes can be distinguished; one of them
is identical with small vacuoles which, in contrast to
large vacuoles, are not destroyed when the tissue is
ground. These vacuoles are not sedimentable in
media which contain even low concentrations of
Tissue
Size of fruit
Exocarp
Endocarp
Exocarp
Exocarp
2— 4 cm
2— 4 cm
6 cm
10 — 15 cm
Leaf
Leaf
Age
1 year
2 years
Colour
of exocarp
dark-green
—
yellowish-green
yellow
Yield Units C1-values
per 100 g
3 419
709
2 787
1 606
0.066
0.066
0.100
0.093
8 666
12 329
0.130
0.093
Table 2. Carboxypeptidase activities in various tissues of
lemon. Experimental conditions are the same as indicated in
Table 1.
also the leaf contains the enzyme: in contrast to the
above findings, more activity is present in extracts
from old leaves. Of all the tissues tested so far the
crude enzyme product from old lemon leaves contained the highest carboxypeptidase activity.
In order to elucidate the functional significance
of the acid carboxypeptidase its intracellular distribution was studied in a suitable object. Preliminary
3
PH. MATILE, Planta 79, 181 [1968].
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Fraction
Specific activity
Cell-free extract
Mitochondrial fraction
Microsomal fraction
Soluble fraction
Light lysosomes (Meristematic vacuoles)
Heavy lysosomes
2.221
1.415
0.641
1.751
11.55
6.10
Table 3. Carboxypeptidase activity in subcellular fractions
from root tips of corn seedlings. The activities are expressed
as /u.M phenylalanine produced in 60 min per mg of protein.
surcrose; the other types of lysosomes, however,
sediment ealisy in 0.4 M sucrose. Hence, two classes
of lysosomes can be obtained from a mitochondrial
fraction which is overlayered on a discontinuous
gradient with steps of 0.4 and 1.17 M sucrose; upon
centrifugation (2 hrs at 39,000 rpm in a Spinco
SW 39 rotor) the mitochondria form a pellet, whilst
heavy and light lysosomes are trapped at the surface
of 1.17 and 0.4 M sucrose, respectively. The data
in Table 3 show that carboxypeptidase is present in
both types of lysosomes. The highest specific acti­
vity is contained in the light lysosomes — that is,
in meristematic vacuoles.
According to early observations of cytologists4
the large vacuole of expanded (differentiated) plant
cells originates from tiny vacuoles of meristematic
cells. It may therefore be assumed that the enzyme
acivity found in the soluble cell fraction is localized
in vacuoles large enough to be disrupted during
homogenization of the cells. This assumption is sup­
ported by the finding that the lysosomal enzymes
4 A. G
5
u i l l i e r m o n d , G . M a n g e n o t , and L. P l a n t e f o l , Traite de
Cytologie vegetale, Le Frangois, Paris 1933.
P h . M a t i l e and A. W ie m k e n , Arch. Mikrobiol. 56, 148
[1967].
present in preparations of isolated vacuoles of yeast
cells are completely soluble if the cells are submitted
to a procedure of homogenization which causes the
rupture of these delicate organelles5. Functionally,
the acid carboxypeptidases seem to be involved in
the intracellular digestion of protein, a process
which, together with protein synthesis, is responsible
for the phenomenon of protein turnover. From the
localization of this and many other lysosomal
enzymes in meristematic vacuoles 6 it must be con­
cluded that this organelle represents the locus of
intracellular digestion.
The acid carboxypeptidases of higher plant cells
seem to represent lysosomal enzymes analogous to
what were formarly termed the cathepsins 7 of ani­
mal cells. In contrast to their alkaline counterparts
(intestinal carboxypeptidase A and B, fungal carb­
oxypeptidase) acid carboxypeptidases seem not to
be metalloenzymes (no inhibition with E D T A ).
They seem to function intracellularly whereas the
neutral or alkaline carboxypeptidases rather re­
present secretion products with an extracellular
function. Acid and alkaline carboxypeptidases have
different active sites, but since they catalyze the
same reaction they are analogous enzymes.
The authors are indebted to D r. P. D o h rn (director
of the Zoological Station, Naples, Italy) for kindly
permitting them to use his laboratory facilities, to the
Swiss National Science Foundation (projects no. 3426
and 4393) for financial support, and to CIBA Ltd.,
Basel, for supplying the strains of fungi employed in
this present study.
6 P h . M a t i l e , Z. Naturforschg. 21b, 871 [1966].
7 J. S . F
r u t o n , G . W. I r v i n g , and
istry 141, 773 [1941] ; L. M .
237, 1082 [1962].
M. B
G
J. biol. Chem­
and R. S h e r m a n
ergm ann,
reenbaum
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