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INTRODUCTION
FOR understanding the regulatory mechanism of protein metabolism in
various tissues, both synthesis and degradation of proteins in a given tissue
are equally important. Our knowledge of the mechanism of protein synthesis
is now fairly extensive, but information on the mechanism of intracellular
protein degradation is rather scarce. The possibility of controlling enzyme
levels by lysosomal protease has been suggested by Coffey and de Duve (l),
but it is rather difficult to explain the specific degradation of individual enzyme
by lysosomal enzymes alone. The possibility of controlling enzyme levels by
changing the degradation rate has been brought out by the work of Schimke
et al. on the effects of altered nutritional states on rat liver arginase (2). Segal
also emphasized the importance of degradation rate for the control of the
enzyme levels in the cell (3).
We discovered three new enzymes which specifically inactivate pyridoxal
enzymes or NAD-dependent enzymes or FAD-dependent enzymes in their
apo-forms in small intestine, liver and muscle of rats. Each inactivating enzyme
does not act on the other types of coenzyme-dependent enzymes or any other
proteins such as albumin and casein. For instance, apo-enzymes of serine
dehydratase, tyrosine transaminase and ornithine transaminase were inacti-
vated by the purified inactivating enzyme for pyridoxal enzymes, but NAD-
dependent enzymes or FAD-dependent enzymes were not inactivated by the
pyridoxal-specific inactivating enzyme. The recently discovered inactivating
enzymes spilt each apo-enzyme to a homogeneous smaller protein and oligo-
peptides in weakly alkaline condition. These inactivating enzymes are
sort of specific endopeptidases. The activities of these inactivating enzymes
increase under the condition of the respective vitamin-deficiency, but the
activity of inactivating enzyme for NAD- or FAD-dependent enzyme is
289
290 N. KATUNUMAet al.
not induced under the B6-deficient condition. The large part of these inacti-
vating enzymes may exist as latent enzymes of inactive forms by binding with
a specific inhibitor under normal dietary conditions. The amount of the inhi-
bitor decreases in vitamin deficiency. These three inactivating enzymes are
different enzymes on many experimental grounds.
We may postulate that these inactivating enzymes serve as the initiator of
enzyme degradation within the cell, and the products formed by the inactivat-
ing enzymes are subsequently degraded into amino acids by lysosomal or
other non-specific proteinases. It is possible that these enzymes play very
important roles in the regulation of degradation of coenzyme-dependent
enzymes.
Protein Determination
Protein concentrations were determined by the method of Folin-Lowry
with crystalline bovine serum albumin as standard (5).
Enzymes as Substrate
In the present study, OTA, serine dehydratase, aspartate transaminase and
tyrosine transaminase were used as substrates of the inactivating enzyme for
pyridoxal enzymes. Glutamic dehydrogenase, lactic dehydrogenase, isocitrate
dehydrogenase and malie dehydrogenase were used as substrates of the
inactivating enzyme for NAD-dependent enzymes. Phosphate independent
glutaminase and urease were also tested as substrates. All of the pyridoxal
enzymes were purified from rat liver. Purification and resolution of PALP
from their enzymes were carried out according to the method of Matsuzawa
et al. (6), Nakagawa et al. (7), and Tomkins et al. (8). Crystalline OTA,
partially purified serine dehydratase or tyrosine transaminase were used as
substrates in the present experiments. Crystalline G D H from bovine liver,
lactic dehydrogenase from rabbit muscle and malic dehydrogenase from pig
heart muscle were obtained commercially. Activity of their dehydrogenases
was assayed at 25C by measuring the rate of the decrease in absorbance of
NADHz at 340 m/~ (9-11). Crystallization of phosphate independent gluta-
minase from rat kidney and the assay of its activity were according to the
method of Katunuma et aL (12).
RESULTS
Total Specific
Volume Protein activity activity Recovery
Fraction (ml) (nag) (units) (units/mg) (%)
3rude extract 420 4410 76,000 17
Mnmoniumsulfate 75 1125 61,000 54 80.5
~.cetone 25 250 37,000 148 48.7
DEAE-cellulose 2.5 37 27,500 680 35.2
~ephadex G-100 0.9 1.9 9540 5050 12.5
S20w 9.9
[
........J L_____J {
...........J \__J
Ornithine Transaminase
36min 45rain
S20w 5. I
Product
36min 45min
FIG. 2. Sedimentation pattern of apo-OTA (upper) and its product split by the inactivating
enzyme (lower).
MODE OF ACTION OF SPECIFIC INACTIVATING ENZYMES 295
TABLE 2, SUBSTRATE SPECIFICITY OF THE INACTIVATING ENZYME
FOR PYRIDOXALENZYMES
apo-forms and PAMP forms to various degrees, but none of the non-pyridoxal
enzymes tested were affected by the inactivating enzyme, as shown in Table 2.
Michaelis constant of the inactivating enzyme for apo-OTA as substrate
was calculated from the double reciprocal plots. Since the molecular weight
of OTA was 132,000 (13), K,, for OTA was estimated to be 1.5 I0 -s M
(Fig. 3). The inactivating enzyme has quite a small K,, for OTA as substrate.
This observation suggests that the inactivating enzyme plays important roles
on catabolism of pyridoxal enzymes in vivo.
10-3/units
I00 l ! I !
200--
v
o
o
50-- 100--
~.
o I [ d
I I I
64 320 0 20 40 60
(Apo OTA) (units/tube) 1/S ( 1/apo OTA ) x 10-3/units
Optimal pH of the inactivating enzyme was studied in both crude and purified
preparations and was observed at 9.0 in these preparations. Effect of vitamin
B6 derivatives and other related compounds to activity of the inactivating
296 N. KATUNUMA et al.
Incubation
concentration
Addition (M) E n z y m e activity 70 of p r o t e c t i o n
None m 4.00 0
PAMP 2.5 10-* 3.81 5.0
2.5 x 10 - a 3.91 2.0
PINP 2.5 10 -4 3.74 6.4
2.5 x 10 -3 3.82 4.4
NAD 2.5 10 -4 4.00 0
2.5 10 - a 4.01 0
FAD 2.5 10 - 4 3.96 1.1
2.5 10 -3 4.01 0
ATP 2.5 10 - 4 4.00 0
2.5 x 10 -3 3.95 1.3
PALP 2.5 10 -5 0.65 83.7
PAL 2.5 10 -4 1.35 66.2
100 I !
~ PALP
PAL
50 -- / ss S'~D'~
/2""
0 ~-tt i I
P A L P (M) 1 x 10 . 5 2 x 10 . 5 3 x 10 .5
I I I
PAL (M) 2 x 10 .4 4 x 10 .4 6 x 10 . 4
CONCENTRATIONS of P A L P or pAI,
enzyme was examined and the results were shown in Table 3 and Fig. 4. The
inactivation was protected markedly by the addition of pyddoxal phosphate
and weakly by pyridoxal. N o protective effect was observed by addition o f
pyridoxamine phosphate, pyridoxine phosphate and other compounds tested
on the inactivating enzyme reaction. Concentration of P A L P or P A L required
for 50 ~ prevention of the inactivating enzyme was calculated as 6.0 x
10 -6 M or 1.6 10 -4 M. It is estimated from the report of Peraino (13) that
58 lysine residues were contained in one molecule of OTA, and only two
lysine residues of them formed Schiff base with pyridoxal phosphate. The
special two lysine residues must be kept free for the inactivation reaction. It
is assumed from the above data that 30 times more P A L than P A L P was
required to protect against the inactivating reaction.
Tissue distribution of the inactivating enzyme in the B6-deficient rat is shown
in Table 4.
Rats were fed on laboratory chow ad libitum before the experiment. Values are expressed
as means i standard errors.
298 N. KATUNUMAet al.
The activity was detected strongly in small intestine and weakly in skeletal
muscle, but no activity was detected in other organs tested. The activities of
the inactivating enzymes increase under the condition of Bt-deficiency, but
the activity of the inactivating enzyme does not increase under the niacin
deficiency or B2-deficiency as shown in Table 5. Furthermore, the activity of
inactivating enzyme for NAD-dependent enzyme is not induced under the
B6-deficient condition.
Total Specific
Volume Protein activity activity Recovery
(ml) (mg) (units) (units/mg) (%)
Erudeextract 370 3700 238 0.0645 100
Ammonium sulfate 105 714 735 1.05 308.8
Acetone 26 161 565 3.50 237.4
~ephadexG-100 0.7 2.1 133.5 64.0 56.1
I I I I
oo
I I I I
Q 10 20 30 40
o
I ~q
I I I I
0 I0 20 30 40 50
FRACTION NUMBER
FIG. 5. Chromatography of the reaction products containing glutamic dehydrogenase and
the inactivating enzyme on Sephadex G-100. Sephadex G-100 column (1.8 55 cm) was
equilibrated with 0.05 M sodium phosphate buffer, pH 7.4. The flow rate was adjusted to
12.0 ml]hr. Ten mg of glutamic dehydrogenase were incubated with 0.5 mg of the purified
inactivating enzyme (specific activity 64.0) for 2 hr. After the reaction was stopped by
standing at 0C, the reaction was terminated at 0 time, then reaction mixture was applied
to the chromatography (A).
onto Sephadex G-100 and the elution pattern was as shown in Fig. 5 (B). The
first peak of the solid line indicates the remaining GDH and the second peak
of the solid line shows one product protein from GDH by the enzyme
reaction. No GDH activity was observed in the second peak and its molecular
weight is obviously smaller than the intact GDH but is still acid insoluble.
The inactivating enzyme itself appears in the third peak indicated by the solid
line and the fourth peak seems to be oligopeptides, derived from the degrada-
tion reaction. The pattern of column chromatography shown in Fig. 5 (A) is
that in the case of zero time incubation. All the GDH activity added was
recovered in the first peak and no other protein or peptides were detected.
We have studied the substrate specificity on the purified inactivating enzyme
and the inactivating enzyme specifically inactivates malic dehydrogenase
(MDH), GDH and lactic dehydrogenase in their apo-forms to various degrees,
but all the non-NAD dependent enzymes tested were unaffected by the in-
activating enzyme, as shown in Table 7. The inactivation of apo-NAD-
dependent enzymes by the inactivating enzyme is inhibited by the addition of
300 N. KATUNUMA et aL
NAD-dependent Non-NAD-dependent
enzymes % enzymes %
Malic dehydrogenase Urease
Glutamic dehydrogenase Glutaminase
Lactic dehydrogenase (phosphate independent)
Isocitric dehydrogenase Ornithine transaminase
(NADP) D-Amino acid oxidase
Albumin
0 )"
I I I I o
0 5 10 15 ZO
T I M E (Day)
FIG. 6. Relationship between the appearance of the inactivating enzyme activity and the
decrease of L D H activity in small intestine during niacin deficiency. Male rats of Wistar
strain, weighing 180-200 g, were fed on niacin-deficient diet for the indicated period.
Acetyl pyridine was also injected daily by intraperitoneal route. The activity of LDH and of
inactivating enzyme in small intestine was assayed. Each point represents the mean of 2--4
rats.
MODE OF ACTION OF SPECIFIC INACTIVATING ENZYMES 301
I I I I I I
i00 m
z
C.)
~ 50--
Z
0
2;
I I I I I I
0 1 2 3 4 5 6
CONCENTRATION OF INHIBITOR (rag/tube)
Fro. 7. Inhibition of the inactivating enzyme for NAD-dependent enzyme by the protein
nature inhibitor. Effect of inhibitor concentration on enzymatic activity. The reaction
mixture contained in 0.5 ml" 0.05 M potassium phosphate buffer pH 7.5, 0.2 mg of glutamic
dehydrogenase, 3 mg of inactivating enzyme and inhibitor as indicated.
ammonium sulfate and is stable to heat at 80C for 5 min, while the
inactivating enzyme for NAD-dependent enzymes precipitate in the 35-60 ~o
ammonium sulfate fraction. The inhibitor is purified by passing through
Sephadex G-100, and this is detected just after the inactivating enzyme on
the chromatography. The mode of inhibition of this inhibitor on the inacti-
vating enzyme is shown with crude inhibitor solution in Fig. 7. After a
preincubation of 30 min in presence of the diverse amounts of inhibitor
indicated, the constant amount of the inactivating enzyme and the con-
stant amount of G D H as substrate were added. N o inhibitor was found
in the case of niacin-deficient rats; however, various amounts of the in-
hibitor were detected in normal rats as shown in Fig. 8 (A). In the content
of the inactivating enzyme in the small intestine, on the other hand, a
reciprocal movement could be recognized. The appearance of the inactivating
enzyme activity was observed in small intestine and liver in niacin deficiency.
The largest parts of these inactivating enzymes exist as latent enzyme in
inactive forms by binding with the specific inhibitor protein in normal rats.
In order to demonstrate this mechanism more directly, the following experi-
ment was performed as shown in Fig. 9. Considerable amounts of the
inactivating enzyme activity appeared from small intestine of normal rats by
302 N. KATUNUMAet al.
1.1 --
3.5 m
0
1.0-- o
. 3.0 m 0.9--
0.8--
2.5~
e~
0.7--
>, 2 . 0 - 0.6--
0.5--
c9 1.5-- N
0
0.4--
(9
1.0-- o 0.3--
(9
0.2--
0.5--
0.I--
am. 0
"
Niacin Normal Niacin Normal
Deficiency Deficiency
ACTIVITY OF THE CONCENTRATIONS OF
INACTIVATING ENZYME INHIBITOR
FIo. 8. Reciprocal relationship between the activities of inactivating enzyme and inhibitor.
The levels of the inactivating enzyme activity for NAD-dependent enzymes in niacin-
deficient and normal rats (A). The levels of inhibitor in niacin-deficient and normal rats (B).
DISCUSSION
We will discuss one of the products derived from ornithine transaminase as
the substrate after the enzymatic degradation reaction in vitro. This product,
oligopeptide, showed the following amino acid composition obtained from
duplicate preliminary analyses (Lys, Asp, Thr, Ser, Gluz, Gly, Arg, Leu). It
might involve very important control mechanisms that PALP form enzyme is
unable to split by the inactivating enzyme but PAMP form enzyme is easily
split by the inactivating enzyme although both forms are freely interconver-
tible during the transaminase reaction. When the concentration of amino
acids in a given tissue increases by feeding high protein diet, the relative
amount of PAMP form enzyme may increase. The extent of decrease of
MODE OF ACTION OF SPECIFIC INACTIVATING ENZYMES 303
1.0
o
t-a.
b~
J
0.8~
0.6~
b~
~ 0o4~
Casein diet
Ornithine transaminase 23 70 60 70
Aspartate transaminase(s) 3070 7470
Alanine transaminase(s) 4700 2570
I ,. =,,= T= TFo==
It
I X // II (Aminoacid) ~ J ~] f non-specffic~
NH2
Amino
acid fnon-specific.]1
Lprteinase
~ 7 b~ ~ I,,f X2(n~
/ \ NAD l ~ ~ "4" Amino
acids
ACTIVEFORM
TT
NAO.,
SUMMARY
We discovered two kinds of new enzymes which specifically inactivate
pyridoxal enzymes or NAD-dependent enzymes in their apo-forms in small
intestine, liver and muscle of rats. These inactivating enzymes were purified
until almost homogeneous protein and some of their enzyme-chemical pro-
perties were reported. Each purified inactivating enzyme does not act on the
other types of coenzyme-dependent enzymes or on any other proteins tested.
These inactivating enzymes in alkaline condition split each apo-enzyme as
substrate to homogeneous smaller protein and oligopeptide. The inactivation
by these enzymes is protected by the addition of each respective coenzyme.
The activities of these inactivating enzymes increase under the condition of
the respective vitamin deficiency. The large part of these inactivating enzymes
exists as latent enzyme of inactive forms by binding with the specific inhibitor
in normal dietary condition. The amount of the inhibitor decreases in vitamin-
deficient condition. These inactivating enzymes might play very important
roles in the control o f degradation of coenzyme-dependent enzymes.
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306 N. KATUNUMA et al.
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(1971).