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MODE OF ACTION OF SPECIFIC

INACTIVATING ENZYMES FOR


PYRIDOXAL ENZYMES AND
NAD-DEPENDENT ENZYMES AND
THEIR BIOLOGICAL SIGNIFICANCE

N. KATUNUMA, E. KOMINAMI, S. KOMINAMI and K. KITO


Department of Enzyme Chemistry, Institute for Enzyme Research,
School of Medicine, Tokushima University, Tokushima, Japan

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.

MATERIALS AND METHODS

Vitamin B6- and Niacin-deficient Rats


Male Wistar strain rats, weighing 90-100 g, were maintained on vitamin
B6-free 20 ~o casein diet for 3-4 weeks. These rats were used as the vitamin
B6-deficient rats. Niacin-deficient rats were obtained by feeding a niacin-
free 9 ~o casein diet for 2-3 weeks. Thirty-five mg per 100 g body weight of
acetyl pyridine were injected intraperitoneally every day to 100 g Wistar
strain rats which are given the diet in order to accelerate the deficiency (4).

Materials of Inactivating Enzymes


The small intestine of B 6- o r niacin-deficient rats was homogenized with
0.05 M potassium phosphate buffer, pH 7.5; the homogenate was sonicated
at 10 kc/s for 2 min, then centrifuged for 10 min at 10,000 x g. The supernatant
was passed through a column of Sephadex G-25 equilibrated with the buffer,
and the protein fractions were collected. The combined fractions were used
as starting material for purification of the inactivating enzyme for pyridoxal
enzymes. Centrifugation at 105,000 x g for 60 min was carried out on the
supernatant centrifuged at 10,000 x g in order to prepare the inactivat-
ing enzyme for NAD-dependent enzymes. The supernatant was fractionated
with ammonium sulfate precipitation and 35-65 ~ saturation fractions were
applied to a column of Sephadex G-25 equilibrated with the same buffer, and
the protein fractions were used for the further purification.

Assay Conditions for Inactivating Enzymes


The usual assay method of the inactivating enzyme for pyridoxal enzymes
was performed as follows: reaction mixture contained in a final vol of 0.5
ml, 0.05 M Tris-HCl buffer pH 9.0, 40-60 units (0.1 mg) of ornithine trans-
aminase (OTA) and various amounts of the inactivating enzyme. After
MODE OF ACTION OF SPECIFIC INACTIVATING ENZYMES 291
incubation at 37C, the reaction was terminated by 10-fold dilution with cold
buffer. Subsequently, the remaining activity of OTA was assayed in the
presence of pyridoxal phosphate (20 /~M) after the reaction was stopped.
Determination of activity of the inactivating enzyme for NAD-dependent
enzymes was carried out by following the disappearance of glutamic dehydro-
genase (GDH) activity during incubation at 37C. The reaction mixture con-
tained the following compounds in a final vol of 0.5 ml: 0.05 M potassium
phosphate buffer, pH 7.5, 0.2 mg of G D H and various amounts of the in-
activating enzyme. Termination of the reaction was carried out by 10-fold
dilution with cold buffer. One unit of the inactivating enzyme was defined as
the amount inactivating 50 70 of substrate enzymes in 30 min. This definition
was applied to both inactivating 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).

Purification of Inactivating Enzymes


(1) Purification of the inactivating enzyme for pyridoxal enzymes. Crude
material of the inactivating enzyme described in the preceding section was
fractionated with ammonium sulfate precipitation. The 40-70 ~o saturation
precipitate was dissolved in 0.05 M potassium phosphate buffer, pH 7.5, and
292 N. KATUNUMAet al.
applied to a column of Sephadex G-25. The proteins were fractionated with
acetone. The 40-70 ~o saturated fractions were collected and dissolved in the
buffer mentioned above. Brief centrifugation was carried out to remove the
insoluble materials. The supernatant was passed through the Sephadex G-25
column equilibrated with the buffer. The protein fractions were applied to
DEAE-cellulose column (2.4 x 15 cm) which had been equilibrated with
0.05 M potassium phosphate buffer, pH 7.5. The column was washed with
200 ml of this buffer at a flow rate of 12 ml per hour. Stepwise elution was
used in order to elute the enzyme. The active enzyme eluted at 0.20 M was
collected and concentrated with 70 ~o saturation of ammonium sulfate. The
precipitate was dissolved in a small vol of 0.05 M potassium phosphate buffer,
p H 7.5, and it was applied to a column of Sephadex G-100 (2.0 x 80 cm)
equilibrated with 0.05 M potassium phosphate buffer, p H 7.5. The fractions
were pooled and stored at 4C.
(2) Purification of the inactivating enzyme for NAD-dependent enzymes.
The 50-75 ~o acetone fractionation was performed on the crude enzyme
solution as described in "preparation of inactivating enzyme". The precipitate
was dissolved in 0.05 M potassium phosphate buffer, pH 7.5, and passed
through Sephadex G-25 column equilibrated with the same buffer. The
protein fractions were applied to a column of Sephadex G-100 (2.0 x 80 cm)
equilibrated with the buffer. The enzyme fractions were collected and stored
at 4C.
Ultracentrifuge analysis of product. Ultracentrifugation was performed on
a Hitachi analytical ultracentrifuge type I. Sedimentation velocity experi-
ments were run at 60,000 rpm at 5C with single sector cells with quary
windows.
Preparation of inhibitor. Crude extract from small intestine of niacin-
deficient rats or normal rats was fractionated with ammonium sulfate precipi-
tation and the fractions of 60-75 ~o saturation were collected. These fractions
were passed through Sephadex G-25 column equilibrated with 0.05
potassium phosphate buffer, p H 7.5. The protein fractions were heated at
80C for 5 rain and the supernatant was used as the inhibitor for the inactivat-
ing enzyme for NAD-dependent enzymes.
Inhibitor studies. One unit of inhibitor was defined as the amount inhibiting
100 ~ of one unit of the inactivating enzyme in 60 min. Conversion to active
form of the inactivating enzyme from the latent form was carried out by
several times freezing-thawing in the presence of 55 ~o ammonium sulfate.
Ammonium sulfate fractionation in the presence of Triton X-100 or deoxy-
cholate is also an effective method for the purpose. The solution was centri-
fuged at 10,000 x g for 10 rain, and precipitate was dissolved in 0.05 M
potassium phosphate buffer, pH 7.5, and then passed through Sephadex G-25
column.
MODE OF A C T I O N OF SPECIFIC I N A C T I V A T I N G ENZYMES 293

RESULTS

(Part I) Inactivating Enzymefor Pyridoxal Enzymes


A summary of purification of the inactivating enzyme for pyridoxal enzymes
was shown in Table 1. A total of four steps resulted in a 300-400-fold puri-
fication with an average yield of 10 ~o. An attempt was made to purify the
inactivating enzyme of control rat using the same procedures as in the Br-
deficient rats. The overall purification produced an enzyme approximately
2000-3000 times more pure than crude homogenate. Both purified prepara-
tions showed a homogeneous band on cellulose acetate membrane electro-
phoresis at pH 8.0. Molecular weight of the inactivating enzyme is assumed

TABLE1. PURIFICATION OF THE INACTIVATING ENZYME FOR PYRIDOXAL ENZYMES


FROM SMALL INTESTINE OF B6-DEFICIENT RATS

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

to be around 30,000 by gel filtration experiment using ovalbumin and


chymotrypsin as the markers. The inactivating enzyme appears between them.
The gel filtration experiments were directed at elucidating the nature of the
products of the inactivating reaction. Crystalline apo-OTA was partially
inactivated by incubation with the purified inactivating enzyme, and sub-
sequently the reaction mixture was applied onto a column of Sephadex G-100.
The elution profile is presented in Fig. 1 (a). The first solid line indicates
remaining apo-OTA and the second peak of the solid line shows the product
protein derived from OTA by the enzyme reaction. No OTA activity is
observed in the second peak. The activity of the inactivating enzyme itself
appears in the third peak indicated by the solid line, and the fourth peak seems
to be an oligopeptide. The elution pattern shown in Fig. 1 (A) is for the case
when holo-OTA was used as substrate. All the OTA activity added was
recovered in the first peak, and no other proteins or peptides were detected.
These results indicate that the inactivation enzyme splits OTA into two pro-
ducts--that is, a homogeneous smaller protein than OTA and oligopeptide.
The inactivation reaction is one kind of specific endopeptidase. Ultracentri-
fuge analysis of the first and second peaks separated by Sephadex G-100
mmll

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

Pyridoxal enzymes % Non-pyridoxal enzymes %


Ornithine transaminase 100 Glutamic dehydrogenase
Tyrosine transaminase 21 Lactic dehydrogenase
Serine dehydratase 140 Urease
Aspartate TAase-s 22 Glutaminase
Aspartate TAase-m 85 (phosphoate independent)
Albumin

Values are the activities of each substrate enzyme expressed as percent-


ages of that of ornithine transaminase.

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

FIO. 3. Effect of increasing concentrations of apo-OTA on the activity of inactivating


enzyme for pyridoxal enzymes (left side) and its reciprocal plots (right side).

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.

TABLE 3. PROTECTIVE EFFECT OF VITAMIN B6-DERIVATIVES AND OTHER COMPOUNDS


ON THE ACTIVITY OF INACTIVATING ENZYME FOR PYRIDOXAL ENZYMES

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,

FI~. 4. Pro tective effect of P A L P or P A L on the activity of i n a c t i v a t i n g e n z y m e for p y r i d o x a l


e n z y m e s at different c o n c e n t r a t i o n s .
MODE OF ACTIONOF SPECIFICINACTIVATINGENZYMES 297

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.

TABLE4. TISSUEDISTRIBUTION OF THE INACTI-


VATING ENZYME FOR PYRIDOXAL ENZYMES IN
B6-DEFICIENT RATS

Organs Activity (units/mg protein)


Small intestine 152.1 4- 18.7
Liver Not detected
Kidney Not detected
Brain Not detected
Heart muscle Not detected
Spleen Not detected
Skeletal muscle 0.15 4- 0.02

TABLE 5. THE LEVELS OF THE INACTIVATING ENZYME ACTIVITY IN SMALL INTESTINE


UNDER THE VARIOUS DIETARY CONDITIONS

Activity of the inactivating enzyme


Diet Rats (units/ms protein)
Chow 18 0.78 -4- 0.21
5 ~ protein diet (5 days) 5 0.12 4- 0.01
70 % protein diet (5 days) 5 115.6 4- 26.5
Starvation (2 days) 3 0.63 4- 0.03
Niacin-deficient diet (2 weeks) 33 1.11
Vitamin Bz-deficient diet (4 weeks) 16 1.50
Vitamin B6-deficient diet (4 weeks) 14 152.1 4- 18.7

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.

(Part II) Inactivating Enzyme for NA D-dependent Enzymes


Almost the same procedure was applied to the purification of inactivating
enzymes for NAD-dependent enzymes as that for pyridoxal enzymes, as
shown in Table 6. A few characteristic aspects of the inactivating enzyme are
pointed out as follows: the inactivating enzymes for pyridoxal enzymes and
for NAD-dependent enzymes are able to be fractionated in different ammon-
ium sulfate concentration and their behavior on DEAE-cellulose column
chromatography is also different. The major part of the inactivating enzyme
activity for apo-NAD dependent enzymes is precipitated between 35 ~o and
55 ~ ammonium sulfate, while the inactivating enzyme for pyridoxal enzymes
is concentrated in the 45-65 ~ fraction. The most striking fact found in the

TABLE 6. PURIFICATION OF THE INACTIVATING ENZYME FOR N A D - D E P E N D E N T


ENZYMES FROM SMALL INTESTINE OF NIACIN-DEFICIENT RATS

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

course o f purification is the phenomenon that marked increases of total


activity are observed after the ammonium sulfate fractionation. This may
suggest the existence of an inhibitor in the 6 0 - 7 5 ~ ammonium sulfate
fraction, separating from the inactivating enzyme fraction of 35-60 ~ , as we
will mention later. The purified inactivating enzyme for NAD-dependent
enzymes showed an almost homogeneous pattern by cellulose acetate mem-
brane electrophoresis in acid or alkaline conditions. The gel filtration experi-
ments were carried out in the same way as in the case of the inactivating
enzyme for pyridoxal enzymes in order to elucidate the pattern of products
after the inactivation reaction. After incubation of apo-glutamic dehydro-
genase (GDH) with the inactivating enzyme, the reaction mixture was applied
MODE OF ACTION OF SPECIFIC INACTIVATING ENZYMES 299

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

TABLE 7. SUBSTRATESPECIFICITYOF THE INACTIVATINGENZYMEFOR


NAD-DEPENDENT ENZYMES

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

NAD or NADH2. A rather high concentration of NAD or NADH2 is


required to protect the reaction, namely 50 ~o protection was observed in the
presence of I mM of NAD or NADH2. The relationship between the appear-
ance of the activity of the inactivating enzyme and the duration of niacin
deficiency are presented in Fig. 6. It should be pointed out that the inactivat-
ing enzyme for pyridoxal enzymes does not increase in the niacin-deficient
condition. We found that the specific inhibitor is fractionated in 60-75

100 ...... "~ "i ' ' , 100

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).

the following treatment: 5 times repeated freezing-thawing in the presence


of 50 ~ ammonium sulfate, or ammonium sulfate precipitation in the presence
of 0.3~o deoxycholate or 0.5~ Triton X-100. From these experimental
results, the activity of the inactivating enzyme is considered to be regulated
by the amount of inhibitor which is controlled by the level of the coenzyme.

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~

To CRUDE AMMONIUM AFTER


EXTRACT SULFATE FREEZING-
FRACTIONATION THAWING
FIG. 9. Appearance of the inactivating enzyme activity for NAD-dependent enzymes from
the latent form. Homogenates of small intestine of the control rats were centrifuged at
105,000 g for 60 rain. The levels of inactivating enzyme activity of the supernatant were
described in the left column. The supernatant was fractionated with ammonium sulfate
and a precipitate of 35-5070 saturation was dissolved in 0.05 M potassium phosphate
buffer, pH 7.5, and then passed onto a column of Sephadex G-25 equilibrated with same
buffer. The activities of the protein fractions are shown in the middle column. In the right
column, the activities after the repeated freezing-thawing in the presence of ammonium
sulfate are given.

TABLE 8. DIFFERENT BEHAVIOR OF PYRIDOXAL


ENZYMES WITH 7070 AND 1070 CASEIN DIETS IN B6-
DEFICIENT CONDITION IN RAT LIVER

Casein diet

Enzyme 7070 10%

Ornithine transaminase 23 70 60 70
Aspartate transaminase(s) 3070 7470
Alanine transaminase(s) 4700 2570

The rats were fed on B6-deficient diet containing differ-


ent percentages of casein for 4 weeks before experiments.
The enzyme activities of rats fed on 7070 and 107o casein
diet with pyridoxin were taken as controls.
304 N. KATUNUMA e t al.

various pyridoxal enzyme activities in B~-deficient rats was compared in


feedings with high protein diet and with low protein diet by Okada and Ochi
(14). As shown in Table 8, marked decreases of rat liver enzymes were observed
in the case of high protein diet. We could demonstrate qualitativelythe existence
of the inactivating enzyme activity for pyridoxal enzymes also in normal rat
liver by treatment with triton. There are some experimental difficulties
because after this treatment rather strong inactivation of the inactivating
enzyme occurred in the present situation. We can assume that the inactivating
enzyme for pyridoxal enzymes also exists in the form of inactive latent
enzyme in the normal state as is seen in the case of NAD-dependent enzymes.
In general, it is well known that the intracellular level of enzymes is deter-
mined by the velocity balance between the synthesis and the degradation of
apo-enzyme protein. The rate of enzyme increase or decrease can be expressed
in the following equation:
d (E) _ K~ --/(2 (E)
dt
Understanding of protein synthesis and its regulation is now fairly extensive
and developed, but information on mechanisms and control of the degrada-
tion at the molecular level, expressed as K2 in the above equation, is rather
scarce. We may postulate that these inactivating enzymes which we have dis-
covered can serve as initiators of enzyme degradation, and the products
formed by these inactivating enzymes are subsequently degraded into amino
acids by lysosomal or other non-specific proteinases. We would like to offer
the following explanation for regulatory mechanisms of catabolism of
enzyme protein as shown in Figures 10 and 11, for pyridoxal enzymes and for
NAD-dependent enzymes, respectively. A preliminary report of this work has
already appeared (15, 16).

I ,. =,,= T= TFo==
It

I X // II (Aminoacid) ~ J ~] f non-specffic~

NH2

Bin. 10. Regulatory mechanism of catabolism of pyridoxal-dcpendent enzymes.


MODE OF ACTION OF SPECIFIC INACTIVATINGENZYMES 305

Amino
acid fnon-specific.]1
Lprteinase

~ 7 b~ ~ I,,f X2(n~
/ \ NAD l ~ ~ "4" Amino
acids

ACTIVEFORM

TT
NAO.,

NADH2 I. Enz. LATENTFORM


FIG. 11. Regulatory mechanism of catabolism of NAD-dependent enzymes.

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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