THI JOURNAL or BIOLOQICAL CREM~STBY Vol. 247, No. 21, Issue of November 10, pp. 6848-6855, 1972

Prinnled in U.S.A.

Properties of a Specific Protease for Pyridoxal Enzymes and Its Biological Role

(Received for publication, April 27, 1972

EIKI KOMINAMI, KEIKO KOBAYASHI, SACHIKO KOMINAMI, AND NOBUHIKO KATUNUMA

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

SUMMARY

A new protease which specifically acts on the apo-form of pyridoxal enzymes was discovered in rat small intestine and skeletal muscle. This enzyme was puriCed about SM-fold from the small intestine of vitamin Be-deficient rats. Some proporties of the enzyme and its action were studied.

The enzyme split the apo-protein of ornithine transaminase into two products, a homogeneous smaller protein and an oligopeptide, and thereby inactivated the transaminase. The substrate enzyme, omithine transaminase, and the larger product have a$,,,, values of 9.9 and 5.1, respectively.

The enzyme inactivated all of the apo-pyridoxal enzymes tested, including serine dehydratase, ornithine transaminase, tyrosine transaminase and aspartate transaminase, but did not affect non-pyridoxal enzymes (glutamic dehydrogenase, lactic dehydrogenase, urease, and glutaminase), bovine serum albumin or some synthetic substrates.

Addition of pyridoxal phosphate or a high concentration of pyridoxal had a protective effect against the specific protease, but another active coenzyme, pyridoxamine phosphate, did not.

The activity of this protease in the small intestine increased during vitamin Be deficiency. A reciprocal relation was found between the activity of this protease and that of ornithine transaminase in the small intestine.

Considerable evidence has accumulated in recent years in- dicating that synthesis and catabolism are both equally impor- tant in the regulation of the intracellular concentrations of enzymes (1, 2). There is now much information available on protein synthesis, but little is known of the mechanism and control of protein degradation.

However, there have been several studies on protein catab- olism in which the degradation of marker enzymes or proteins was measured in whole animals, isolated organs, slices, homog- enates, and subcellular fractions. Grossman and Mavrides (3), Kenney (4), and Schimke (5) observed that inhibitors of protein synthesis prevented enzyme inactivation in whole animals. Levitan and Webb (6) also observed this in isolated perfused liver. These observations suggest that protein synthesis may

be required for protein degradation. The requirement of energy for this process was demonstrated by Hershko and Tomkins (7) in cell cultures and by Bromstom and Jeffay (8) in homog- enates. In these systems the processes involved were probably nonspecific intracellular degradations, but their nature was not clarified.

Coffey and de Duve (9) suggested that enzyme levels might be controlled by lysosomal proteases, but it seems unlikely that these would cause specific degradation of individual enzymes.

Another mechanism of protein catabolism was shown by the work of Bonsignore et al. (10) who demonstrated a specific en- zyme for the inactivation of glucose 6-phosphate dehydrogenase. However, this mechanism does not seem likely to explain the continual replacement of all proteins.

During studies on the mechanism of degradation of pyridoxal enzymes in vitamin Bs deficiency, we found a new enzyme which may be important in the regulation of the intracellular concen- tration of pyridoxal enzymes in rat small intestine and skeletal muscle.

Rapid and marked decrease of pyridoxal enzymes was ob- served in the small intestine of rats in vitamin Be deficiency (11). To investigate this mechanism further, we began our work ila vitro by looking for the material which affects the decrease of the activity of ornithine transaminase. Purified ornithine trans- aminase was incubated with tissue homogenates prepared from several organs of vitamin Ba-depleted rats, and the change of ornithine transaminase activity was followed. The results showed that homogenates of small intestine and skeletal muscle contained some material which inactivated ornithine transam- inase. Furthermore, we demonstrated that this material had the nature of an enzyme and that it attacked not only ornithine transaminase but also other pyridoxal enzymes. This new en- zyme split the apo-protein molecule into a smaller protein and an oligopeptide under alkaline conditions.

This paper reports the purification and some properties of this enzyme, and the function of the enzyme in the regulation of the degradation of pyridoxal enzymes. A preliminary report of this work has appeared (12).

EXPERIMENTAL PROCEDURE

MaterialsPyridoxal phosphate was obtained from Chugai Pharmaceutical Co. Ltd., Tokyo, Japan. Other vitamin Bc derivatives were obtained from Sigma and flavin adenine di- nucleotide was from Boehringer-Mannheim. The enzymes

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purchased commercially were : glutamic dehydrogenase (Boehringer), lactic dehydrogenase (Sigma), urease (Wako Pure Chemical Industries Ltd., Osaka, Japan) and trypsin (Merck Co. Germany). The following compounds were purchased from Peptide Center, Institute for Protein Research, Osaka: N-benzoyl L-arginine ethyl ester, L-arginine methyl ester, p-toluene sul- fonyl+a.rginine methyl ester, L-lysine methyl ester, p-toluene sulfonyl-L-lysine methyl ester, L-glycine ethyl ester, N-acetyl-n- tyrosine, and L-tyrosine ethyl ester. The marker proteins used in determination of molecular weights were bovine serum albu- min, ovalbumin, chymotrypsin, and myoglobin and these were obtained from Mann Research Laboratories.

Vitamin-deficient Rats-Male Wistar strain rats, weighing 90 to 100 g, were maintained on a 20% casein diet without vitamin Bs for 3 weeks before use as vitamin BG-deficient rats. Similarly niacin-deficient and riboflavin-deficient rats were obtained by feeding rats on niacin-free or riboflavin-free diets for 2 weeks. To accelerate the onset of niacin deficiency, animals were in- jected intraperitoneally with 35 mg per 100 g of acetyl pyridine at 9:00 a.m. every day (13).

Isolation of Splitting Enzyme Speci$c for Pyridoxal Enzymes- Animals were killed by decapitation and the small intestine and various other organs were removed and weighed. Then 20% (w/v) homogenates were prepared in 0.05 M potassium phosphate buffer, pH 7.5, with a Potter-Elvehjem type homogenizer. The homogenates were sonicated at 10 kc for 2 min and then cen- trifuged for 10 min at 10,000 x g. The supernatant was passed through a column of Sephadex G-25 equilibrated with the same buffer, and the fractions containing protein were collected. These fractions were combined and used as material for assay of splitting enzyme.

Assay of Splitting Enzyme Specific for Pyridoxal Enzyme- The enzyme was assayed as reported previously (11) with some modifications. The reaction mixture (final volume, 0.3 ml) con- tained 30 pmoles of Tris-HCl buffer, pH 8.6, 40 to 60 units (0.1 mg) of substrate enzyme, and a suitable amount of the splitting enzyme preparation. The mixture was incubated at 37” and the reaction was stopped by lo-fold dilution with cold buffer. Then the activity of the substrate enzyme remaining was assayed and the percentage of inactivation was calculated. One unit of splitting enzyme was defined the amount inactivating 50% of the substrate enzyme in 30 min under these conditions.

Protein Determination-Protein concentrations were deter- mined by the method of Lowry et al. with crystalline bovine serum albumin as standard (14).

Preparation and Assay of Substrate Enzymes-All of the pyr- idoxal enzymes used as substrates were purified from rat liver. Crystalline ornithine transaminase and partially purified tyro- sine transaminase, serine dehydratase, aspartate transaminase, and homoserine deaminase were obtained by the methods of Matsuzawa et al. (15), Valeriote et al. (16), Nakagawa et al. (17), Huzino et al. (18), and Matsuo et al. (19), respectively. The enzymes were converted to the apo-forms before use as sub- strates (15-17, 20, 21). These enzymes were assayed by the methods of Katunuma et al. (22), Rosen et al. (23), and Sayer and Greenberg (24) as modified by Nakagawa et al. (17)) Karmen (25), and Matsuo and Greenberg (19), respectively. Crys- talline glutamic dehydrogenase from bovine liver and lactic dehydrogenase from pig heart muscle were obtained commer- cially and were assayed by measuring the rate of decrease in absorbance of NADH at 340 nm at 25’ (26, 27). Preparation of crystalline phosphate-independent glutaminase from rat kidney and assay of glutaminase activity were performed by the methods

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of Katunuma et al. (28, 29). Jack bean type III urease was obtained commercially and assayed by measuring the rate of ammonia formation with Nessler’s reagent (30). Proteolytic activity of the splitting enzyme with synthetic substrates was assayed by the method of Roberts (31).

Estimation of Molecular Weights-Molecular weights were determined using a Sephadex G-100 column (2 x 80 cm) equil- ibrated at 4” with 0.2 M potassium phosphate buffer, pH 7.5. The test sample was applied in a volume of 1.5 ml, and 3-ml fractions were collected at a flow rate of 15 ml per hour. Blue dextran marked the void volume and bovine serum albumin, ovalbumin, chymotrypsin, and myoglobin were used as markers.

Disc Gel Electrophoresis-Electrophoresis in polyacrylamide gels (7.5%) was carried out as described by Davis et al. (32). Samples containing 50 bg of protein in 10 ~1 of a 10% sucrose solution were layered over the upper gel. The buffer used was 0.05 M Tris-0.38 M glycine. Electrophoresis was carried out for about 3 hours at 4” at a constant current of 2 ma per tube. Proteins were detected by staining with Amido black. The gels were destained in 7% acetic acid.

Ultracentrifugal Analysis of Reaction Products-Ultracentrif- ugation was performed in a Hitachi analytical ultracentrifuge, type 1, at a speed of 66,000 rpm at 5” in single sector cells with quartz windows. Sedimentation coefficients were determined by the means of the schlieren optical system with Fuji spectrographic plates. The movement of the refractive index gradients was determined by the use of Fuji microcomparator. Analysis was done as described by Schachman (33).

Immunological Procedures-Antiserum against ornithine trans- aminase was prepared as described previously (15). Ouchterlony double diffusion gel analysis was performed as described by Clausen (34).

RESULTS

Purification of Splitting Enzyme Specific for Pyrdoxal Enzymes from Rat Small Intestine

Representative data on the purification of the enzyme from the small intestine of vitamin Be-deficient rats are summarized in Table I. All procedures were carried out at 4” using 0.05 M

potassium phosphate buffer, pH 7.5. Step l-Acetone powder extract. Four hundred f i f ty grams

of small intestine of vitamin Bs-deficient rats were homogenized in a Waring Blendor in 450 ml of 0.05 M potassium phosphate buffer, pH 7.5, for 1 min at full speed. The homogenates were mixed with acetone (previously cooled to - 15’; 20 ml of acetone

TABLE I Purification of pyridoxal enzymes .speciJic splitting enzyme from

small intestine of vitamin Be-deficient rats

Fraction I I

Volume TOtal protein

ml mg

Acetone powder extract. . . 1,100 13,090

Ammonium sul- fate.. . 1,000 6,190

Acetone.. . . . . 200 4,220 Acetone.. . 41 1,730 DEAE-cellulose 2.4 48 Sephadex G-100. 1.0 4

-

Total activity

units units/mg %

4,090,000 310 100

4,100,000 660 100 5,040,000 1,190 123 4,080,OOO 2,360 100 1,240,ooo 25,800 30

620,000 130,000 15

Specific activity

R.XOV- -Y

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per g of small intestine), and then the suspension was ground in a large mortar until the preparations were free from large lumps. The mixture was poured onto a Buchner funnel, and the residue was washed twice on the funnel with cold acetone. The material was sucked dry and the powder was crumbled and allowed to dry in the air at 4” for a few hours. The dried powder was stored at -20”. About 50 g of acetone powder were obtained from 450 g of small intestine. The acetone powder was mixed with 1,000 ml of 0.05 M potassium phosphate buffer, pH 7.5, and the mixture was stirred at 0” for 20 min. It was then centrifuged at 10,000 x g for 10 min. At this step, increase of specific ac- tivity to 5- to lo-fold was observed.

Step .9-Ammonium sulfate fraction. Solid ammonium sulfate, 176 g per liter was added to the supernatant and the mixture was stirred for 30 min. The mixture was centrifuged at 10,000 x g for 10 min, and the precipitate was discarded. Then further ammonium sulfate was added to give 70% sat- uration (472 g per liter). The mixture was stirred and centri- fuged and the precipitate was dissolved in 0.05 M potassium phosphate buffer, pH 7.5 (Buffer A) and dialyzed overnight against this buffer.

Step %-First acetone fractionation. Ice-cold acetone (70 ml per 100 ml) was then added to the dialyzed suspension wit.h stirring over a period of 5 min and the mixture was immediately centrifuged at 10,000 x g for 5 min, and the resulting precipitate was collected. This precipitate was dissolved in Buffer A and entrifuged briefly to remove insoluble material.

Step &-Second acetone fractionation. The ice-cold acetone (40%, v/v) was added to the supernatant with stirring over a period of 5 mm, and the mixture was immediately centrifuged at 10,000 X g for 5 min. To the supernatant solution additional cold acetone was added to give a final concentration of 60% (v/v), and the resulting precipitate was collected by centrifu- g&ion. This precipitate was dissolved in a minimum volume of Buffer A and centrifuged briefly to remove the insoluble ma- terial. The supernatant was passed through a Sephadex G-25 (5.5 x 20 cm) column equilibrated with Buffer A.

Step b-DEAE-cellulose chromatography. The enzyme solu- tion was applied to a DEAE-cellulose column (3.5 x 20 cm) equilibrated with Buffer A and the column was washed with 500 ml of the same buffer at a flow rate of 60 ml per hour. The column was then &ted step-wise with 300 ml of 0.05 to 0.20 M potassium phosphate buffer, pH 7.5, at a flow rate of 40 ml per hour; fractions of 6 ml were collected. The splitting enzyme was eluted in 0.20 M buffer. Fractions with high specific activity were pooled and solid ammonium sulfate (472 g per liter) was added to 70% saturation. After stirring for 30 min, the pre- cipitate was collected by centrifugation at 10,000 X g for 10 min and dissolved in a small amount of Buffer A.

Step 6--Gel filtration on Sephadex G-100. The enzyme solu- tion was applied in a Sephadex G-100 column (2.5 X 90 cm) equilibrated with Buffer A (Fig. la). The enzyme was excluded from the gel and was collected and concentrated to a final volume of about 1 ml in a collodion bag. Finally, the preparation was

0 P

10 20 30 40 50 60 70 s

Fraction Number 0 -+::

I

8

(cl

2 200 -

Bovine Serum Albumin c

Ovalbumin

4 150 - G.S,S. Enzyme

2 Chymotrypsin

F 2 100 . A:

>$yoglobin

5 Gi

50 ,“’ I I

4.0 4.2 4.4 4.6 4.8

Yolecular Weight (log)

FIG. 1. a, Sephadex G-166 chromatography. The enzyme was columns were equilibrated with 0.1 M potassium phosphate buffer eluted from the column with 0.05 M potassium phosphate buffer pH 7.5, at a flow rate of 15 ml per hour. The purified splitting at a flow rate of 55 ml per hour. Fractions of 6 ml were collected enzyme was applied to columns in combinations with blue dextran and assayed for the absorbance at 266 nm and the splitting enzyme (mol wt 2,000,OOO) and one of following markers: sperm whale activity. b, polyacrylamide gel electrophoresis of the purified myoglobin (mol wt 17&W), beef pancreatic chymotrypsinogen pyridoxal enzymes specific splitting enzyme. Fifty micrograms A (mol wt 25,666), ovalbumin (mol wt 45,666), and bovine serum of enzyme were applied and migration was from top to bottom of albumin (mol wt 67,666). The elution patterns were measured the gel. Each gel was 6 cm long. C, determination of the molecu- by determination of protein, and that of the splitting enzyme lar weight of the pyridoxal enzymes specific splitting enzyme by (G.S.S. Enzyme) was determined by assay of activity. gel filtration on columns of Sephadex G-166 (2.0 X 80 cm). The

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Experiment I

Experiment II

1Sxperiment III

-_

Ornithine transaminase Tyrosine transaminase Serine dehydratase Homoserine dehydratase Ornithine transaminase Aspartate transaminase(s)’ Aspartate transaminase(m)m Glutamic dehydrogenase Lactic dehydrogenase Glutaminase Urcase

subjected to disc gel electrophoresis at 4” with a constant current of 2 ma per tube in 0.05 M Tris-0.38 M glycine buffer. The prep- aration gave one major protein band and one minor protein band which stained with Amido black (Fig. lb). Elution of slices of an unstained control gel permitted demonstration of the splitting enzyme activity coincident with the major protein band. The

TABLE II

Substrate speciJicity of pyridoxal enzymes specific splitting enzyme Various substrate enzymes were prepared from rat tissues or

obtained commercially as described under “Experimental Pro- cedure.” Residual activity was assayed in mixtures (final volume, 0.5 ml) containing 50 mM potassium phosphate buffer, pH 7.5, the indicated concentration of substrate enzyme, and the splitting enzyme (2 units in Experiments I and II, 18 units in Experiment II). Incubation was performed at 37” for 10, 20, and 30 min in Experiments I and II, and for 2 hours in Experiment III. Values indicate the inactivation of substrate enzymes taking that of ornithine transaminase as 100%.

Substrate

ElUyIIle

- Relative COW3l- activity tration

- units/ tubea %

60 100

50 7.6 60 62.2 16 84 50 100

100 21.8 18 81.9 75 <O.l 75 <O.l 74 <O.l 81 <O.l

0 Units per tube; products formed in micromoles per hour per tube.

* The supernatant form of aspartate transaminase. c The mitochondrial form of aspartate transaminase.

100

80

Crude enzyme

6 7 a 9 10

PH

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purified enzyme was fairly stable on the storage at -20” at least for 2 weeks.

Some Properties of Purified Enzyme

The molecular weight of the purified splitting enzyme was estimated by gel filtration on a Sephadex G-100 column as 31,000 using the method of Andrews (35), as shown in Fig. lc.

The purified preparation of the splitting enzyme showed high specificity for pyridoxal enzymes as shown in Table II. Even with prolonged incubation times the activity of non-pyridoxal enzymes tested (glutamic dehydrogenase, lactic dehydrogenase, urease, and phosphate-independent glutaminase) was not affected. The protease activity of the splitting enzyme was assayed with synthetic substrates. Larger amounts of enzyme were used in this experiment since, in general, synthetic sub- strates are less sensitive than protein substrates to proteases. No activity was observed with trypsin substrates (iV-benzoyl-L- arginine ethyl ester, L-arginine methyl ester, p-toluene sulfonyl- L-arginine methyl ester, L-lysine methyl ester, p-toluene sulfonyl-L-lysine methyl ester) or chymotrypsin substrates (iV- acetyl-L-tyrosine ethyl ester, L-tyrosine ethyl ester).

The optimum pH for inactivation of ornithine transaminase was studied with both crude and purified preparations and both pH curves had an optimum at pH 9.0 (Fig. 2).

The relationship between the substrate concentration and reaction velocity was studied, the results of which are shown in Fig. 3. The Michaelis constant of the splitting enzyme for the substrate, ornithine transaminase, was calculated as 1.52 X

low5 M, assuming that, the molecular weight of ornithine trans- aminase is 132,000 as reported by Peraino et al. (36).

Eflect of Vitamin Bg Derivatives on Activity of Splitting Enzyme

The apo-form of the enzymes was inactivated by the splitting enzyme. The effects of vitamin 136 derivatives and other vi- tamins on the inactivation of these enzymes were studied by incubating apo-ornithine transaminase with compounds before adding the splitting enzyme. Pyridoxal phosphate and pyridoxal protected the enzymes against the splitting enzym

Apo-ornithine transaminase f 10M5M I

FIG. 2 (left). pH-activity curve of the pyridoxal enzyme spe- cific splitting enzyme. Buffer was used at a final concentration of 50 mM potassium phosphate buffer, pH 6.0 to 8.0; Tris-HCl buffer, pH 8.0 to 9.0; glycine-sodium hydroxide buffer, above pH 9.0.

FIG. 3 (center). Effect of concentration of substrate enzyme (ornithine transaminase) on the activity of pyridoxal enzymes specific splitting enzyme. Enzyme activity was assayed under standard conditions except that the concentration of the sub-

L- --r---C--r--.,---y-- -. 1 2 3 4 5

PAL ( x lO-4 MI

Concentrations of PLP or PAL

strate, ornithine transaminase, was varied. The inset shows a Lineweaver-Burk plot of data from this figure.

FIG. 4 (right). Effect of concentration of pyridoxal phosphate and pyridoxal on the activity of the pyridoxal enzymes specific splitting enzyme. Reaction mixtures containing the indicated amount of pyridoxal phosphate (PLP) or pyridoxal (PAL) and 50 units of apo-ornithine transaminase were previously incubated at 37” for 30 min, before the reaction was started by the addition of 4 units of the splitting enzyme.

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TABLE III Protective effects of vitamin Be derivatives and other vitamins against

inactivation of pyriclozal enzymes by splitting enzyme

Reaction mixtures containing the indicated amounts of vitamin Ba derivatives and other vitamins, 50 units of apo-ornithine transaminase, and 100 mM Tris-HCI, pH 8.6, were incubated for 30 min at 37” before the reaction was initiated by the addition of 4 units of the splitting enzyme.

1.0

(NH4)2504

Addition

None Pyridoxal phosphate Pyridoxamine phosphate

Pyridoxine phosphate

Pyridoxal NAD

FAD

Final concentration

-- M units/tubes

2.5 X 1O-6 2.5 X lo-; 2.5 X 1O-3 2.5 X lo-’ 2.5 X 1O-3 2.5 X 1O-4 2.5 X 1O-4 2.5 X lo-* 2.5 X lo-* 2.5 X 10-8

10 0 43.6 83.4 12 5.0 10.8 2.0 12.6 6.4 11.8 4.4 36.5 66.2 10 0 10 0 10.4 1.1 10 0

$ Protection

(Table III). Other compounds, including pyridoxine phos- phate and pyridoxamine phosphate, did not have protective effects. It is interesting that pyridoxamine phosphate had no protective effect since functionally the pyridoxamine phosphate form of the enzyme is as active as the pyridoxal phosphate form in the transaminase reaction. The same result was recognized when the pyridoxamine phosphate form was made by the in- cubation of the pyridoxal phosphate form with ornithine in the absence of ar-ketoglutarate.

The protection was most effective when apo-ornithine trans- aminase was incubated with pyridoxal phosphate and thus con- verted to the holo-form. Pyridoxal phosphate also protected tyrosme transaminase and serine dehydratase from inactivation. The concentrations of pyridoxal phosphate and pyridoxal re- quired for 50% protection were calculated as 6.0 x 10-e M and 1.6 x 10-a M, respectively (Fig. 4). This concentration of pyr- idoxal phosphate is almost the same as the K, value of ornithine transaminase for pyridoxal phosphate (37). Thus, the addition of pyridoxal phosphate or 30 times more pyridoxal than pyridoxal phosphate should be equally effective in protecting the enzyme against inactivation.

The effect of pyridoxal phosphate on a purified preparation of the splitting enzyme was studied by incubating the enzyme with 0.5 mM and 5 mM pyridoxal phosphate for 30 min, and then passing the reaction mixture through a column of Sephadex G-25. Then, apo-ornithine transaminase was added to the eluted en- zyme solution, and the splitting enzyme activity was assayed. Pyridoxal phosphate was found to have no effect on the splitting enzyme, suggesting that the splitting reaction is not dependent on pyridoxal phosphate.

Analysis of Reaction Products

To examine the products of the reaction, crystalline apo-orni- thine transaminase was partially inactivated by incubation with purified splitting enzyme and then the reaction mixture was immediately applied to a Sephadex G-100 column. The elution profile is shown in Fig. 5b. The jirst so&? line represents intact ornithine transaminase and the second solid line is the protein

10 20 30 40

Fraction Number

FIG. 5. Identification of the products of the splitting reaction on a Sephadex G-100 column. Twelve milligrams of crystalline holo- (a) or apo-ornithine transaminase (b) were partially inac- tivated by incubation with 2860 units of purified splitting enzyme (G.S.S. Enzyme) for 10 min. The reaction was stopped by cooling the mixture to 0”. Then the mixture was applied to a column of Sephadex G-100, previously equilibrated with 0.05 M potassium phosphate buffer, pH 7.5, and material was eluted with the same buffer at a flow rate of 15 ml per hour. Fractions of 3.0 ml were collected. Ammonium sulfate was applied with the mixture as a marker.

split from ornithine transaminase by the enzyme. The second peak did not have ornithine transaminase activity. The splitting enzyme gave the third peak indicated by a solid lip. The fourth peak is due to a second product derived from ornithine trans- aminase, which seems to be an oligopeptide. The elution pat- tern shown in Fig. 5a was obtained with holo-ornithine trans- aminase as substrate. All of the ornithine transaminase activity added was recovered in the first peak, and no other protein or peptide liberated from substrate. Ornithine transaminase was detected on the chromatogram. These results indicate that the enzyme splits ornithine transaminase into two products, a smaller protein and an oligopeptide. Therefore, the splitting enzyme seems to be a specific endopeptidase. To examine these products further, the first peak, apo-ornithine transaminase, and the second peak eluted from the Sephadex G-100 column were sub- jected to ultracentrifugal analysis (Fig. 6).

Both proteins appeared to be homogeneous on ultracentrif- ugation and in the standard buffer had Sag+, values of 9.9 and 5.1, respectively. On electrophoresis on a cellulose acetate membrane they also both appeared homogeneous and had similar mobilities. The second peak had no ornithine transaminase activity but could not be distinguished from the latter anti- genitally . On analysis on Ouchterlony double diffusion plates, the antibody specific for native ornithine transaminase gave a single precipitation line with the material in the second peak.

Further Degradation of Ornithine Transaminase by Trypsin

It was of interest to study whether degradation of pyridoxal enzymes by the splitting enzyme could be followed by the action of intracellular nonspecific proteases. Apo-ornithine trans- aminase was first inactivated by the splitting enzyme and then the larger product was isolated by chromatography on Sephadex G-100. This fraction was then incubated with trypsin in 0.1 M potassium phosphate buffer, pH 7.5. Simultaneously, native apo-ornithine transaminase was subjected to trypsin.

As shown in Fig. 7, apo-ornithine transaminase and bovine serum albumin were scarcely degraded by trypsin, while the product was markedly degraded.

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(a)

FIG. 6. Sedimentation patterns of ape-ornithine transaminase and its product released by the splitting enzyme specific for pyri- doxal enzymes. The product wan obtained by applying the reac- tion products of apo-ornithine transaminase released by the Bplit- ting enzyme to a Sephadex G-100 column, a~ shown in Fig. 5. Both peaks on the chromatogram, apo-ornithine transaminase (10.6 mg per ml) in 0.1 M potassium phosphate buffer, pH 7.5, and the product (11.5 mg per ml) in the same buffer (a) were cen- trifuged independently in a Hitachi analytical centrifuge, type 1, at 5”. Sedimentation is from left to right. Pictures were taken at Q-min intervals after attaining the maximum Bpeed of 60,OOQ rpm.

Tissue Distribution of Enzyme

The distribution of the splitting enzyme was studied using crude preparations of tissues from vitamin Ba-deficient rats. Among the tissues examined, the small intestine had high ac- tivity and skeletal muscle low activity. Extremely low activity was observed in other organs (liver, kidney, heart, spleen, brain, stomach, large intestine) compared with that of two organs de- scribed previously. However, it is possible that the enzyme may be present in a latent form in other organs.

Effect of Dietary Conditions on Enzyme Activity

The activity of the splitting enzyme in the small intestine of animals under various dietary conditions was investigated. Ac- tivity increased in vitamin Be deficiency, as shown in Table IV, at a time when ornithine transaminsse activity in the small intestine, determined as an indicator of Be deficiency, was greatly

Product split by G.S.S. Enzyme

j ‘v ;tactApo-OTAt

0 30 60

Time (min 1

Fro. 7. Digestion of native apo-ornithine transaminase and its product by trypsin. The product wan obtained by same pro- cedure a~ Fig. 6. The incubation mixture contained 1.5 mg of protein a~ BUbBtrate and 1 mg of trypain in a final volume of 1.0 ml (0.1 Y potassium phosphate buffer, pH 7.5). Aliquots of 0.3 ml were removed at the times indicated, trichloroacetic acid wan added, and protein in the Bupernatant WBB measured by method of Lowry et al. (12). Values indicate the percentage of breakdown of BUbBtrate protein, taking breakdown of all of the substrate protein a8 100%. G.S.S. Enzyme represents the pyridoxal en- zymes specific splitting enzyme.

TABLE IV Splitting enzyme specific for pyridoxal enzymes activity in small

intestine under various dietary conditions

Rats weighing 100 g were maintained on various diets for the indicated periods before experiments. Crude preparations were obtained as described under “Experimental Procedure,” and activities were measured with the standard assay system. Ac- tivities are expressed BB meanB f standard errors.

N”FEi Of Activity

Chow. . . . . . . . . . . . . . . . . . . . . 18 0.78 f 0.21 5% Protein diet (5 days). . . . . 5 0.12 f 0.01 700/, Protein diet (5 days). 5 115.6 f 26.5 Starvation (2 days). . . . 3 0.63 f 0.03 Niacin-deficient diet (2 weeks). 33 1.11’ Vitamin Bz-deficient diet (4 weeks). . 16 1.500 Vitamin Be-deficient diet (4 weeks). 14 152.1 f 18.7

a Activities were assayed with collected samples of crude preparations.

reduced. The activity of the splitting enzyme did not increase in vitamin Br, or niacin deficiency but was increased in rats fed on high protein diets.

Reldan between Activity of Enzyme and of Ornithine Transaminase in Small Intestine

In order to get information on the roles of the splitting enzyme in Go, activity of both the splitting enzyme and of ornithime transaminase was assayed in crude extracts of small intestine of rats fed on vitamin Be-deficient diet for various periods. As shown in Fig. 8, a reciprocal relation between both activities was observed. A double logarithmic plot of both activities yields a straight line with the range of ornithine transaminase activity from 1.0 to 0.2 unit per mg of protein. Linearity of the double logarithmic plots permits application of the following equations to express the relation between both activities.

log [Orithine transaminase] = log 2.2-0.5 log [Splitting enzyme]

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Splitting enzyme activity I units/ mg protein 1

FIQ. 8. A reciprocal relation between the splitting enzyme specific for pyridoxal enzymes activity and ornithine transaminase activity in small intestine during the development of vitamin Be deficiency. Rats weighing 100 g were maintained on vitamin BB-deficient diets for the various periods before experiments. Crude preparations from small intestine were obtained as de- scribed under “Experimental Procedure,” and both activities of the splitting enzyme and of ornithine transaminase were assayed. The inset shows a double logarithmic plot of data.

or

[Ornithine transaminasel = 2.2

d/[Splitting enzyme]

This equation suggests the possibility that concentration of orni- thine transaminase as a representative of pyridoxal enzymes is controlled by the splitting enzyme. In considering this prob- lem, the intracellular compartmentalization of the splitting en- zyme and that of pyridoxal enzymes must be taken into con- sideration.

DISCUSSIOiS

We named the new enzyme described in this paper a group- specific splitting enzyme for pyridoxal enzymes on the basis of the following four experimental observations.

1. The enzyme showed strict group specificity for pyridoxal- dependent apo-enzymes and did not affect any other non-pyr- idoxal enzymes or proteins or synthetic substrates tested (Table II).

2. Addition of pyridoxal phosphate completely protected apo- pyridoxal enzymes from inactivation by this enzyme (Table III).

3. The activity of this enzyme increased in vitamin B6 defi- ciency, but not in vitamin Bz or niacin deficiency (Table IV). The exact reason for this is unknown but it indicates that there must be some relation between changes in the activity of this enzyme and the intracellular concentration of vitamin Bg.

This enzyme inactivated apo-ornithine transaminase, (s&,+ = 9.9) by splitting it into an oligopeptide and a smaller protein (s&+ = 5.1).

The enzyme has an alkaline pH optimum. In this property it differs from lysosomal proteases which usually have an acidic pH optimum.

As indicated in Table III, a large amount of pyridoxal had a protective effect against the splitting enzyme, although it did not act as coenzyme of pyridoxal enzymes. Peraino et al. (36) and Matsuzawa et al. (17) found that 1 molecule of ornithine transaminase contains about 60 lysine residues and 2 special

lysine residues among them are sites to bind with pyridoxal phos- phate. It is expected that addition of 30 times more pyridoxal than pyridoxal phosphate should form a SchifI base with all of the lysine residues including these two special lysine residues in the ornithine transaminase molecule.

On the other hand, the pyridoxamine phosphate form of pyr- idoxal enzymes, as well as the apo-form, is susceptible to the splitting enzyme (Table III). Therefore, these two observations suggest that special lysine residues, which only form a Schiff base with pyridoxal phosphate, must remain free when pyridoxal enzymes are available as substrate for the splitting enzyme. The latter observation also suggests that transaminases may be susceptible to degradation by the splitting enzyme at some time during resonance, since the pyridoxamine phosphate form is as active as the pyridoxal phosphate form in the transaminase reaction

A highly purified preparation of the enzyme split ornithine transaminase into two products (Fig. 5). The larger product was isolated as a small homogeneous protein. It had no orni- thine transaminase activity so it seemed to be an inactive subunit. The smaller product liberated from ornithine transaminase by the splitting enzyme appeared as a homogeneous oligopeptide on paper chromatography with acetate-butanol-water as solvent Preliminary analysis showed that it contained the following amino acids: lysine, aspartic acid, serine, glutamic acid, glycine, alanine, and leucine. These results indicate that the splitting enzyme attacks a quite limited region in the substrate enzymes, although the exact point of attack is uncertain.

It is thought that, in general, denaturation of protein is a pre- requisite for enzymic breakdown by the usual, nonspecific pro- teases. Coffe and de Duve (9) reported that many proteins are resistant to lysosomal proteolysis in their native form at an al- kaline pH value. Our results also indicated that native apo- ornithine traneaminase was scarcely broken down by trypsin (Fig. 7), but the splitting enzyme actively split native apo-orni- thine transaminase in alkaline conditions, and the resulting product was readily degraded by trypsin (Fig. 7). These ob- servations suggest that the reaction of the splitting emyme is the initial step in intracellular degradation of pyridoxal enzymes. It seems likely that the splitting enzyme converts pyridoxal enzymes to a form which is more susceptible to nonspecific pro- teases. The in oioo finding that there is a reciprocal relation between the activity of the splitting enzyme and that of ornithine transaminase suggests that the splitting enzyme is important in regulation of the intracellular levels of pyridoxal enzymes.

There are several possible explanations of the mechanism and biological teleology of the increase of activity of the splitting enzyme in vitamin B6 deficiency. However, further work is required before any conclusions can be made on these problems.

High activity which inactivates ornithine transaminase was observed in rats fed on high protein diets (Table IV). But the activity of ornithine transaminase was not increased or decreased in small intestine. Therefore, the inverse rule between the splitting enzyme activity and ornithine transaminase activity observed in vitamin Bs deficiency was not held on a high protein diet. However, we have not determined whether the nature of this enzyme is the same as that of the splitting enzyme prepared from the small intestine in vitamin Ug deficiency and whether the mechanism of the increase of activity of the splitting enzyme is the same as in vitamin Bs deficiency. The properties of the splitting enzyme presented in this paper were studied with the preparation purified from vitamin 136 deficient rats.

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6855

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Eiki Kominami, Keiko Kobayashi, Sachiko Kominami and Nobuhiko KatunumaProperties of a Specific Protease for Pyridoxal Enzymes and Its Biological Role

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