Metabolism of lysine in rat liver

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Vol. 29, No. 1, 1967

NEXADOLEX OF LYSINE IN RAT LIVER Kasuya Higashino,

Rotoji

F'ujioka,

Takakasu Aoki,

and Yuichi

Yamamura

Third Department of Internal Medioine and Department of Biochemistry Osaka University Medical School, Osaka, Japan

ReceivedAugust29,

1967

It was shown previously

that lysine

in the presence of&-ketoglutarate accunniLs.ted (1).

!!'he obligatory

and, under these conditions, requirement

breakdown as well as for saccharopine bolized, its

in mammalian liver,

biosynthesis

to o(-aminoadipic

ofci-ketoglutarate

formation

for lysine

suggest that lysine

by a reversal of reaction

describes

that

saccharopine

acid in the mitochondria

is meta-

sequence described

for

is further

and that NAD rather

metaboliaed than NADP is

in this reaction.

I%iTERIALS

LU-Aminoadipic

was obtained

AND METHOE

acid was purchased from Calbiochem,

FAD from Sigma Chemical

%-Lysine

Compaq.

from Radiochemical

(unifofials

by the action

of sxtracts

by chromatogrsphy previously

as exsminedby

radioactivity

of bakers'

yeast (2, 6).

over Dowex 50 ~8 (H-form)

described

(1).

and NAD, NADP and labeled,

1% mC/mmole)

and unlabeled

oc-ketoglutarate

Centre.

4 C-Saccharopine was prepared from 4 C-lysine

purity

saccharopine

in yeast (2-6).

The present report

required

was degraded in the mitochondria

lh C-Saocharopine

chromatography

The product

was isolated

and Dowex 1 Xl0 (formate-form) thus obtained

onDouex orpaper,

was free from imandhad

a specific

of 1.4 mC/mmole.

Male albino

rats nere obtained

locally

95

and fed on laboratory

chow 5

as

Vol. 29, No. 1, 1967

libitum.

BIOCHEMICAL

Mitochondria

were obtained

O.OClh EDTA by centrifugation was prepared by sonically (10 kc., natant

Sacchsropine nM-@rin

obtained

disrupting

the mitochondria the sonicate

extract

in 0.02k Tris-HCl,

at high speed.

pH 8.3

The super-

was used as the enzyme solution.

andO(-sminoadipate

(8) and radioactivity

in a Packard Tri-Carb

from homogenates prepared in 0.2514 sucrose-

in the usual manner (7). Hitochondrisl

1 min) and by centriguging solution

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

liquid

were determined

calorimetrically

with

measurements were made in a dioxane mixture scintillation

spectrometer.

RESULITS

With a suspension of mitochondria, occurred. preparation

little

or no saccharopine

The breakdown, however, was demonstrated or the sonic extract

ments described

of the mitochondria

below, the sonic extracts

breaMown

by the frozen and thawed (Table I).

In the experi-

were used.

Table I Effect

of Freezing

and Thawing on Sacchsropine

Degradation

pH 8.3, The reaction mixture (1.0 ml) contained 50 oles of Tris-HCl, 4 C-saccharopine (ll,OOC c.p.m.,l.4 mC/mmole), r S~moles of DAD, lymole of EIYTA, 1O~moles of 2-mercsptoethanol and a mitochondridl suspension or the same frozen andthawedtentimes (5.7 mgprotein). Themixtures were placed in the main compartments and 0.2 ml of 0.2 N KOH in the stoppgrs of the Thunberg tubes. The tubes were evacuated and incubated at 37 for 30 min. At the end of the incubation time, 0.25 ml of 2.5N IiClO was ingroduced through the side arms, and the sealed tubes were incu b ated at 60 for30min. The radioactivity of CO evolved was determined by counting the KOH fraction. Radioactive sacchsxop&e was estimated after its isolation bychromatographyonDowex50~8. 4 C-Saocharopine disappeared

Treatment

4 co2 evolved I

c.p.m.

c.p.nl.

5852

396

Jhezing

and Tbbling I

I

96

Vol. 29, No. 1, 1967

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Table II Cofactor

Requirement

for Saccharopine

Sonic &tract

Breakdown

by

of Mitochondria

The complete reaction w contained, in a volume of 1.0 ml, 50 moles of Tria-HCl, pH 8.3, C-aaccharopine (15,OCXI c.p.m., 1.4 mC/sanole), 2. !! rmoles of NAD and a sonic extract of mitochondri.a which had been dialyzed agast 0.02M Tria-HCl, pH 8.3 containing In@1EDTA and 1OmM 2-mercaptoethanol (6.8 mg protein). Incubation was at 37' for 30 min. Saccharopine was estimated after its isolation by Dowex 50 chromatography. l4 CSaccharopine disappeared c.p.m. Complete

12153

n

-NAD

11

-NAD, +MDP (2.5rmolea)

,,

-NAD, tFAD (0.25rmole)

243

Cofactor Requirements-

4753 588

Table II shows the cofactor

degradation.

The reaction

showed an obligatory

nucleotide.

NA@ was about 30% as active

requirement

requirement

for aaccharopine

for a pyridine

as HAD under the conditions

of Table

II. Produots

of Saccharopine

Catabolism-

degraded by the mitochondria added (1).

Incubation

mftochondrial

extract

Fig. la.

&ure

with an evolution

of aaccharopine also yielded

supports the view that Iysine the reaction

It uas previously

labeled

radioactive

on Dower 50 revealed

of the authentic

sample (Fig.

aaccharopine.

No lyaine

produot

from Dowex $3 was chxsnatographed

eluted

in the lyaine CO2 (Table I).

two radioactive

lb).

was

moiety with a This observation chromatography

of

peaks as shown in

acid by rechromatography

after

Peak B was the remaining

was formed under these conditions.

97

was

of CO2 wheno(-ketoglutarate

is degraded via aaccharopine.

Peak A was shonn to be d-atninoadipic

the addition

shown that lysine

When the reaction

on paper in four solvent

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIObJS

Vol. 29, No. 1, 1967

30 NUHBEH

50

40

Chromatography of the reaction product on Dower 50 x8. The reaction zu%contained, in a volume of 4.0 ml, 2OOpmole& of R-is-HCl, pH 7.4, C-saccharopine (1.2 x l0' 4rmoles of KDTA, 40 moles of 2-mercaptoethanol, a.p.m., 1.4 mC/mmole and a sonic extract of mitochondria (15.6 mg protein). After incubation at 37' for 2 hours, the reaction was terminated by the additior of perchloric acid to the final concentration of 5%. The mixture was then neutralized with KOH and the insoluble materials were removed by centrifugation. The supernatant was chromatographed on Dowex 50 x8, H-folm (1 x 20 cm). The column was washed with water to remove the radioactive material which was not held on the column. Gradient elution was then applied with H 0 in the mixing vessel (280 ml) and 4 N HCl in the resemir. Five ml fractl 3 ns were collected. The figure shows the elution profile from the beginning of gradient elution.

systems, The radioactivity

coincided

The Rf values in pyridine:

acetone:

formic acid: water (8: 1: l), formic

aaid: water (70:

tithermore, adipate

15: 15) were 0.18,

a constant

evidence for the identity

specific

color of&-aminoadipate.

ammonia: water (93: 30: 5: 19,

phenol saturated

repeated crystallization

yielded

with the ninhydrin

with water,

the addition

activity

(Table III).

of the product

and tert-butanol:

0.46, 0.27 and 0.54,

after

respectively.

of unlabeled

with o(-aminoadipic

98

isopropanol:

W-amino-

This provides acid.

further

Vol. 29, No. 1, 1967

BIOCHEMICAL

Fig. lb. Co-ohromatograpby Peak A of Fig. la was taken an aliquot containing l,!%O Chromatograp& was on Douex

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

of the reaction product with L-OQuminoadi.pic acid. to dryness and the residue dissolved in H 0. To c.p.m. were added 1Spmoles of L-u-amino &pate. 50 X8 under the same conditions as in Fig. la.

Table III Specific

Radioaotivities

of Reaotion

Product after

Repeated Crystalli~ations To an sliquot of the degradation product Fig. la) containing about 18,000 c.p.m. were L-of-aminoadipic acid. iiecrystallization was eaoh crystallization, the compound was dried usedforradioaotivitymeasurements. Crystallieation

of I.4 C-saccharopine (Peak A, added 100 mg of unlabeled from ethanol-H 0 mixture. At over P20s and P mg portions were

specific activity of owuninoadipic aoid c.p.m./mg

mg 499.3 16.2

99

Vol. 29, No. 1, 1967

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

DISCUSSION

Lysine breakdown was shown to occur in liver supplemented

with&-ketoglutarate.

of o(-ketoglutarate

mitochondria

This fact end the obligatory

for sacoharopine

formation

suggest that lysine

proceeds by a reversal

of the pathway of its biosynthesis

and Neurospora 12-6).

The possibility

exlste,

glutarate

participates

somewhere inthe

process of lysine

formation

is only a side reaction.

The data described is further immediate

metabolized products

in this

communication

to c&minoadipate

of saccharopine

aldehyde and glutamate,

neither

(A'-piperideine-6-carboxylic

only when

catabolism

in Saccharortvces

however, that although a-ketobreakdown, saccharopine

present evidence that saccharopine

in liver

catabolism

requirement

mitochondria.

Although

the

would be o(-aminoadipic-&s-seni-

the aldehyde nor its

cyclization

acid) was detected under the conditions

product used in

this investigati.on. To understand metabolism which lysine determination

the importance

of lysine,

and saccharopine

1. 2. 3. 4.

5. 6. 7.

8.

formation

pathway in manrmslian

be necessary to lcnow the comparable rates at are degraded in mitochondria.

of these rates is rendered difficult

pine does not penetrate saccharopine

it will

of the saccharopine

the mitochondrisl is not effected

However, the

by the fact that saccharo-

membrane (Table I) and conversely ny disrupted

mitochondria.

Higashino, K., Tsukada, K., end Lieberman, I., Biochem. Biopws. Bes. Coxmn., 20, 285 (1965) Kuo, M. II., Saunders, P. P., and Broqtist, H. P., J. Biol. Chem., 239, 508 (1964) Trupin, J. S., and Broquist, H. P., J. Biol. Chem.,2@, 2524 (1965) Jones, E. E., and Broquist, H. P., J. Biol. Chem., m, 2531 (1965) Jones, E. E., and Broquist, H. P., J. Biol. Chem., m, 3430 (1966) Saunders, P. P., and Broquist, H.P.,J. Biol. Chean.,m, 3535 (1966) Hogeboom, G. H., Schneider, W. C., and Striebich, M.T, J. Biol. Chem., 196, I-U., (1952) Ibsen, II., Arch. Biochem. Biopws., 2, 10 (1957)

100