Transport of 5-oxoproline into rabbit renal brush border membrane vesicles

Vol. 105, No. 1, 1982

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Pages 28-35

March 15, 1982

TRANSPOrt OF 5-OXOPROLINE INTO RABBIT RENAL BRUSH BORDER MEMBRANE VESICLES VadiveL

Ganapathy,

R.

August

Roesei* and Frederick H. Leibach

Department of Cell and Molecular Biology Medical College of Georgia Augusta, Georgia 30912 and *Gracewood State School and Hospital Gracewood, Georgia 308[2

Received January 19, 1982

5-Oxoproline has been shown to be +transported intact into rabbit renal brush border membrane vesicles by a Na -gradient dependent mechanism. The transport occurs against a concentration gradient. The equilibrium transport of 5-oxoproline decreases with increasing medium osmolarity. The predominant portion of transport occurs into an osmotically responsive intravesicular space, the non-specific surface binding being negligible. Competition experiments show that transport of 5-oxoproline is inhibited by most of the neutral amino acids whereas acidic, basic and imino acids do not have any effect.

Introduction: 5-Oxoproline,

also known as pyrrolidone carboxylic acid or pyroglutamic

acid is a cyclic amino acid present in free form in all animal tissues and also at the N-terminus of a number of naturally occurring, biologically active peptides and proteins.

The metabolic formation and utilization of 5-oxoproline

in the y-glutamyl cycle have been reviewed (i).

This amino acid is produced

in free form independently by the action of at least three enzymes - y-glutamyl cyclotransferase

(I), pyrrolidonyi carboxypeptidase (2,3) and y-glutamylamine

cyclotransferase

(4).

most

5-oxoproline as the N-terminal residue of proteins is

likely formed late in protein synthesis by cyclization of N-terminal

glutamine

(5).

Utilization of 5-oxoproline is initiated by the action of

5-oxoprolinase which converts 5-oxoproline to glutamic acid (6). 5-Oxoproline is present in all animal tissues and plasma. 5-oxoproline in the mouse tissues is 20-50 ~M (7).

The level of

It is also a major water

soluble nitrogen compound in the epidermis where its concentration is at least ten times higher (8).

The urinary excretion of 5-oxoproline is 1 ~mole per mg

0006-291X/82/050028-08501.00/0 Copyright © 1982byAcadem~ Pre~,lnc. AHrigh~ofreproduction m anyform reserved.

28

Vol. 105, No. 1, 1982 creatinine

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

in the mouse

(7) and 820 ~mole per mg creatinine

in man (9).

The

tissue and plasma levels of 5-oxoproline and its excretion in urine increases many

fold in patients with 5-oxoprolinuria

synthetase deficiency An interesting

(i0) or 5-oxoprolinase feature

associated with either glutathione deficiency

of 5-oxoprolinuria

(11,12).

is the accompanying

level of proline and other amino acids in the plasma

(13,14).

elevated

It was suggested

that there may be competitive

interactions between proline and 5-oxoproline

metabolism

However, no data are available on the transport

or transport

of 5-oxoproiine across

and its interaction with other amino acids during transport

any cellular

teristics

of

(7).

in

membrane.

5-oxoproline

We report here for the first time the charac-

transport

in kidney,

using purified

renal brush

border membrane vesicles from rabbit.

Methods and Materials Rabbit renal brush border membrane vesicles were prepared as described previously (15,16). The purity of the memb;anes was routinely evaluated by specific marker enzymes (16) and occasionally by electron microscopy. Only the membranes completely free from basal-lateral membrane contamination as assessed by the absence of (Na+-K +) ATPase activity were used. Transport was assayed by the Millipore filtration technique described in detail elsewhere (15). When the effect of various amino acids on transport was studied, the pH of the solutions in each case was adjusted to 7.5 with either HC1 or Tris base. Plasma 5-oxoproline was determined as glutamic acid after hydrolysis as described by Hagenfeldt et al. (17). 200 DI of deproteinized plasma was passed through a column (6 x 0.6 cm) of Dowex 50 W x 4 (50-100 mesh) and the 5-oxoproline eluted with 3 ml of water. The eluate was mixed with 3 ml of 5 N HCI and hydrolyzed for i hr at 100°C. The solution was evaporated to dryness and I ml of sodium citrate buffer (200 mM, pH 2.1) added and analyzed on the amino acid analyzer (Beckman Model 120-C). Urine 5-oxoproline was measured as previously described (ii). The organic acids in the urine were isolated on a DEAE-Sephadex column and eluted with 1.5 M pyridinium acetate buffer, pH 5.0. The eluate was evaporated to dryness and the organic acids esterified with 14% boron trifluoride in n-butanol. The butyl esters were then separated and quantified by gas chromatography (Varian, model 3700) using a 6% diethyleneglycol succinate column. 5-Oxoprolinase activity was determined by the method of Stromme and Eldjarn (18). The intravesicular contents were extracted as described in (19) and analyzed for 5-oxoproiine and glutamic acid (17). [14C(U)]-5-oxoproline (286 mCi/mmol) was purchased from New England Nuclear. Unlabeled 5-oxoproline was from Sigma. Other amino acids were from California Corporation for Biochemicals. All other reagents were of analytical grade. 29

Vol. 105, No. 1, 1982

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Results and Discussion Plasma and urinary levels of 5-oxoproline. the plasma and urinary analyzed

the

levels

of 5-oxoproline

There is no data available on in the rabbit.

rabbit plasma and urine for their 5-oxoproline

concentration

of 5-oxoproline

We, therefore, content.

The

in the plasma was 30 ~M and the urinary excretion

was 0.36 ~mole/mg creatinine. 5-Oxoprolinase activity. is catalyzed by 5-oxoprolinase contain very high activities nmol/hr/mg protein

protein

in an ATP-dependent

Mammalian kidneys

of this enzyme.

the 5-oxoprolinase

as

in the purified

activity

of 5-oxoprolinase

nmol/hr/mg

5-oxoprolinase

protein

Since

in

the transport

version of 5-oxoproline

of giutamic

activity

and

border

in the homogenate

membrane

the trace activity

to glutamic

acid during

vesicles.

added

of 5-oxoproline

to glutamic

was maximal

the

Fig.

The amount

Glutamic acid in However, with

of glutamic acid (0.0006 nmol) were clear that there is no appreciable

acid under the normal transport assay

1 represents

the

typical pattern of

(20 ~M) into brush border membrane vesicles in NaCI

+ a Na -gradient.

vesicles

traces

It is, therefore,

5-0xoproline

of

with

We followed the formation

levels after 30 sec incubation.

(30 minute),

of 5-oxoproline

presence

Since

associated

to the incubation medium was 3 nmol.

in the medium.

KCI media.

and

the regular transport assay

would be negligible.

conditions. TTansport of 5-oxoproline. transport

protein

The specific

assays were carried out in the absence of ATP, con-

membranes

incubation

conversion

vesicles.

the cytosolic contamination.

the medium was below detectable

detected

In the present study, we

in the rabbit renal cortex homogenate

the brush enzyme,

and il nmol/hr/mg

acid in the incubation medium as a function of time.

of 5-oxoproline

prolonged

(20,21)

(8).

was 13 nmol/hr/mg

represents

the purified

The reported value is about 30

brush border membrane

is a soluble

purified membranes

reaction.

in the rat kidney homogenate

as well

using

to glutamic acid

in the guinea pig kidney homogenate

determined

0.03

The conversion of 5-oxoproline

exhibited The

the

'overshoot'

accumulation

of 5-oxoproiine

at 30 sec and then decreased

30

phenomenon

in the

inside the

due to the effiux of the

Vol. 105, No. 1, 1982

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

c

915 E o

E

c

0

'

,//-r-/~r-

Incubation Time (minutes) Fig. L

Time

course

of 5-oxoproLine

trausport

in NaCi and KCi media.

The vesicles were incubated with labeled 5-oxoproline varying periods of time in NaCi and KCi media. -

(20 pM) for

- NaC~ medium

o---o KCi medium

amino acid. of

No overshoot

5-oxoproline

in KCI

phenomenon

medium

was

was observed

in KCI medium.

lower than the transport

The transport

in NaCI m e d i u m

at

+

all

the

initial

transport line

(30 min)

the vesicles

and At

this

final

the peak

5-oxoproline

was

min

the

incubation.

transport

was

incubation

was found

medium

reached

The

Na -dependent

the

overshoot,

twice

space,

to be

of the results

the

the

was

The

of

non-specific

to infinite

osmolarity

3]

at prolonged

incubation

in both NaCi and KCI concentration

transport the m e d i u m to

binding

of

value.

to the membranes

proportional

of

of 5-oxopro-

intravesicular

equilibrium

stimulation

The amount

the same

binding

as a function

inversely

2).

value

that of the equilibrium

non-specific

measured

(Fig.

an equilibrium

level of transport

between

intravesicular

incubation)

polation

of

almost

To distinguish into

of

was 13 fold at 15 sec and 6 fold at 30 sec.

inside

media.

periods

the

and

transport

of 5-oxoproline osmolarity. osmolarity

calculated

by

(30 The

of

the

extra-

was 15% of the transport

observed

VOI. 105, No. I, 1982

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

1,5-

,= x

~I0o c

05-

Osmolarity"1 Fig.

Effects of mediu~ osmolarity on the initLal transport of 5-oxoproiine in NaCi medium.

2

and equilibrium

To calculate the initia$ rates of transport, the vesicles were first preequilibrated in Na -free medium of varying osmolarities for 30 min. The vesicles were then incubated with labeled 5-oxoproline (20 uM) for 30 see. in respective osmolar solutions containing NaCI. To calculate the equilibrium transport, the vesicles were directly incubated with labeled 5-oxoproline (20 ~M) for 30 min in NaCI medium of varying osmoiarities. Initial rates of transport o----o Equilibrium transport

at

300 mosm.

studied

using

+

Na -free assay. but

The

effect

a 30

media

of osmolarity

sec

incubation.

of varying

The initial

the binding

was

less

predominant

portion

osmotically

responsive

Intravesicular 30 sec incubation

of

The vesicles

osmolarities

transport

before

rates varied

than

of transport

total

was

pre-equilibrated

in the transport

with osmolarity

transport

at

therefore,

in

(Fig.

300 mosm. occurs

2),

The

into

an

space.

Analysis

of the vesicles

rates

they were used

transport,

intravesicular

were

inversely

5% of the

5-oxoproline

contents.

on the initial

with

of

the

20 u M

intravesicular

contents

[±4C]-5-oxoproline

after

in the presence

+ of a Na -gradient form

of

glutamic

showed

5-oxoproline. acid was

that more

Only

trace

than

99% of the radioactivity

amounts

detected.

32

of radioactivity

was

in the

in the

form

of

Vol. 105, No. 1, 1982

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Tab Le 1

Effects of other amino acids on the initial rates of Na -~ependent 5-oxoproiine transport

Addition

Relative Transport

None Isoleucine Alanine Valine Tryptophan Serine GLutamine 5-o×oproiine Pheny[aianine Threonine Methionine Leucine Pro[ine Hydroxyproline Glycine Lysine Arginine Giutamic acid Aspartic acid Pipecolic acid Glycyl-proiine

i00 37 ± 7 40 ± 8 46 ± 7 49 i 4 51 ± i3 57 i 2 57 i 9 58 ± 2 61 i 5 67 i 2 80 i 5 75 i 8 102 ± 5 104 ± 5 i13 i 7 116 ~ 9 i02 ± 9 i04 i 4 i04 i 3 112 ± Ii

Renal brush border membrane vesicles were incubated with labeled 5-oxoproline (20 ~M) at 25°C for 30 sec in NaCI and KCI media. The concentration of unlabeled amino acids in the incubation medium was 2 mM. The Na -dependent 5-oxoproline transport was calculated by substracting the transport observed in KCI medium from the transport in NaCI medium. The control value for the Na+-dependent transport was 18.4 i 1.8 pmol/mg protein and this was taken as i00.

Effects the

effects

transport

of

of

markedly caused

of other amino acids acidic

5-oxoproline.

inhibited

The

basic

Na+-dependent

transport

amino

a small but highly

reproducible

inhibition while

aspartic

acid

transport. hydrolysis inhibition. acids

basic amino acids and

glutamic

Pipecolic by

renal These

share

border

results a

common

of

membrane

clearly

show

transport

except

glycine.

vesicles that

33

Proline

hydroxyproline

and the acidic

had no

amino acids

on 5 - o x o p r o l i n e

a d i p e p t i d e h i g h l y resistant (19)

did

5-oxoproline

mechanism

border membrane.

I shows

5 - o x o p r o l i n e was

also did not have any effect

acid and g l y c y l - p r o l i n e , brush

acids

lysine and arginine

acid

Table

amino acids and imino acids on the

the neutral

The

by

and

transport.

all

effect.

amino

neutral,

on 5 - o x o p r o l i n e

in

the

not

and

rabbit

cause

the renal

any

neutral brush

to

Vol. 105, No. I, 1982

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

The inhibition, Because would

though weak,

of the structural

expect

Glycine,

a greater

proline

by proline

similarities

inhibition

of

and hydroxyproline

system in the kidney.

However,

is an interesting observation.

between proline and 5-oxoproline, 5-oxoproline

are known

transport

by proline.

to share a common

transport

a number of recent investigations

show that

proline also interacts with many neutral amino acids during transport. has been shown to inhibit membranes

(22).

phenylalanine,

surprising.

Proline

alanine transport in the rabbit renal brush border

Similarly,

a number

cause significant

kidney (23,24).

one

of

neutral

amino

acids,

especially

inhibition of proline transport

in the rabbit

The inhibition caused by proiine is, therefore,

not altogether

This may be one of the possible reasons for the elevated plasma

concentration of proline in 5-oxoprolinuria. Pipecolic animals. acid,

acid has

There

proline

and

5-oxoprolinuria competition present

are

long been known as a product of lysine metabolism in

significant

structural

5-oxoproline.

associated

with

between proline

study shows

Our

similarities

recent

findings

5-oxoprolinase

and pipecolic

that pipecolic

between pipecolic

with a patient with

deficiency

acid during

(ii)

suggested

transport.

acid does not compete with

But,

the

5-oxoproline

during transport. The data presented ciently

reabsorb

tration

gradient,

Once inside

5-oxoproline thus

the tubular

of 5-oxoprolinase

in this paper show that the rabbit kidney can effifrom the glomerular filtrate against a concen-

effectively cells,

avoiding

its elimination

in the urine.

it can be utilized because of the high levels

activity in the renal tissue.

Acknowledgements: This study was supported in part Research Grants AM[3L50 and FR-5365.

by National

Institutes

of Health

References: I. 2. 3. 4.

Van der Werf, P. and Meister, A. (1975) Adv. Enzymol., 4 3 Arme~Itrout, R.W. and Doolittie, R.F. (1969) Arch. Biochem. 132 80-90. Szewczuk, A. and Kwiatkowska, J. (i970). Eur. J. Biochem., Fink, M.L., Chung, S.I. and Folk, J.E. (1980) Proc. Natl. (USA), 77 4564-4568. 34

519-566. Biophys., 1 5 92-96. Acad. Sci.

Vol. 105, No. I, 1982 5. 6. 7. 8. 9. I0. ii. i2. 13. 14. 15. 16. [7. i8. 19. 20. 21. 22. 23. 24.

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Abraham, G.N. and Podell, D.N. (1981) Mol. Ceil. Biochem., 3 8 181-190. Van der Werf, P., Orlowski, M. and Meister, A. (1971) Proc. Natl. Acad. Sci. (USA), 68 2983-2985. Van der Werf, P., Stephani, R.A. and Meister, A. (1974) Proc. Natl. Acad. Sci. (USA), 71 1026-1029. Wolfersberger, M.G. and Tabachnick, J. ([974) J. Invest. Dermatol., 62 587-590. Oberholzer, V.G., Wood, C.B.S., Palmer, T. and Harrison, M.B. (1975) Clin. Chim. Acta., 62 299-304. Wellner, V.P., Sekura, R., Meister, A. and Larsson, A. (1974) Proc. Natl. Acad. Sci. (USA), 71 2505-2509. Roesel, A., Hommes, F.A. and Samper, L. (1981) J. Inher. Metab. Dis., 4 89-90. Larsson, A., Mattsson, B., Wauters, E.A.K., Van Gool, J.D., Duran, M. and Wadman, S.K. (1981) Acta Paediatr. Scand. 70 301-308. Eldjarn, L., Jellum, E. and Stokke, O. (1972) Clin. Chim. Acta, 4 0 461-476. Marstein, S. and Terry, T.L. (1981) Clin. Chim. Acta., 109 13-20. Ganapathy, V., Mendicino, J. and Leibach, F.H. (1981) J. Biol. Chem., 256 i18-i24. Ganapathy, V., Mendicino, J. and Leibach, F.H. (1981) Biochim. Biophys. Acta., 642 381-391. Hagenfeldt, L., Larsson, A. and Anderson, R. (1978) N. Eng. J. Med., 299 587-590. Strophe, J.H. and Eldjarn, L. (1972) Scand. J. Clin. Lab. Invest., 29 225-342. Ganapathy, V., Mendicino, J., Pashley, D.H. and Leibach, F.H. (1980) Biochem. Biophys. Res. Commun., 97 I133-Ii39. Wendel, A. and Flugge, U.I. (1975) Noppe Seyler's Z. Physiol. Chem., 356 873-880. Van der Werf, P., Griffith, O.W. and Meister, A. (1975) J. Biol. Chem., 250 6686-6692. Fass, S.J., Hammerman, M.R. and Sacktor, B. (1977) J. Biol. Chem., 252 583-590. McNamara, P., Ozegovic, B., Pepe, L.M. and Segal, S. (1976) Proc. Natl. Acad. Sci. (USA) 73 4521-4525. Hammerman, M.R. and Sacktor, B. (1977) J. Biol. Chem., 252 591-595.

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