Proton NMR spectroscopic studies of dipeptidase in human erythrocytes

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 305-312

Vo1.110, No. 1,1983 January 14, 1983

PROTON NMR SPECTROSCOPIC STUDIES OF DIPEPTIDASE IN HUMAN ERYTHROCYTES Glenn F. King, Michael J. York, Bogdan E. Chapman and Philip W. Kuchel Department of Sydney,

University Received October

of Biochemistry, Sydney, NSW, 2006, Australia

27, 1982

In studies on human erythrocyte metabolism in situ, high resolution (400 MHz) IH spin-echo N M R spectroscopy was used to follow the time dependence of hydrolysis of glycylglycine and L-cysteinylglycine in intact cells and their lysates. The concentration dependence of the hydrolysis of L-cysteinylglycine was described by a rectangular hyperbola with K m, 3.5 ± 0.6 mmol/£ lysate and Vmax, 64.2 ± 3.2 mmol/Z lysate/h. We demonstrated that glycylglycine readily enters the erythrocyte and we introduce a means of analysing the data from the coupled reaction sequence; the sequence consists of transport followed by enzyme catalysed hydrolysis.

Human erythrocytes and the cytosol;

contain proteolytic

and peptidases

a range of endopeptidase, activities

(3).

in both the membrane

most reports on the subject have dealt with activities

associated with the membrane proteinases

enzymes

(1,2).

The few reports on cytosolic

in the mature erythrocyte dipeptidylaminopeptidase

The function of proteolytic

suggest that there is

and aminopeptidase

enzymes in the erythrocyte

has

not been fully elucidated. It has been reported

(4) that rabbit erythrocytes

~-glutamyltranspeptidase-cyclotransferase over of erythrocyte of a dipeptidase work

glutathione.

to hydrolyse

(5), however,

the L-cysteinylglycine

has shown that neither

unrelated

thus the presence

to glutathione

dipeptidase

activity

differentiating

cell.

accounting

This cycle is dependent

cytes contain y-glutamyltranspeptidase white cells;

pathway

contain a functional

on the presence

formed. - More recent

rabbit nor human mature erythro-

in preparations

of dipeptidase

that are free of

activity appears

turnover in these cells.

are not,

to be

It is possible

is the residuum of activity present As dipeptides

for the turn-

in general,

that the

in the substrates

for

0006-29]X/83/0]0305-0850].50/0

305

Copyright © 1983byAcadem~ Pre~,Inc. Aft righ~ of reproductmn m any form rese~ed.

Vol. 110, No. 1, 1983

metabolic

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

reactions

the dipeptidase

the role of assisting amino acids

are h y d r o l y s e d

than by erythrocyte cells appears erythrocytes. spectroscopy intact monitor cytes,

lysates

to be rate In this (NMR)

erythrocytes

pool

(6).

limited

against

of dipeptides

to studying

Magnetic

dipeptidase

We also show h o w N M R has been

of an earlier

of g l y c y l g l y c i n e procedure

by intact

of the dipeptide

we show that N u c l e a r

adapted

MATERIALS

the loss of

lower rate by intact erythrocytes

by the transport

(and hydrolysis)

an e x t e n s i o n

could serve

(6).

The h y d r o l y s i s

communication

and lysates.

in the erythrocyte

of the o r g a n i s m

at a m u c h

can be easily

the transport using

in the p r o t e c t i o n

from the m e t a b o l i c

Dipeptides

activity

into the Resonance

activity

in

used to

in intact erythro-

(7).

A N D METHODS

L - C y s t e i n y l g l y c i n e w a s obtained from V e g a B i o c h e m i c a l s , Tucson, Arizona, USA, g l y c y l g l y c i n e from Sigma, Saint Louis, Missouri, USA, d e s f e r r i o x a m i n e mesylate from Ciba Laboratories, Horsham, W e s t Sussex, UK and E 2 , 3 , 4 , 6 , 6 , 2 H 5 ~ D - g l u c o s e from Merck, Sharp and Dohme, Point ClaireDorval, Quebec, Canada. A l l other reagents were of A R grade. Human erythrocytes w e r e p r e p a r e d from freshly drawn venous b l o o d by w a s h i n g twice in isotonic saline in IH20 and once in Krebs bicarbonate (8) in 2H20. The Krebs solution contained antibiotics (sodium p e n i c i l l i n G 27 mg/i, s t r e p t o m y c i n sulfate 50 mg/~ and a m p h o t e r i c i n B 2.5 mg/i) and i0 mM n i c o t i n a m i d e (9). The h e m a t o c r i t (Hc) of the cell suspensions was m e a s u r e d and they were then lysed by freezing and thawing. L - C y s t e i n y l g l y c i n e w a s reduced w i t h m e r c a p t o e t h a n e sulfonic acid and p u r i f i e d on a Dowex 2-X8 anion exchange resin b e f o r e use. The p a r a m a g n e t i c ion Fe 3+ was liganded by addition of FeCI 3 to d e s f e r r i o x a m i n e (i:i mole ratio). In some e x p e r i m e n t s the rate of hydrolysis of dipeptide by lysate p r e p a r e d from Hc = 0.8 cells was too rapid to follow by NMR. In these cases the lysate was diluted w i t h Krebs b i c a r b o n a t e solution to reduce the activity. Just prior to N M R measurements, 0.5 ml of lysate or cells was p l a c e d in a 5 m m o.d. N M R tube and h e a t e d to 37°C. V a r i o u s reagents were then added (see text). On addition of reagents the samples w e r e p l a c e d in a h e a t i n g b l o c k at 37°C for 1 min f o l l o w e d by N M R measurements. The zero time of the reaction was taken as the time reagents w e r e added. Measurements were started w i t h i n 3 h of drawing blood. 1

H N M R spectra were m e a s u r e d at 400 MHz in the F o u r i e r mode using a B r u k e r WM400 s p e c t r o m e t e r at 37°C. The inversion recovery spin-echo sequence (i0) 1 8 0 ° - T - 9 0 ° - T - 1 8 0 ° - T w i t h T = 0~06 s, and a repetition time of 1.0 s was used. A total of 64-256 transients were a v e r a g e d using a sweep w i d t h of 5000 Hz and 8192 data points. Chemical shifts are q u o t e d relative to t e t r a m e t h y l s i l a n e (TMS)/CCI 4 p r e s e n t in a small capillary inside the N M R tube. Glycine concentrations w e r e d e t e r m i n e d from the intensity ratio of the glycine ~CH 2 resonance to the TMS signal or the ergothioneine q u a t e r n a r y amine m e t h y l signal. C a l i b r a t i o n curves of glycine concentration vs intensity ratios w e r e p r e p a r e d by addition of known amounts of glycine to lysate samples.

306

Vol. 110, No. 1, 1983

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

RESULTS AND DISCUSSION Expanded spectra of erythrocyte lysate with i0 mM L-cysteinylglycine, various incubation times are shown ~n Fig.

i.

The group of resonances

marked a arise from the glycyl of L-cysteinylglycine, and the glycyl of glutathione.

at

showing an AB pattern,

Peak b is free glycine, peak c the C-5

proton of 2H5-glucose and peak d the quaternary amine methyl signal of ergothioneine.

Signals from the ~-CH 2 of cysteinyl and free cysteine are

upfield of the ergothioneine peak; assignment

in this region of the spectrum

is difficult as there is overlap of peaks with lysate resonances.

Peak e

is the ~-CH2-glutamyl signals of glutathione. It is evident from the spectra that the glycyl peak of L-cysteinylglycine fell

and the free glyc~ne peak increased with time.

From these

spectra, plots of free glycine concentration vs time g&ve initial velocities for the hydrolysis of L-cysteinylglycine by the lysate.

The dependence of

e

4.0

30

2.0

ID

PPM

Fi 9. i. 400 MHz IH NMR spectra of L-cystcinylglycine (i0 mM) in erythrocyte lysate.

307

Vol. 110, No. 1, 1983

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

80

..~

60

rn

f

40 e ,-4 ,-.4 o

20

0

,

I

.

I

10

•

,

I

20

.

I

30

,

I

40

,

I

50

60

L-CYSTEINYLGLYCINE(mM) Fig. 2. The rate of release of glycine versus L-cysteinylglycine concentration in erythrocyte lysate at 37°C.

the rate of release of free glycine g l y c i n e is shown in Fig. regression

(Ii)

K m of 3.5

±

intensities

2.

on the c o n c e n t r a t i o n

The data set was a n a l y s e d by n o n - l i n e a r

on the M i c h a e l i s - M e n t e n

relative

concentrations

by r e f e r e n c e

for h e m a t o c r i t

and dilution.

to be 4 to 5 days

of g l u t a t h i o n e 3000 times

to a g l y c i n e

therefore

(12).

reaction

the d e t e c t i o n

limit of our N M R e x p e r i m e n t s

(Fig.

2).

Since

species m u s t be less than ~ 0.i mM;

w h i c h is still g r e a t l y

catalysed

of

and c o r r e c t e d

has been

of the 2 m m o l / i

cells w h i c h is

of the L - c y s t e i n y l g l y c i n e

in v i v o L - c y s t e i n y l g l y c i n e the c o n c e n t r a t i o n s

at this s u b s t r a t e

i.i x 10 -6 M

the

cells/h

in excess of the rate of net G S H t u r n o v e r (13).

is b e l o w

of the free

concentration

r e a c t i o n w o u l d be 1.8 m m o l / Z

but n o t of that in rat k i d n e y

concentration

curve

has a net rate of % 2 x 10 -5 mol/I/h,

less than the m a x i m a l v e l o c i t y

rate of the d i p e p t i d a s e

The peak

c o n v e r t e d to

in human e r y t h r o c y t e s

Synthesis

an a p p a r e n t

lysate/h.

calibration

(dipeptidase)

cytes,

and y i e l d e d

to a TMS r e f e r e n c e w e r e

The t u r n o v e r time of g l u t a t h i o n e estimated

equation

0.6 mM and a V m a x of 64.2 ± 3.2 m m o l / i of glycine

of L - c y s t e i n y l -

in erythro-

Only at an L - c y s t e i n y l g l y c i n e

(which m a y be less than t h a t of the enzyme

308

concen

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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

b a

4

3

2 PPM

1

0

Fig. 3. 400 MHz IH NMR spectr~n of glycylglycine (8 mM) in a suspension of human erythrocytes (Hc= 0.8) after incubation at 37°C for 4 h.

tration) would the enzyme, assuming no interference by effectors, become rate limiting.

Therefore the high activity of the enzyme in erythrocytes raises

the possibility that it has a role in addition to L-cysteinylglycine turnover for glutathione synthesis.

Fig. 3 shows an expanded of intact erythrocytes

1

H NMR spectrum of glycylglycine in a suspension

(Hc = 0.8) washed in Krebs bicarbonate buffer in 2H20.

The peak marked a is from the glycine residues of glycylglycine and that marked b from free glycine.

The transport of glycylglycine can be followed

relatively easily using ]H NMR.

The method depends on molecules inside and

outside the cell having different specific intensities

(7).

In the present

system this was arranged by changing the bulk magnetic susceptibility of the outside medium, which had the effect of enhancing magnetic field gradients outside the cell and broadening and suppressing signals from molecules outside the cell. complex Fe (14).

3+

This waS achieved by the addition of the paramagnetic

-desferrioxamine which is known to not cross the cell membrane

The complex is effective at low concentration

alter the metabolic properties of the cell.

309

(~ 1 mM) and does not

Vol. 110, No. 1, 1983

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10

0

8

• g

0 0

0

0

6

4

0

| 20

0

| 40

* 60

| 80

* 100

| 120

* 140

TIME(min) Fig. 4. Plots of intensity relative to ergothioneine of the glycylglycine (O) and free glycine (X) signals with time. Sample conditions; Hc = 0.8, I0 mM glycylglycine and 1 mM Fe3+-desferrioxamine.

Fig.

4 shows the signal

as a function transported

of time.

also i n c r e a s e d

by the dipeptidase.

rate of glycine

release

Under identical

conditions

rate of glycine

release

Hydrolysis times

was

By reference

of h e m a t o c r i t

by intact

and substrate

cells was

following

for e x t r a c e l l u l a r

gl and g2 are the glycine

and ~l,i and ~2,o are the c o e f f i c i e n t s extracellular

glycylglycine

from a standard glycylglycine

curve

peaks

which

to peak intensity;

for glycine

in a lysate,

at the c o m m e n c e m e n t

310

these

ii

into

by using the

concentration:

- 2~l,i)

intensities

relate

lysate/h.

of glycylglycine

(4gl + g2 - 2 g i ~ i , ~ / ( ~ 2 , o and g l y c y l g l y c i n e

the

approximately

and was q u a n t i f i e d

glycylglycine

cells/h.

concentration

therefore

was

curve the

mmol/Z

18.9 ± 0.8 mmol/i

was

environment.

glycylglycine

to a calibration

The rate of t r a n s p o r t

=

as the peptide

intracellular

to be 1.7 ± 0.i

than its hydrolysis,

where

increased

homogeneous

the cells was faster

Eglycylglycine~outside

and free glycine

as the i n t r a c e l l u l a r

calculated

than b y lysates.

expression

peak

by lysed cells was

of g l y c y l g l y c i n e

slower

of g l y c y l g l y c i n e

The g l y c y l g l y c i n e

into the m o r e m a g n e t i c a l l y

The free glycine p e a k hydrolysed

intensity

'

in the spectrum

intracellular values were

glycine obtained

and from the initial

of the reaction.

and

combined

The formula was

Vol. 110, No. 1, 1983

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

14

v

0

10

20

30

40

50

60

70

80

90

100

110

120

TIME(min) Fig. 5. Extracellular (O), intracellular glycylglycine (•) and intracellular glycine (A) in a suspension of erythrocytes (Hc = 0.8) with 1 mM Fe3+-desferrioxamine. Data from Fig. 4 analysed according to the formula in the text. developed assumed efflux

from the expression that ~2,i = 2~i,i

5 shows

the time course

from the data of Fig. regression

analysis

± 0.3 mmol/~

of e x t r a c e l l u l a r

glycylglycine

glycine

acid and p e p t i d e erythrocyte

peptidases

glycylglycine activation

in the gut

is unknown,

angiotensin

may possibly

constituent

acids.

amino

(16).

after

linear

the concent-

Interestingly,

the

that of the c o r r e s p o n d i n g demonstrated

for amino

The total p h y s i o l o g i c a l

in p l a s m a II)

role of

of

during hormone

or during

inflammation

enter erythrocytes and be broken

311

7.2

the concentration

but in v i e w of the rapid entry released

analysed

30 min was

free glycine

inferred.

I + angiotensin

clot dissolution,

and

and

reaction From

between

that has been

it, and other peptides

(e.g.

was

is many times

(15), a feature

absorption

influx

and i n t r a c e l l u l a r

rate of the dipeptide

acid,

coupled

expression.

the difference

glycylglycine

verified)

mass

the time of the experiment.

of the o v e r a l l

the p r e v i o u s

by taking

of i n t r a c e l l u l a r

transport amino

4 using

during

the rate of g l y c y l g l y c i n e

cells/h;

of g l y c y l g l y c i n e

(which w a s e x p e r i m e n t a l l y

from the cell is n e g l i g i b l e

Fig.

ration

for the c o n s e r v a t i o n

and

down to the

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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

ACKNOWLEDGEMENTS: This work was supported b y the National Health and Medical Research Council of Australia. Valuable technical and computing assistance was obtained from Mr. W.G. Lowe and Mr. B.T. Bulliman respectively. REFERENCES i. 2. 3.

4. 5. 6. 7. 8. 9. i0. Ii. 12. 13. 14. 15. 16.

Moore, G.L., Kocholsty, W.F., Cooper, D.A., Gray, J.L. and Robinson, S.L. (1970) Biochem. Biophys. Acta. 212, 126. Tokes, Z.A. and Chambers, S.M. (1975). Biochem. Biophys. Acta. 389, 325. Pontremoli, S., Milloni, E., Salamino, F., Speratore, B., Michetti, M., Benatti, A., Morelli, A. and de Flora, A. (1980). Eur. J. Biochem. Ii0, 421. Palekar, A.G., Tate, S.S. and Meister, A. (1974). Proc. Nat. Acad. Sci. USA. 71, 293. Srivastava, S.K., Awasthi, Y.C., Miller, S.P., Yoshida, A. and Beutler, E. (1976). Blood 47, 645. Krzysik, B.A. and Adibi, S.A. (1977). Amer. J. Physiol. 233, E450. Brindle, K.M., Brown, F.F., Campbell, I.D., Grathwohl, C. and Kuchel, P.W. (1979). Biochem. J. 180, 37. Krebs, H.A. and Henseleit, K. (1932). Z. Physiol. Chem. 210, 33. Kuchel, P.W., Jarvie, P.E. and Conyers, R.A.J. (1982). Biochem. Med. 27, 95. Rabenstein, D.L. (1978). Analytical Chem. 50, 1265. Osborne, M.R. (1976). Aust. Math. Soc. B. 19, 343. Dimant, E., Landsberg, E. and London, I.M. (1955). J. Biol. Chem. 213, 76. Orlowski, M. and Meister, A. (1970). Proc. Nat. Acad. Sci. USA. 67, 1248. Jones, A.J. and Kuchel, P.W. (1980). Clin. Chim. Acta. 104, 77. Young, J.D. and Ellory, J.C. (1977) in Membrane Transport in Red Cells (Ellory, J.C. and Lew, V.L., eds.) pp.301-325, Academic Press, New York. Adibi, S.A. and Morse, E.L. (1971). J. Clin. Invest. 50, 2266.

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