Subcellular distribution and some properties of homocarnosine-carnosine synthetase from chick red blood cells

Comp. Biochem. Physiol., 1976, Vol. MB, pp. 523 to 525. Peroamon Press, Printed in Great Britain

SUBCELLULAR DISTRIBUTION A N D SOME PROPERTIES OF H O M O C A R N O S I N E - C A R N O S I N E SYNTHETASE FROM CHICK RED BLOOD CELLS RONALD H. NO AND FINLEY D. MARSHALL* Department of Biochemistry, The University of South Dakota School of Medicine, Vermillion, SD 57069, U.S.A.

(Received 2 October 1975) Abaract--1. Subcelhilar fractionation of homogenate of chick red blood cells was achieved by means of differential centrifugation. 2. The results indicate that most of the activity of homocarnosine-carnosine synthetase can be recovered in the soluble fractions while the nuclear fraction contains most of the particle-bound enzyme activity. 3. The enzymes from two of the soluble fractions containing highest activity were partially purified and their properties were compared. 4. Mter centrifuging the homogenate of red blood cells at 100,000 g to yield a supernatant and a pellet fraction, repeated washing of the latter resulted in release of enzyme activity into soluble form.

INTRODUCTION The presence of homocarnoaine--carnosine synthetase has been demonstrated in brain, heart, liver and muscle of rat, mouse, frog and chick (Ng & Marshall, 1976). Several physiological functions for carnosine have been suggested including as a neurotransmittcr in the primary olfactory pathway (Margolis, 1974) and as a component in muscle contraction (Severin et al., 1963). Recently, the presence of carnosine in nucleated erythrocytes of chick and frog was reported by Van Balgooy et al. (1974). However, it was not shown if the synthetic enzyme was actually present in the erythroeytes. The purpose of this study was to detect the presence of homocarnosine-carnosine synthetase in chick red blood cells (RBC), to study its subeelldar distribution and to compare properties to those reported on the rat brain enzyme .(Skaper et al., 1973; Ng & Marshall, 1974). The results of these studies are reported here. MATERIALS

L-[-ring-2-t4C]histidine (specific activity 55.4 mCi/mmole) was purchased from Amersham-Searle, Des Plaines, Illinois. I-l-x'~c]-/~-alanine (specific activity 3.6mCi/mmole) was purchased from ICN Pharmaceuticals, Inc.. Irvine. California. All other chemicals were obtained from Sigma Chemical Company, St. Louis, Missouri. Chicks, GaUus domesticus of Babcock B-370 strain, 12 days old, were obtained from Simpson Hatchery, Yankton, South Dakota. METHOD

Blood was drawn from the chicks by means of cardiac puncture, using sodium citrate as an anticoagulant. All subsequent steps were carried out at 0--5°C. The blood was centrifuged at 1500 f for 10 vain to separate the red blood cells from the plasma. The red blood veils were washed once in Ringer's solution and then centrifuged at * To whom requests for reprints should be directed.

1500g for 10 min. The cells were resuspended in 4 vol of a sucrose medium containing 0.25 M sucrose, 0.03M MgCI2, I mM EDTA, 5 mM dithioerythritol and 10raM TrisvHCl, p H 7.4. The suspension was homogenized in a glass homogenizer using a teflon pestle. The homogenate was used to perform the subcellular fractionation by means of differential centrifugation as described by Dowben (1971). Carnosine and homoearnosine synthesis was assayed in each of the suheellular fractions. The assay method was the same as that reported on the distribution of homecarnosine--carnosine synthetase in tissues of rat, mouse, chick and frog, except that sodium bicarbonate and v-81ucose were omitted in the assay medium since they decrease the enzyme activity slightly if present in the medium. In order to compare the properties of the enzyme from 'two of the soluble fractions, a partial pUrifiCation was 'rt~ade. The two fr~tions were first shell-frozen and lyophilized. The ly0philized powder was dissolved and subjetted to avamonium sulfate fraetionation (30%0 saturation). After centrifugin8 at 10,000g for 15 min, the pellet was suspended in 50 mM Tris-HCI buffer, pH 7.4, containing 5 mM dithioerythritol, and served as the source of enzyme to be assayed. In a separate experiment red blood cells were washed once in l~inger's solution and then homogenized in 10 mM Tris-HC1 buffer, pH 7.4, containing 5 mM dithloerythritol, 50 mM sodium bicarbonate and 31 mMD-glucose. Sodium bicarbonate and D-glucoSe were used only in the homogenization step and not in the enzyme assay. This procedure resulted in highest enzyme activity. The homogenate was centrifuged for I hr at 100,(D00. Half of the pellet was suspended in the buffer (10 mM Tris--HCl, pH 7.4) containiug 5 mM dithioerythritol. Another half of the pellet was suspended in the buffer containing 0.2 M KCI and then added Triton X-100 (1.5%, v/v). The suspensions were centrifuged at 100,000 1 for 1 hr. The supernatants and the original suspensions were assayed for enzyme activity.

RESULTS AND DISCUSSION

SubceUular fractionation o f RBC As shown in Fig. 1, plasma had no detectable enzyme activity. Howeve=, cgrnosine and homocarnosine synthesis was detected in RBC suspension, homo523

524

RONALD H. NG AND FINLEY D. MARSHALL Total activity*

blood RBCe'~Plasma (p) Chick

Washed ~ S u p e r n a t a n t

Carn.

($3)

Specific activity**

Homocarn.

Carn,

Homocarn.

P

0

0

0

0

S~

0.31

0.12

0.005

0.002

RBC Susp!ension (C)

C

532.99

225.73

0,08

0.03

Homogenke (H)

H

472.74

256.45

0.06

0.03

S~

266.09

139.01

0.08

0.04

N Mt

83.14 0.89

26.46 0.61

0.03 O.ll

O.Ol 0.08

S~

0.02

O.Ol

0.08

0.04

L

O.Ol

0.004

0.12

0.04

5u

25.06

6.10

0.20

0.05

Mc

0.06

0

0.06

0

SupernatantU~Pellet i Iresuspended ~-~Supernatant(S~) Nuclear (N) Mitochondrial (Mr) Supernatant I

~ Pellet resuspended --~Supernatant (S~)

I

~

Lys6somal (L) Supernatant

Micro

oma > Supernatant (Su) l (Mc)

Fig. 1. Carnosine and homocarnosine synthesis in subcellular fractions of chick RBC homogenate. * nmoles/hr per fraction. ** nmoles/hr per mg of protein. genate and subsequent subeellular fractions. The specific activities of the synthetase in RBC before homogenization are 0.08 nmole carnosine/h per nag protein and 0.03 nmole homocarnosine/h per nag protein. These values correspond to 8.16nmoles carnosine/h per ml blood and 3.46 nmoles homocarnosine/li per ml blood. This large amount of activity is comparable. to those reported on chick muscles (Ng & Marshall, 1976). After subcellular fractionation, most of the enzyme activity appeared in the soluble fractions. The soluble fraction $2, which was associated with the nuclear fraction, contained more than half of the total homogenate activity. The 100,000 g supernatant (Su) also exhibited significant amount of activity. Among the particulate fraction, the nuclear fraction had the most activity and mitochondrial, microsomal, and lysosomal fractions are in the order of decreasing activity. The ratio of carnosine and homocarnosine .synthesis is not the same for all fractions. This is probably due to the fact that these are relatively crude enzyme preparations. Presently only nucleated erythrocytes have been found to contain carnosine. In the present investigation, at least half of the total synthetase activity was found in the supernatant derived from the nuclear fraction. This may be significant since this amount of activity cannot be accounted for by trapped supernatant. The next highest amount of enzyme activity was found in the nuclear fraction. The distribution of the dipeptide synthetase in chick RBC is different from that in rat brain and in chick muscle where the enzyme activity is associated only with the 100,000 g supernatant. Further experiments are needed to determine if a unique function of earnosine in the nucleated RBC is the reason for the distribution of enzyme activity.

The enzyme was partially purified by ammonium sulfate fractionation prior to assay. As seen in Table 1, additions of p-chloromercurial benzoate (PCMB) and iodoacetamide inhibited the synthetases in both $2 and Su fractions. Addition of dithioerythritol, in one manner or another, stimulated the enzyme activity in both $2 and Su fractions. Therefore, it appeared that the enzymes from S~ and Su both had sulthydryl groups essential for enzyme activity. ATP, NAD and MgC12 are eofactors required for rat brain synthetase (Skaper et al., 1973). These compounds were tested in the present investigation and the results are shown in Table 2. In both $2 and Su fractions, ATP but not NAD was needed for enzyme activity. However, in the case of MgCI2, it seems to be required only by fraction $2. This phenomenon was unchanged when the experiment was repeated with fractions $2 and Su, regardless whether ammonium sulfate fractionation was carried out prior to assay. In Table 3, the ability of $2 and Su to form dipeptides with histidine and fl-alanlne was compared. It

Properties of the synthetases from fractions $2 and Su

*Carnosine synthesis was assayed.

In order to gain information to answer the question whether the synthetases from different snl~,,etlular fractions of RBC are identical, some properties of the synthetase were studied using fractions $2 and Su.

Table 1. The effect of sulfhydryl reagents on homocarnosine-earnosine synthetase from fractions $2 and Su Treatlllent

Final concentration (raM)

Control

%of control activity* S~ lO0

Su I00

+ pC~

OmOI5

13

+ Iodoacetamide

3.0

18

7

26

+ Dithioerythritol

2.0

74

148 ,

+ DithioePythritol**

I. 5

14g

86

A11 sulfhydryl reagents were added to the reaction mixture in the enzyme assay except in the case of ** where dithloerythritol was present throughout the enzymepreparatlon. Results are meansof two determinations.

Homocarnosine-cltrno sine synthetase Table 2. Cofactor requirements for homocarnosine-carnosine synthetase from fractions $2 and Su

525

Washed RBC

I

Total

Homogenate Reaction mixture

Carnosine

%of the control activity* S2

Su

~'-~-~Supernatant

(si)

activity* Homocarnosine

Sl

46.70

20.58

P2

91.38

57.50

S~

312.26

122.60

DP2

208.06

99.56

DSz.

298.21

150.46

Pellet (lO0,O00 g) Complete systm

I00

100

ATP omitted

I

5

NAD omitted

116

II0

1

I08

MgCl2 omitted

buffer

Suspension (Pz) ~Centrifugation Supernatant (S2) + buffer and KCl

*Carnosine synthesis was assayed.

Suspension (DP2)

Results are means of two determinations.

appears that for both $2 and Su, fl-alanine is a better substrate than GABA to form dipeptide with histidine, while L-alanine had no activity at all. In the synthesis of fl-alanyl dipeptide by $2, L-histidine appears to be a slightly better substrate than the substituted histidines. In the case of Su, where low activity was detected, there seems to have been no signiticant difference in enzyme activity among L-histidine, L-methylhistidine and L-3-methylhistidine. Based on the results from the present investigation, the synthetases from fractions $2 and Su appear to be very similar in properties. However, it cannot be concluded that the enzymes are identical since much more biochemical studies will be needed for this purpose.

Release of homocarnosine-carnosine synthemse from 100,000 g pellet of RBC homogenate In view of the presence of large amounts of enzyme activity in the supernatant fraction $2 which was associated with the nuclear fraction and the similarities in properties between the syntbetases of $2 and Su, a study was made to show whether or not the synthetase was loosely bound in the particulate fractions. As seen in Fig. 2, after washing the 100,000 g pellet with buffer containing KC1 and Triton X-100, the resultant supernatant (DS2) showed large amounts of activity. Moreover, approximately the same amount of activity was obtained in the supernatant ($2) if the 100,000 g pellet was washed with the buffer containing no KCI or Triton X-100. This is an indication Table 3. Substrate specificity of homocarnosine--camosine synthetase from fractions $2 and Su Substrate

/~

Dipeptide synthesized* S2

Su

~-alanine**

2.13

0.65

GABA**

0.57

0.23

L-alanine*t*

0

0

L-histidine****

3.45

0.42

L-l-methylhistidine****

2.75

0.48

L-3-methylhistidlne****

2.65

0.48

*nmoles/h/mg protein **L-[ring-2-1~C] hlstidine (specific activity 55.4mCi/mmol) was used. ***[U-~C] L-alanine (specific activity 58 mCi/mmol) and unlabeled histidine wereused. ****[l-l~C] B-alanine (specific activity 3.6 mCi/mmol) was used.

la Triton X-IO0 ed centrifuged Supernatant (DS~I

Fig. 2. Release of carnosine-homoeamosine synthetase from 100,000 g pellet of RBC homogenate. * nmoles/hr per fraction. that the synthetase is loosely associated with the particulate fraction and that the enzyme may be released during differential centrifugation of RBC homogenate. In conclusion, the detection of a large amount of activity of homoearnosine-carnosine synthetase in RBC in an animal which has activity in other tissues, particularly brain and muscle, suggests an important function requiring the synthesis of carnosine in RBC. It is also possible that these cells may supply carnosine to tissues which have little synthetic activity and where carnosine or one of its metabolic products may serve an important function. Preliminary investigations in this laboratory showed that drugs such as reserpine (Sandrill) and chlorpromazine (Thorazine) inhibited the synthetase in RBC. Further investigation will be carried out to study the effects of various agents on homocarnosine--carnosine synthetase in RBC in an effort to determine the function of carnosine in these cells.

Acknowledgements--This work was supported by NIH Grant NS-06137 and a grant from Huntington's Chorea Foundation. The authors wish to thank Mrs. Betty Hogan for technical assistance. REFERENCES DOWB~,~ R. M. (1971) Cell Biology, p. 174. Harper & Row, New York. M^ROOLISF. L. (1974) Carnosine in the primary olfactory pathway. Science, N.Y. 184, 9139-911. NG R. H. & M^RSI-Ud.LF. D. (1974) The effects of various agents in vitro on homoearnosine--carnosine synthetase from rat brain. Trans. Am. Soc. Neurochera. S, 170. No R. H. & M^RsrlAt~ F. D. (1976) Distribution of homocarnosine-carnosine synthetase in tissues of rat, mouse, chick and frog. Comp. Biochem. Physiol. ~wIB, 519-521. SEVBa~INS. E., BOC~L~RNIKOVAI. M., VUL'FSONP. L., GrtlooRowcn Y. A. & SOLO'EVAG. A. (1963) The biological role of carnosine. Biokhimiya 28, 510-516. SF,XP~R S. D., DAS S. & MARSI-~II F. D. (1973) Some properties of a homocarnosine-carnosine synthetase isolated from rat brain. J. Neurochem. 21, 1429-1445. VAN B^LOOOy J. N. A., MARS~^LL F. D. & ROBERTSE. (1974) Carnosine in nucleated erythrocytes. Nature, Lond. 247, 226-227.