151

Clinica Chimica Acta, 85 (1978) 151-157 0 ElsevierlNorth-Holland Biomedical Press

CCA 9273

EFFECTS OF MACROMOLECULAR LEUKOCYTE fl-GALACTOSIDASE

KAORU

KIGUCHI

*, YOSHIKATSU

COMPOUNDS ACTIVITY

ET0 and KIKUMARO

ON HUMAN

AOKI

Department of Pediatrics, Tokyo Jikei University School of Medicine, Nishishinbashi, Minato-ku, Tokyo (Japan) (Received

October

21st, 1977)

The enzyme properties of leukocyte P-galactosidase and the effects of macromolecular compounds on its activity were studied in detail. It was demonstrated that various macromolecular compounds markedly influence the activity of P-galactosidase. These phenomena should be taken into account when considering the biochemical abnormalities of tissues of patients with mucopolysaccharidoses and mucolipidoses.

Introduction Congenital deficiency or hypoactivity of any lysosomal enzyme causes some metabolites to accumulate in the lysosomes and induces a so-called “storage disease”. A congenital deficiency of fl-galactosidase (EC 3.2.1.23), one of the lysosomal enzymes, occurs in GM1 gangliosidosis and Krabbe’s disease. Several studies indicate that GM, gangliosidosis may be generalized fl-galactosidase deficiency [ 11, while Krabbe’s disease is due to a specific /3-galactosidase (galactocerebroside fl-galactosidase) deficiency [ 21. Non-specific /3-galactosidase deficiency has not been observed in tissues of patients with Krabbe’s disease [ 21. On the other hand, a remarkable reduced activity of fl-galactosidase is seen in tissues of patients with various types of mucopolysaccharidosis and mucolipidosis, type II. In 1969, Ho and O’Brien [3] found the deficiency of a specific component of P-galactosidase in the case of the Hurler syndrome and regarded Later, Matalon and Dorfman [4] it as the etiology of this syndrome. reported that the Hurler syndrome was due to a deficiency of a-L-iduronidase. In the other types of mucopolysaccharidosis, various kinds of specific enzyme * To

whom

correspondence

should

be addressed.

152

deficiency were found. Therefore, reduced activity of P-galactosidase in mucopolysaccharidoses is assumed to be caused by a secondary inhibitory effect by the accumulated compounds. Kint et al. [5,6] suggested that the decrease of fl-galactosidase activity in tissues of these disorders was induced by the inhibitory action of accumulated mucopolysaccharides (MPS); the reduction in enzyme activity in tissues of Hurler and Hunter disease could be neutralised by cetylpyridinium chloride (CPC) which is known to form complexes with mucopolysaccharides. In the present communication, investigation of the enzyme properties and the effects of various compounds on leukocyte fl-galactosidase are reported, since leukocytes can be collected with relative ease and have diagnostic value. Materials and methods Human leukocytes were prepared by the method of Snyder and Brady [7] and subjected to the estimation of fl-galactosidase activity by the method of Gatt and Rapport [ 81. Leukocyte /3-galactosidase was separated by isoelectric focusing using the method of Vesterberg and Svensson [9] and also separated on Sephadex G-200. The column (2.0 X 40.0 cm) was equilibrated with 10 mM citrate phosphate buffer (pH 6.0). The sample was collected in portions of 0.5 ml at a column speed of 0.1 ml per minute. Results Fig. 1 shows the results of separation of the activity by isoelectric focusing. Isoelectric points (~1) of two peaks were pH 3.9 and 4.5, respectively. When the activity was estimated at pH 4.2 and 6.0, it exhibited a pattern as shown in Fig. 1. At the buffer pH of 4.2, the peak of P-galactosidase activity with ~13.9 was inhibited. When estimated at pH 6.0, the activity at both peaks was decreased.

5

10

15

20

25

30 fraction

35 No

40 0 5 ml/tube

Fig. 1. The activity profile of leukocyte P-galactosidase. by isoelectric focusing (Ampholyte PH 3.5-10.0). a--., pH; l -------m. 0.1 M citrate phosphate buffer, PH 5.0; ‘b- - -;, 0.2 M NaCl in 0.1 M citrate phosphate buffer, pH 4.2: ‘1pn, 0.1 M citrate phosphate buffer, PH 6.0.

153

r

-

~

void .

rc

10

20

30

40

-1

60

60

fraction No 0.5~1 /tube Fig. 2. The activity

profile

of leukocyte

&galactosidase,

by Sephadex

G-200

gel filtration.

When Sephadex G-ZOO was used to separate the enzyme, two peaks were obtained similarly in two fractions, tube No. 26 and No. 30, as shown in Fig. 2. Leukocyte fl-galactosidase had a IT, value of 3.1 X 10e3 M. The two peaks, fraction I and II were separated by Sephadex G-200. Fraction I had a somewhat larger molecular weight and a KNI value of 2.3 X 10s3 M (Fig. 3). The peak of fraction II had a somewhat small molecular weight and a KM value of 0.9 X 10d3 M (Fig. 3). Various concentrations (25, 50, 250 and 500 Erg/O.4 ml) of chondroitin sulfate (Ch-s)A, B and C were added to the incubation medium and their effects on /3-galactosidase activity were examined. They revealed a mild inhibitory effect on the enzyme activity, although there was some fluctuation in this effect (Fig. 4). When MPS (Ch-s A and B) and leukocytes were first incubated and then mixed with the substrate to estimate the enzymic activity, the inhibitory effect was enhanced in proportion to the incubation time (Fig. 5).

l/v

(1)

(3)

(2)

0,

0. e

/ /

4 -2

~

A 2 4 6 8 10 * l/s mM

2 4 6 8 10

Fig. 3. KM of leukocyte R-galactosldase: Sephadex G-200; (3) KM of the fraction

-2/

2 4 6 8 IO

(1) KM of the sU!XEmatant; (2) KM of the fraction II separated on Sephadex G-200.

I separated

On

/ 100

1

l

300

ZOO

400 MPS

Fig. 4. The effects sulfate A; ~.------~

of mucopolysaccharides , chondroitin sulfate B;

I

1

30

45

I

500

15

mtn.

..ug

on leukocyte fl-galaetosidase activity. chondroitin sulfate C. n ---------•,

l-----•,

chondroitin

Fig. 5. The effects of mucopolysaccharides on leukocyte &Mactosidase activity. (Chondroitin sulfate is added and incubated with leukocyte enzyme before the addition of substrate into incubation mixture.) chondroitin sutfate B (250 ~gt0.4 mIf. A-, ~hondro~t~~lfate A (250 ~&Z/0.4ml): LJ-------I,

Then the effect of serum (10, 25 and 50 p1/0.4 ml) on the P-galactosidase activity was examined. When IO ~1 of serum was added to the incubation medium, the inhibition of ~-gaia~tosidase was nearly 90% of that of control. When serum was added to fractions I and II, its inhibitory effect was more distinct on fraction I than that on fraction II (Fig, 6).

10

I

20 pi

I

/

1

30

40

50

Fig. 7. The effects after dialysis; b-L,

25

10

pl serum

serum

Fig. 6. The effects of serwn on the activity fraction II. m-------a, fraction I: 0 -fl,

on leukocyte

l 50

/

I l

P-galaetosidase

of the addition of dialyzed serum on leukocyte before dialysis.

isoenzyme

&galactosidase

fraction

activity.

I and II.

A,

serum -0 OIlI) I

10

1

I

23

50

Fig. 8. The effects

of the addition

Fig. 9. The effects of the addition dase activity (serum: pi/incubation

L”

1”

,III serum

iJI of heparin on leukocyte

fl-galactosidase

activity

(heparin;l

ml = 10 mg).

of concanavalin A on the inhibition by serum of leukocyte medium, Con A: pglincubation medium).

P-galactosi-

To verify the presence of the factor inhibiting /3-galactosidase activity in the serum, serum was dialyzed against 10 mM phosphate buffer (pH 7.0) at 4°C for 24 h. The inhibitory effect was almost removed after dialysis (Fig. 7). As an example of another high molecular substance, heparin was used in the experiment. Various concentrations (0.1, 0.25 and 0.5 mg/O.4 ml) of heparin I-

5: 5’ E: 8

B M

g’

st : 8

:.

3”

2””

13”

,ug Con A

_I’

,uug Con A

IL-’

Fig. 10. The effects of the addition of concanavalin A only and with serum on leukocyte fi-galactosidase Con A + serum 10 ~1; a-------m, Con A only @g/incubation medium 0.4 ml); 7’1, activity. ~-0 Con A + serum 20 ~1. Fig. 11. The effects of the addition of concanavalin A on the inhibition with mucopolysaccharide (chonCon A only (@g/incubation medium droitin sulfate B) for leukocyte @galactosidase activity. l-, Ch-s B 125 pglincubation medium. 0.4 ml); -v, Chs B 25 pglincubation medium; 0 --O,

inhibited /3-galactosidase activity as shown in Fig. 8. When 50, 150 and 500 iig, 0.4 ml of Concanavalin A (Con A) was added to each tube, P-galactosidase activity was activated in proportion to the concentration of Con A. When Con A and serum were added simultaneously, the remarkable inhibitory effect with serum disappeared (Figs. 9 and 10). Thus, correlation between the Con A and MPS (Ch-s B) was observed. When Con A and MPS (Ch-s B) were added at the same time and incubated for 30 min, P-galactosidase activity was restored and activated to approx. 150-180% of original activity. Therefore, the profound inhibitory effect of MPS disappeared after the addition of Con A (Fig. 11). Discussion Although leukocyte a-galactosidase activity has been measured often, enzyme characteristics of leukocyte P-galactosidase have not been studied well. The present study reveals by the use of either isoelectric focusing or Sephadex G-ZOO that P-galactosidase activity in a leukocyte preparation consists of at least two major fractions. It is well documented that /3-galactosidase activity is deficient or reduced in various tissues of patients with mucopolysaccharidoses or mucolipidoses. The reduced activity of this enzyme may be caused by a secondary phenomenon probably due to accumulated MPS in patients’ tissues, since their primary enzyme defects were recently clarified in these disorders [lo]. However, there have been few systemic investigations of the mechanism of reduced P-galactosidase activity in these disorders. Kint et al. [5,6] reported that the accumulated MPS in patients’ tissues might be bound to fl-galactosidase and thus decrease the activity of P-galactosidase. An abnormal isoenzyme pattern may be produced by accumulated MPS. In the present investigation, it is also demonstrated that leukocyte P-galactosidase activity is reduced when both Ch-s and leukocytes enzyme are incubated together. Furthermore, this inhibitory effect is enhanced according to the incubation time with leukocytes. On the other hand, serum itself acts as a strong inhibitor for fl-galactosidase. The inhibitory effect of serum is removed after dialysis of serum. These results indicate that the unidentified substance exhibiting an inhibitory effect in serum may be a relatively small molecular compound. Kint 1113 pointed out that albumin bound to MPS and had a stimulating effect on the /3-galactosidase activity of liver obtained from patients with Hurler syndrome. Various high molecular compounds were examined for effect on leukocyte P-galactosidase. Heparin was an inhibitory substance. Heparin is also an MPS and its inhibitory effect was enhanced by increasing concentrations of heparin. Therefore, full consideration should be given to the concentration of heparin used when collecting blood samples. Con A itself activated /3-galactosidase activity. When it is added with Ch-s and serum which alone possess remarkable inhibitory effects on /3-galactosidase activity, the effects disappeared. It is probable that Con A bonded with a terminal residue of sugar in the inhibitory factors of serum, or to a portion of the @-galactosidase enzyme molecule (lysosomal enzyme is known to be glycoprotein) to prevent binding with the inhibitor.

157

In conclusion, MPS and other macromolecular substances could have inhibitory or activating effects on P-galactosidase by themselves or in combination with other substances such as those in serum. When discussing lipidoses and mucopolysaccharidoses it is essential to take these findings mentioned into consideration. References 1 2 3 4 5 6 7 8 9 10 11

Okada, S. and O’Brien, J.S. (1968) Science 160.1002 Suzuki. K. and Suzuki, Y. (1970) Proc. Natl. Acad. Sci. U.S.A. 66, 302 Ho, M. and O’Brien, J.S. (1969) Science 165.611 Matalon, R. and Dorfman. A. (1972) Biochem. Biophys. Res. Commun. 47.959 Kint, J.A., Dacrr?mont. G.. Carton. D., Drye, E. and Hoof, C. (1973) Science 181. 352 Kint, J.A. (1973) FEBS L&t., 36. 53 Snyder, R.A. and Brady. R.O. (1969) Clin. Chim. Acta 25.33 Gatt, S. and Rapport, M. (1966) Biochim. Biophys. Acta 113, 567 Vesterberg, 0. and Svensson, G. (1966) Acta Chem. Stand. 20, 920 Dorfman. A. and Matalon, R. (1976) Proc. Natl. Acad. Sci. U.S.A. 73,630 Kint, J.A. (1974) Nature 250, 424