Biochimica et Biophysics Acta 877 (1986) 1-8

Elsevier

BBA 52204

Optimal assay conditions for enzymatic characterization of homozygous

and heterozygous twitcher mouse

Srinivasa Raghavan * and Allan Krusell

Department of Biochemistry, Eunice Kennedy Shriver Center for Mental Retardation,

200 Trapelo Road, Waltham, MA 02254 (U.S.A.)

(Received September 24th, 1985)

Key words: Galactosylceramide /3-galactosidase; Lactosylceramide P-galactosidase; Heterozygote detection;

(Twitcher mouse)

The neurological mouse mutant twitcher is characterized by a genetic deficiency of galactosylceramide p-galactosidase (galcerase) (EC 3.2.1.46) which also represents lactosylceramide jl-galactosidase I (lactosi- dase I) activity. The assay conditions for both these activities in several mouse tissues have been optimized to facilitate the enzymatic characterization of homozygous and heterozygous twitcher mice. Galcerase in mouse tissues is optimally activated by 7.0 mg/ml of sodium taurocholate (pure) and 1.5-2.0 mg/ml of oleic acid in this system. When lactosylceramide is used as the substrate, no more than 1 mg/ml of taurocholate is appropriate in the assay, since higher concentrations of this pure bile salt stimulate another enzyme, lactosylceramide &galactosidase II (lactosidase II), which is unaffected in twitcher mice. At the optimized condition, lactosidase I in the twitcher mouse amounts to 3-495 of control activity in agreement with the residual galcerase (2%) in this mouse mutant. These assay conditions provide better sensitivity to discriminate heterozygotes from controls until 40 days of age from measurement of this activity in clipped tail samples.

Introduction

The genetic, clinical and pathological features in the neurological mouse mutant, twitcher closely

resemble the human and canine forms of globoid

cells leukodystrophy known as Krabbe’s disease [l] although there are some apparent differences [2]. Kobayashi et al. [3,4] have shown that galcerase

(and lactosidase I) activity is profoundly deficient in the brain and liver of twitcher mice as in the

human and canine forms of Krabbe’s disease. To

date, it is the first and only neurological mouse mutant in which the primary genetic defect of enzyme deficiency has been demonstrated. We

* To whom correspondence should be addressed.

have, therefore, characterized and optimized assay

conditions for galcerase (and lactosidase I) activity

in several mouse tissues, including tail clippings

which facilitate the identification of homozygous

and heterozygous twitcher mice. These conditions

optimized for the mouse tissues are different from that commonly employed for this enzyme in hu-

man tissue samples [5]. Also, the level of enzyme activity determined at optimal conditions by our method in tail clippings at any particular age is at

least two-fold higher than that reported by Kobayashi et al. [6]. Using our assay conditions, mice heterozygous for this mutation can be dis- tinguished from homozygous normal mice until 40 days of age from measurements of levels of galcerase or lactosidase I activity in homogenates of a small piece of clipped tail. This relatively

0005-2760/86/$03.50 0 1986 Elsevier Science Publishers B.V. (Biomedical Division)

2

non-invasive technique for identifying 1 hetero-

zygotes is very helpful in establishing a breeding

colony for the production of a large number of

twitcher mice. Affected animals can be identified

by the enzymatic deficiency in embryonic stage or

soon after birth, long before clinical and patho-

logical changes become evident, so that they can

be used in experiments to understand the develop-

ment pathobiologically at the cellular level and to

design therapeutic approaches to this genetically

determined neurological disorder.

Materials and Methods

Twitcher mice on C57BL/6J background, ob-

ligate heterozygotes and homozygous controls were

obtained from Dr. Richard Sidman, Children’s

Hospital, Boston. Sodium taurocholate from

Calbiochem, La Jolla, CA (Cat. No. 580217, pur-

ity greater than 96% by TLC) or from Sigma, St.

Louis, MO (synthetic, 98% pure, Cat. No. T-4009)

and oleic acid (99 + W) from Supelco, Inc., Belle- fonte, PA (Cat. No. 4-5520) were used in the assay

of galcerase and lactosidase. Nonhydroxy

galactosylceramide (kerasin) was obtained from

Supelco, Inc. (Cat. No. 4-6045). N-Stearoyl-DL-di-

hydrolactosylceramide was purchased from Miles

Labs, Elkhart, IN (Cat. No. 91-072-l).

Substrate preparation. The two glycolipids were

labeled with tritium in the C-6 position of galac-

tose according to Radin and Evangelatos [7]. They

were stored as solution in chloroform/methanol

(2 : 1) at - 20°C. Each time before use, suitable

aliquots were taken to dryness, redissolved in 5 ml of chloroform/ methanol (2 : 1) and washed according to Folch et al. [8] to remove water-solu- ble radiochemical decomposition products that are

formed during storage [9]. The radioactive lipid in the chloroform phase was appropriately diluted with either unlabeled galactosylceramide or lacto- sylceramide to a specific activity of about 2000 cpm/nmol. The substrates were dispersed by sonication in 0.1% Triton X-100 in water to about 2 mM concentration as previously described [lo]. 20+1 aliquots were taken for orcinol assay [ll] to determine the exact concentration of the glyco- lipid in the dispersion. Another aliquot was counted to determine the specific activity. The substrate dispersion (prepared for about 200 as-

says) was kept frozen and sonicated each time

before use to obtain homogeneous dispersion.

Taurocholate-oleic acid mixture. An aliquot of

oleic acid in hexane (100 mg/ml) was taken to

dryness and dispersed by sonication in aqueous

solution of sodium taurocholate containing 0.1%

Triton X-100. For galcerase assay, this dispersion

contained 20 mg/ml of oleic acid in a solution of

70 mg/ml of sodium taurocholate (solution A).

For lactosidase I assay, the same amount of oleic

acid was present in a solution of 10 mg/ml of

sodium taurocholate (solution B). Solutions A and

B prepared for about 200 assays were kept frozen

and sonicated each time before use to obtain

homogeneous dispersion of oleic acid.

Enzyme extracts. Aqueous homogenates of tis-

sues (10%) were prepared in a Potter-Elvehjem

homogenizer with a Teflon pestle. A small portion

of the tail (0.5-1.0 cm long) was clipped from the

tip and homogenized in 0.5-l ml of water. The

homogenate was centrifuged at 600 X g for 10 min

at 0-4°C to remove unbroken tissue, bone, col-

lagen and adhering hair. The clear supernatant

was used as the enzyme source. The tail clippings

from younger mice could easily be homogenized

and very little material spun down as a pellet with this low-speed centrifugation. Protein was quanti-

tated according to Lowry et al. [12] using bovine

serum albumin as the standard.

Enzyme assays. For galactosylceramide hydrol-

ysis, the reaction mixture in a final volume of 100

~1 contained 10 ~1 each of substrate, solution A

and citrate-phosphate buffer (pH 4.0 and 1 M in

citrate) with 50-100 pg of protein from tissue homogenates. For lactosylceramide hydrolysis by

the same enzyme, the reaction mixture in a final

volume of 100 ~1 contained 10 pl of substrate, solution B and citrate-phosphate buffer (pH 3.5 and 1 M in citrate) with 50-100 pg of protein

from tissue homogenates. Boiled enzyme controls or blanks without enzyme were always used with each set of incubation. The samples were incubated for 2 h and the reaction was terminated by the addition of 5 ml of chloroform/methanol (2 : 1). After the addition of 0.1 ml of carrier galactose (100 pg) and 0.8 ml of water, the tubes were shaken vigorously in a vortex mixer and briefly centrifuged at 600 x g to clarify the organic and aqueous phases. The aqueous upper phase con-

taining [ 3 Hlgalactose released during the course of

the reaction was transferred to a second tube,

washed with 2 ml of chloroform and centrifuged.

One half of the upper phase (0.9 ml) was trans-

ferred to a scintillation minivial and taken to

dryness at 4O“C with a stream of nitrogen. The

residue was dissolved in 0.25 ml of water and

mixed with 4 ml of Scintiverse (Fisher Scientific

Co., Medford, MA). Radioactivity in the vial was

measured in a Packard liquid scintillation counter

(Tricarb 2660). Several other lysosomal hydrolases

such as P-hexosaminidase, /3-galactosidase and

P-glucuronidase were assayed according to

Kolodny and Mumford [13]. j%Glucosidase and

sulfatidase were assayed as previously described

[14,15].

A

Results

Our assay conditions optimized for galcerase and lactosidase I in mouse tissue homogenates

show profound deficiency of the two activities in

the brain, kidney, liver and spleen of twitcher mice

(Table I) as originally demonstrated by Kobayashi

et al. [3,4]. Several other lysosomal hydrolases

such as sulfatidase, P-glucosidase, fl-glucuroni-

dase, P-galactosidase and P-hexosaminidase

exhibited normal activity in the mutant mice. Only

in the brain did we notice a three-fold elevation of

/?-glucuronidase and &hexosaminidase in the

twitcher compared to controls.

We have evaluated the optimal conditions for

the hydrolysis of galactosylceramide and lactosyl-

ceramide in tail clippings also, so that hetero-

zygotes can be accurately identified and bred to

Effect of Sodium Taurocholate on Galactosylceramide p Galactosidase E

7

E

ii \

2- TAIL

I -

0 I 1 I I I

400 600 800 500 700 900 200 400 600 600 1000

pg Taurocholate

E \ Effect of Oleic Acid on Galactosylceramide p Galactosidase

5 g

KIDNEY

z 16 -

12

2

1.5

1.0

0.5

0 100 200

pg Oleic Acid

TAIL

3-

2-f

I’ ’ ’ ’ ’ 100 200

Fig. 1. Optimal amounts of pure sodium taurocholate (A) and oleic acid (B) required for galactosylceramide P-galactosidase activity in homogenates of kidney, brain and tail clippings from control mice.

4

TABLE I

LYSOSOMAL HYDROLASES IN TISSUES OF TWITCHER AND CONTROL MICE

Enzyme activities are expressed as nmol hydrolyzed/mg protein per h. Values from two animals are given either separately or mean

activity

Enzymes Kidney

control twitcher

Spleen

control twitcher

Liver

control twitcher

Brain

control twitcher

Galactosylceramide- 14.5 0.15

/3-galactosidase 13.4 0.46

Lactosylceramide- 22.6 0.76

&galactosidase I 19.8 0.72

Sulfatidase 2.25 1.85

P-Glucosidase 39.4 36.4

/%Glucuronidase 91.0 73.0

/%GaIactosidase 272.0 291.0

fi-Hexosaminidase 2100.0 2200.0

1.46

0.87

3.11

82.0

255.0

364.0

1260.0

0.03 1.59 0 1.36 0

0.04 1.41 0.04 1.43 0 _ 2.57 0.11 2.61 0.13

2.44 0.12 2.47 0.09

4.5 2.8 4.13 1.75 1.24

84.1 151.0 146.0 50.0 56.0

272.0 215.0 271.0 29.5 88.5

369.0 141.0 155.0 100.0 126.5

1030.0 1330.0 1010.0 1885.0 5320.0

generate a large number of twitcher mice for vari- the hydrolysis of ga~a~tosylceramide in tail com- ous experiments. Fig. 1 shows the optimum amount pared with kidney and brain at 30 days of age. of sodium taurocholate and oleic acid required for Addition of taurocholate is essential for this en-

Effect of Substrate on Lactosy~ce~amide

)%zq pzzlzq

p Ga~actosidase I 6

TAIL Krn=TIpM

Effect of Sodium Tauro~holate on Lactosylceramide p Galactosidase 1.

24 - KIDNEY

20 -

100 300 500

pg Taurochotatg

30

t

TAIL f

,I

Fig. 2. Substrate saturation kinetics with lactosylceramide (A) and amount of pure sodium taurocholate (B) appropriate for lactosylceramide fl-galactosidase I activity in homogenates of brain, kidney and tail clippings from control (O------- 0) and twitcher (A------A) mice.

5

zymatic reaction. Maximal hydrolysis was ob- tained with 700 pg taurocholate when its con- centration was varied at a fixed concentration of oleic acid (50 pg for tail and kidney and 200 I-18 for brain) in 0.1 ml of reaction mixture (Fig. 1A). Beyond this concentration, taurocholate was in- hibitory in kidney but not in brain and tail. Ad- dition of oleic acid further stimulated the hydrol- ysis obtained with 700 pg taurocholate alone in the reaction mixture (Fig. 1B). Maximal stimu- lation is obtained with 200 pg of oleic acid in our assay system with tail, kidney and brain homo- genates. The K, for the hydrolysis of galactosyl- ceramide is 56 PM in all these tissues.

Fig. 2 shows the conditions for lactosidase I as a function of substrate concentration and taurocholate concentration. With 100 pg of taurocholate and 200 pg of oleic acid in 0.1 ml reaction mixture, lactoside concentration was varied from 5 to 50 nmol to show saturation kinetics in tail, kidney and brain (Fig. 2A). The twitcher showed deficiency in the whole range of substrate concentrations, demonstrating that lactosidase I is measured in this entire substrate concentration. But if taurocholate concentration is increased in the reaction mixture at a fixed saturating concentration of the lactoside substrate (20 nmol/O.l ml), the enzymatic deficiency for the hydrolysis of this substrate in the twitcher mouse begins to disappear by 200 pg and the activity

1’ 0 10 20 10 40 50 60

Age of Toil in Days

0

Fig. 3. Variations with age in the activities of galactosylcera- mide P-galactosidase (o- - -0) and Iactosylceramide /?-

galactosidase I (0- - - 0) in tail clippings from control

mice. Tails from four animals were individually assayed for

each time point and the mean value is represented.

tends to be normal by 500 pg of taurocholate in the incubation mixture (Fig. 2B). This is because lactosidase II, which is unaffected in the mutant mice, is activated beyond 100 pg of pure taurocholate at this fixed substrate concentration, whereas increase in substrate concentration at a fixed amount of 100 pg of taurocholate in the reaction volume does not result in any significant lactosidase II activity. Oleic acid stimulates lactosidase I as shown for galactoscleramide hy- drolysis in Fig. 1B.

Fig. 3 shows that galcerase and lactosidase I in tail decline as a function of age. Tails from four mice were individually assayed for each time point. The specific activity (+ SD.) of galcerase was 4.54 k 0.19, 3.51 L- 0.36, 2.58 + 0.15, 2.23 + 0.08 and 1.38 f. 0.18 at 10, 20, 30, 40 and 60 days of

age, respectively. The specific activities of lactosidase I for the same respective time periods were 8.14 k 0.47, 7.40 _+ 0.49, 5.32 f 0.54, 3.91 k 0.10 and 2.29 k 0.51. From these results, it is evident that heterozygote detection requires age- matched controls and preferably corresponding litter mates for comparison of control values.

Fig. 4 shows our screening data for hetero- zygote detection based on galcerase activity. Con- trols at 20 days show higher activity than at 30 days, showing that it is important to use age- matched controls for comparison. The values for obligate heterozygotes that have produced twitcher mice upon mating do not overlap with controls or with affected animals. It is, usually, very easy to establish the cut-off limit for distinguishing homo- zygous controls from heterozygotes within a litter born of heterogygous parents. In a few rare cases,

TABLE II

GALACTOSYLCERAMIDE /3-GALACTOSIDASE IN THE

TAIL CLIPPINGS OF NEONATAL AND SUCKLING

MICE BORN OF PARENTS HETEROGYGOUS FOR

TWITCHER MUTATION

The activity is expressed as nmol hydrolysed/mg protein per h.

Values in parenthesis represent the number of animals.

Age of Controls

mice Heterozygotes Twitchers

Day 1 5.95; 5.18 2.83 +0.15 (5) 0.28; 0.22 Day 5 5.18+0.34 (5) 2.54+0.10 (4) 0.18; 0.28; 0.19

Galactosylceramide p Galactosidase

MEAN f SD.

3.58 f .39

:OWTROLS 30-40 MW

( 14)

HEfEROZY6OTE8

30 - 40 DAYS

(2 21

2.46 f .21

DlltASE 30 - 40 DAY8

(11)

UNKOWN 30-40 DAY!

(43)

I .04 +_ .35 ,044 f 038,

Fig. 4. Galactosylceramide &alactosidase activity in tail clippings for distinguishing heterozygotes from homozygous normal and

affected twitcher mice. Values in parenthesis in each column represent the number of animals.

Lactosylceramide p - Galactosidase I

3-

3-

? -

j-

j-

b-

s-

> _

I -

h

.

.

a

; .

.

0 ; -

.

em -

s I! ;T :

8 . .

”

CONTROL 20 DAYS CONTROLS 30-40 MIS HETCROLYe0TLs DISEASE 30- ‘0 DAYS UNKOWN 30-40 DAYS

(8) (II 1 30 - 40 DAYS (IO) (431 Q4)

MEAN + SD.

7.1 IT.66 4.34 Z.84 I.82 z .59 24Z.12

Fig. 5. Lactosylceramide /3-galactosidase I activity in tail clippings for distinguishing heterozygotes from homozygous normal and affected twitcher mice. Values in parenthesis in each column represent the number of animals.

the value may fall on the border of being incon-

clusive. Heterozygote detection is further con- firmed from lactosidase I activity as shown in Fig.

5. Presumptive heterozygotes detected by the two

biochemical assay procedures have been shown to be true heterozygotes by mating experiments to

obtain twitcher offspring. In these experiments,

heterozygotes were identified at 30-40 days of age

because, at this age, diseased twitcher mice in a

litter are easily recognized by clinical symptoms.

Unaffected mice are then characterized by this

enzymatic assay as heterozygotes or homozygous

controls. Using this assay technique, one can go

earlier than 20 days to identify the heterozygotes

and can diagnose the mutants with the twitcher

trait soon after birth and long before the ap-

pearance of clinical symptoms (Table II).

Discussion

There are two genetically distinct P-galactrosi- dases in human tissues and both can hydrolyze

lactosylceramide in vitro [16-181. One of these, lactosidase I, is identical to galcerase and is defi-

cient in Krabbe’s disease [5,19]. The other,

lactosidase II, is associated with /3-galactosidase

activity determined with non-specific synthetic

substrate and G,, ganglioside and is deficient in

GM,-gangliosidosis [19]. The assay conditions for

the two enzymes are quite different. Sodium

taurocholate activates only lactosidase I, while

lactosidase II is activated by taurodeoxycholate,

glycodeoxycholate and taurochenodeoxycholate [18,20,21]. Addition of oleic acid causes further

stimulation of lactosidase I as originally demon-

strated for galcerase from rat brain by Bowen and Radin (221. However, the assay conditions opti-

mized for the two &galactosidases in human tis- sues may not hold for murine tissues. Thus, unlike the Wenger system [5,21] for human galcerase the

murine enzyme in our assay system is optimally

activated by 700 pg of taurocholate and 150-200

pg of oleic acid in 0.1 ml reaction mixture. Simi-

larly,the amount of taurocholate optimized for assaying human lactosidase I in the Wenger sys- tem (2.5 mg/ml) would begin to activate the murine lactosidase II activity in our system. Thus, no more than 1 mg/ml of taurocholate is ap- propriate in our assay for lactosidase I activity in

7

mouse tissues. Using this condition, we have found

that lactosidase I activity in the twitcher mouse amounted to 3-4% of activity in control mice, in

agreement with the residual galcerase (2%) in these

mutant mice. Using the Wenger system, Kobayashi

et al. [3,4] obtained about 14% residual lactosidase

I activity in the brain and liver of twitcher mice

which is much higher than the residual galcerase

in these mice. This higher residual activity is prob-

ably due to some interference by the unaffected

lactosidase II activity in their assay system. Under

the conditions chosen by us to eliminate the inter-

ference by lactosidase II activity, the activity ob-

tained for lactosidase is only about 1.5-2-fold

higher than that for galcerase.

Our system also yields higher activity for

galcerase in tail clippings than that of Kobayashi

et al. [6] at any particular age. This activity de-

clines with age in tail clippings. Yet, our value for

controls and heterozygotes at 30-40 days of age is

similar to their value at 7 days of age. For the

same age, our system provides at least 2-fold higher activity and thus better sensitivity to dis-

criminate heterozygotes from controls. Further-

more, we have shown that lactosidase I activity in

tail is also useful in the identification of het-

erozygotes. We routinely carry out the assay with

galactosylceramide as the substrate. If the values

fall on the border of being heterozygotes and

controls, we use the system for lactosidase I to

clarify the results. Thus, with our two assay sys-

tems, heterozygotes can be identified until 40 days

of age and bred to produce twitcher mice for

various experiments.

Acknowledgement

This work was supported by a grant from the National Institutes of Health, HD 05515.

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