Enzymatic determination of galactosylceramide galactosidase in tissues by NAD cycling

ANALYTICAL

BIOCHEMISTRY

126,44-S

1 (1982)

Enzymatic Determination of Galactosylceramide in Tissues by NAD Cycling’ TARAHIKO

KATO*

AND YOSHIYUKI

Galactosidase

SUZUKI**

*Department of Biochemistry, Institute of Brain Research, and **Deparfment of Pediatrics, Fact&y of Medicine, University of Tokyo, Hong0 7-3-1, Bunkyoku. Tokyo, Japan Received January 18, 1982 An enzymatic determination method for galactosylceramide galactosidase (EC 3.2.1.46) was devised by using an enzymatic amplification reaction, NAD cycling. Gaiactose released by crude enzyme samples (tissue homogenates and cell suspensions) from galactosylceramide quantitatively reduced NAD to NADH by the galactose dehydrogenase reaction; then the NADH was amplified 6000-lO,OOO-fold by NAD cycling and determined fluorometrically. A higher sensitivity of assaywas obtained compared with the previous radiometric method. The present method was successfully applied to tissues from patients with Krabbc’s disease, whose organs are deficient in galactosidase. The galactosidase reaction rate with a crude sample was not proportional to its concentration. However, the double-reciprocal plot of the reaction rate against the sample concentration became linear and provided a unique value of specific activity to each sample.

higher sensitivity than the radiometric method, was developed in terms of an enzymatic amplification reaction, NAD cycling (16). In the galactosidase reaction, the reaction rate was not proportional to the concentration of crude tissues, which were supposed to contain some inhibiting factor(s). Therefore a unique specific activity could not be calculated and a quantitative comparison of the specific activities was not feasible among different samples. In this work, the quantitative comparison was undertaken by using the double-reciprocal plot of the reaction rate against the tissue concentration. Part of this paper was reported in a preliminary form (17).

Although galactosylceramide galactosidase is known to hydrolyze several natural lipid substrates: galactosylceramide ( 1,2), lactosylceramide (3,4), galactosylsphingosine (5), and monogalactosyldiacylglycerol(6), the radiolabeled galactosylceramide has been used as an original substrate in the radiometric determination method (7-l 1). The radiolabeled substrate is now not commercially available and one has to prepare it (12,13). This enzyme is deficient in patients with Krabbe’s globoid cell leukodystrophy ( 14,15), therefore, its assay method is important for diagnosis using crude enzyme samples such as homogenate of human organs, leukocytes, and cultured cells. The available amounts of these samples are generally small, and high sensitivity of the assay method is required, especially when amniotic fluid cells are analyzed for prenatal diagnosis. Thus, considering this requirement and the inconvenience in obtaining radiolabeled substrate, a new nonradiometric method, having a

MATERIALS

Crude samples. Cerebral, liver and kidney homogenates (20%, w/v) of rat (SpragueDawley, male, 230 g) were prepared in 20 mM Tris-HCl (pH 8.0) by using a motordriven Teflon pestle homogenizer. To prepare the cerebral supernatant, a portion of homogenate was diluted to 10% with the

’ This work was supported in part by Grant 211204 from the Ministry of Japan. OOOZ2697/82/150044-08%02.00/O Copyright 0 1982 by Academic Press, Inc. All rights of reproduction in any form reservd.

AND METHODS

44

GALACTOSYLCERAMIDE

GALACTOSIDASE

ASSAY

45

same medium and sonicated in ice for 1 min was used as blank. As standards, galactose was added at five appropriate concentration with a dipping probe at the maximum output of an ultrasonic sonifier (Tomy UR-150, levels to the reaction mixture with a blank of 20 mM Tris-HCl, pH 8.0, or boiled kidney Tokyo, Japan). The sonicated homogenate was centrifuged at 10,OOOg for 10 min in the homogenate. The mixture was cooled to 0°C cold to separate the cerebral supematant as and 10.4 ~1 of 1 M NaOH was added to adjust a sample containing lysosomal membrane. the pH to 5.6 to 6.8. The neutralized mixture Human leukocytes were collected from was heated in boiling water for 3 min to dehealthy adults (six males and two females) stroy enzymes and cooled again to 0°C. Then according to the method of Snyder and Brady 62.6 ~1 of the galactose assay reagent was ( 18). The leukocytes were sonicated as above added to the cooled mixture. The reagent and suspended in physiological saline at 3 to consisted of 200 mM Tris-HCl (pH 9.0), 0.1 5 mg of protein/ml. mM GSH, 100 PM NAD+, and 100 &ml Human cerebrum, liver, and kidney were galactose dehydrogenase. Following the inobtained at autopsy or an abortion of a fetus cubation, the pH of the mixture was made and stored at -20°C for 6 months to 4 years 11 to 12byadding31.9rlofl MNaOH,and until use. Cerebral cortex and white matter this alkaline mixture was heated at 70°C for were separated from the slices of cerebra. 30 min to destroy unreacted NAD+. An alHomogenates (10%) of human organs were iquot ( 1.38 ~1) of the alkaline mixture, cooled prepared in 20 mM Tris-HCl, pH 8.0. again to 0°C was added to 4b ~1 of NAD Galactosylceramide galactosidase reaction cycling mixture; NADH formed in the aliand standard assay conditions. The standard quot was amplified 6000- to 1O,OOO-fold and assay mixture (46.8 ~1) was a modification determined fluorometrically (16). The cyof that of Bowen and Radin (9) and consisted cling reaction rate was not influenced by adof 100 mM citrate-phosphate buffer (pH 4. I), dition of the aliquot containing tissue sam165 pM Tris base, 200 pM galactosylceramide ples corresponding to 20 mg wet wt/ml of the (about 165 &ml), 2.35 mM oleic acid, 3.72 standard assay mixture. The enzyme activity mM sodium taurocholate (2 mg/ml crude was given in micromoles (or mmol) of gapreparation), 1 mg/ml Tween 20,0.5 mg/ml lactose released from galactosylceramide per Myrj 56, and an appropriate amount of a kilogram of wet weight (or of protein) per sample, whose reaction rate was apparently hour. proportional to its concentration (see below). Reaction on a microscale. In place of the Tris base was included according to the origglass tube, a disposable plastic microtube inal method to compare the present results (Eppendorf, Hamburg, Germany) was used with the original work. One of the above to reduce the reaction volume to 4.41 ~1. To crude samples (4.68 ~1) was directly added prevent evaporation of the reaction mixture, at 0°C to 42.1 ~1 of the reaction mixture in the microtube was plugged with a Teflon rod, a glass tube (8 X 10 mm) containing other whose tip was slightly sharpened so as to fit concentrated components, whose final con- tightly in the tube and placed as closely as centrations were made as above. The enzyme possible to the surface of the assay mixture. reaction was carried out by incubation at The volume of the mixture was constant at least for 3 h. In proportion to the reduction 38°C for 2 or 3 h. A blank was run by adding in the assay volume, the volumes of the sam20 mrvr Tris-HCl (pH 8.0) in place of samples except for the assay of kidney homogenate, ple and the other reagents were reduced as since the boiled tissues gave the same blank follows: assay sample, 0.475 ~1; 1 M NaOH value as the suspension medium. Kidney for neutralization, 1.13 ~1; galactose assay homogenate contained a detectable amount reagent, 6.59 ~1; and 1 M NaOH for pH adof galactose ( 17); boiled kidney homogenate justment to destroy NAD+, 3.45 ~1.

46

KATO

AND

Other determinations. Radiometric assay of galactosylceramide galactosidase was performed as previously described (9) in 200 ~1 of the reaction mixture including 200 pM [3H]galactosylceramide (866 mCi/mol) and the same concentrations of other components as in the standard assay mixture. MUP-Galactosidase activities of samples were determined with 4-methylumbelliferyl P-galactoside as substrate ( 19). Galactosylceramide was separated from the cerebral homogenate (60 mg wet wt of original tissue) by preparative silica gel thin-layer chromatography (developing solvent, chloroform: methanol:water, 70:30:4) after extraction by the method of Folch et al. (20). About 0.5 pg of galactosylceramide separated as above was dried at the bottom of a capped glass tube ( 10 X 100 mm) and hydrolyzed for 3 h at 100°C in 1.O ml of 1 M HCI. The released galactose (about 10 nmol) was determined fluorometrically after incubation at 38°C for 30 min in the assay mixture (1 .O ml) containing 100 mM Tris-HCl (pH 9.0), 1 I’IIM GSH, 200 PM NAD+, and 30 &ml galactose dehydrogenase. The galactosylceramide concentrations of the stock solutions for 3H-labeled and nonlabeled substrates were standardized in the same way as above. Protein was determined by the method of Lowry et al. with bovine serum albumin as standard (21).

Materials. /3-Galactose dehydrogenase (Dgalactose:NAD oxidoreductase, EC 1.1.1.48) from Pseudomonasjluorescens was supplied by Boehringer-Mannheim, Mannheim, Germany; Myrj 56 by Atlas Chemical Industries, Wilmington, Delaware; and galactosylceramide from beef brain by Serdary Research Laboratory, Ontario, Canada. Other chemicals except for the crude preparation of sodium taurocholate were of analytical grade. RESULTS

Reaction Conditions The reaction rates observed with enzyme samples from rat organs were constant for 4 h in the concentration range up to 20 mg wet v&/ml in the standard reaction mixture. Sim-

SUZUKI

ilarly, without addition of galactosylceramide, all samples except liver produced galactose from endogenous substrate constantly for 3 h (see below). These observations indicated that the average reaction rate during incubation for 2 or 3 h could be considered to be the initial velocity in the presence or absence of substrate exogenously added. The activity of the sample from each source reached a plateau at a unique concentration of substrate; and 200 pM galactosylceramide,enough over each saturation level, was adopted in the standard reaction mixture (Fig. 1). Galactose formation was observed with the samples of rat cerebrum, rat kidney, and human leukocytes (for leukocytes, data were shown in Fig. la of Ref. (22)) when only these samples were incubated without substrate. This suggested that in the absence of added substrate the endogenous galactosylceramide was utilized by the galactosidase and/or other galactosidase(s) included in the samples produced galactose from other substrate(s) (e.g., GM, ganglioside, trihexosylceramide, etc.) Rat cerebral homogenate, which had been the starting material for partial purification of the galactosidase (8), was mainly used as an enzyme sample to find the optimal reaction conditions. For the activity of cerebral homogenate, the optimal pH and the optimal concentrations of components other than the substrate were the same as those in the standard assay mixture. The cerebral enzyme was activated by oleic acid as described previously (9). Sodium taurocholate activated the enzyme maximally at 3.72 mM and efficiently reduced the turbidity of the reaction mixture. Tween 20 and Myrj 56 had no marked effect on the activity but clarified the reaction mixture at the low concentrations. These detergents were thought to promote formation of substrate micelles and not to solubilize the enzyme from membrane particles, since the enzyme activity was recovered 97% in the pellet when the reaction mixture containing cerebral homogenate was centrifuged at 150,OOOg for 30 min after incubation at 38°C for 30 min.

GALACTOSYLCFBAMIDE

GALACTOSIDASE

ASSAY

47

Comparison between Standard, Microscale, and Radiometric Assays

RG. 1. Rate curves of galactosylccramide gaiactosidase activities in rat organs as a function of substrate concentration. The reaction was carried out in the standard assay mixture, in which the concentration of exogenous galactosylceramide was varied as indicated on the abscissa: 0, cerebral homogenate, 1.72 mg wet wt/ ml; 0, cerebral supematant, 1.69 mg wet wt/ml; n , kidney homogenate, 1.72 mg wet wt/ml; and A, iiver homogenate, 3.04 mg wet wt/ml.

The reaction rates of samples from all sources were not linear functions of sample concentrations (Fig. 2a). However, the initial rising parts of the rate curves could be regarded as linear below 4 mg of tissue/ml within an error range of 5%.

The results from both standard and microscale assays are not statistically different (Table 1). In the standard assay mixture, the final blank value resulting from NAD cycling reaction corresponded to the fluorescence of NADH originated from the galactose produced by 0.4 mg wet wt of cerebral homogenate/ml (Fig. 2a). Thus, 18.7 pg wet wt of cerebral homogenate was the minimum amount of the sample accurately assayed in the standard reaction mixture (46.8 ~1). In the microscale assay (4.89 pl), 1.95 pg wet wt of the sample was the minimum assayable amount of sample. Since 200 jd of assay volume was used in the radiometric assay, the amount of sample could be reduced in the microscale assay to less than 1/4Oth of that for the radiometric assay. Compared with the present method, a 6.2% lower average activity of rat cerebral homogenate was obtained in the radiometric assay, though the activity difference between the two methods was statistically not different (Table 1). This was thought to be due to

FIG. 2. Rate curves of galactosylcemmide galactosidase activities in rat organs as functions of tissue concentrations. (a) The samples were incubated in the standard reaction mixture for 3 h at 38°C. The initial rising parts of the curves were regarded as linear below 4 mg wet wt/ml (indicated by arrow on the abscissa). The average blank value is also indicated by arrow on the ordinate. (b) Double-reciprocal plots (see the last section of Results) were produced based on the results given in a. The regression lines were determined by a computer according to the least-square method. 0, Cerebral homogenate; 0, cerebral supematant; W, kidney homogenate; A, liver homogenate.

48

KATO AND SUZUKI TABLE

1

GALAC~OSLYCERAMIDE GALACTOSIJXSE ACIWITIES OF RAT CEREBRAL HOMOGENATE A=Y method Standard

Galactose released (amol/kg wet wt/h) 503 t 11”

(8) Microscale

501% 11

Radiometric

472 I 8*

(8) (8) Note. The sample concentration was I .88 mg wet wt/ ml. ‘Average + SEM. The number of samples is in parentheses. * Statistically not different (p > 0.01) from standard and microscale assays.

dilution of radiolabeled substrate by endogenous galactosylceramide in the sample. Galactosylceramide concentration in rat cerebral homogenate was determined as described under Materials and Methods (6.56 f 0.03 mmol/kg wet wt, average + SEM for four samples). Under the assay condition to obtain the results in Table 1, the concentration of nonradiolabeled substrate originating from endogenous galactosylceramide was 12.3 PM at 1.88 mg wet wt/ml of the sample. Thus, the ratio of the radiometric activity to the true activity would be 200/ (200 + 12.3); therefore the reduction was calculated to be 5.8% of the true activity. This reduction agreed well with the actual difference (6.2%, see above). This suggests that almost all the endogenous substrate was utilized by the enzyme and that the amount of other contaminating galactosidase(s), if any, contributed to a negligible portion of galactose formation in the present assay method. Activities of Rat Organs and Human Leukocytes Among rat tissue samples, kidney homogenate had the highest activity and liver homogenate had the lowest activity (Table 2, second colum). Cerebral supematant, pre-

pared by centrimgation at 10,OOOg for 10 min, contained 96% of the activity of cerebral homogenate (see also Fig. 2a). This result suggested that the insoluble enzyme (see above) was largely present in lysosomal membranes in the crude mitochondrial fraction as reported by Bowen and Radin (1,8). Activities of Control Human Krabbe Patients’ Organs

Organs and

Each human organ seemed to have dried to a different extent, depending on storage period and condition, so the specific activity was expressed on a protein basis. The MUfl-galactosidase activities were well retained in all samples and no difference was detected in the activities between control and Krabbe patients’ organs (Table 3). Patients’ organs had markedly lower activities of galactosylceramide galactosidase than control organs; similarly lower activities were found with the radiometric assay method. These results indicated the validity of the present method for application to pathological samples. Double-Reciprocal Plot of Reaction Rate against Sample Concentration Although the reaction rate of galactosylceramide galactosidase was constant during the incubation period, the reaction rate was not proportional to the sample concentration (Fig. 2a). This fact suggested that some inhibiting factor might be present in crude samples. In the radiometric method, the endogenous galactosylceramide acts as a competitive, substrate and was thought to contribute in part to this nonlinearity (8,9). Since the endogenous substrate was utilized in the present method (Table I), the improportionality may be due to unknown inhibitor(s). The concentration of inhibitor (I) is proportional, as well as enzyme concentration (E ), to the concentration of crude tissue sample (T) in the reaction mixture (E = cuT and Z = /3T; (Yand B are constant coefficients representing the amounts of enzyme and inhibitor per unit weight of tissue, respectively). When the enzyme reaction obeys the Mi-

GALACTGSYLCERAMIDE

GALACTOSIDASE

49

ASSAY

TABLE 2 GALACT~SYLCERAMIDE

GALAC~OSIDASE

ACTMTIE~

OF CRUDE

Galactose Slope of

released l/Slope

initial part (4

Sample

SMPLES

of

double-reciprocal plot (B)

&‘A

pmol/kg wet wt/h + SEM Rat Cerebral homogenate (6)* Cerebral supematant (6) Kidney homogenate (6) Liver homogenate (6)

485 467 870 149

+ f f +

9 10 18 5

537 485 951 199

+ f f f

27 27 24 13

1.11 1.04 1.09 1.34

mmol/kg of protein/h + SEM Human leukocytes (8)

3.25 + 0.24

3.76 + 0.77

1.16

Note. The difference between any two of the samples from rat organs is statistically significant (p < 0.01) except for that between cerebral homogenate and supematant. ’ The specific activity of each sample was calculated based on the maximal activity estimated from the slope of the initial rising part of the rate curve (A; see Fig. 2a) and from the reciprocal of a slope of the double-reciprocal plot (B; see Fig. 2b). * Number of samples.

chaelis-Menten equation, the double-recip rocal plot of the reaction rate (v) against T gives the following equation irrespective of the type of inhibition (23) l/v = (K,,, + S)fak$-

l/T + C,

where S is substrate concentration; Km and k3 are the Michaelis constant and turnover number, respectively; and C represents a term including S, the dissociation constant of Z(KJ and the above constants (a, /3, K,, k3). C takes a different form depending on each type of inhibition (details not described here). When S is invariable, C becomes constant and the above equation expresses a straight line with a constant slope. Actually the double-reciprocal plots for the samples from alI sources provided straight lines (Fig. 2b). Under the standard assay condition, the contribution of endogenous galactosylceramide to the substrate concentration was less than 6% of the total amount and was negligible compared with that of exogenous substrate (i.e., Scan be regarded as constant),

as exemplified by cerebral homogenate (Table 1). Thus, the reciprocal of a slope of a straight line [ak3S/(Km + S)] gave a unique value to each sample and approximated its specific activity (ak3) where no inhibitor was present. In practice, since Km values have been reported to be 10.8 and 27 PM for partially purified enzymes from human liver (24) and rat cerebrum (l), respectively, the reciprocal value may be nearly equal to ak3 under the reaction condition. Therefore the average specific activities, calculated from the reciprocal values, allow a quantitative comparison of specific activities among the samples from all sources (Table 2, third column). Furthermore the ratio (B/A) between the reciprocal of the slope of double-reciprocal plot and the slope of initial part of the rate curve reflects the degree of contamination by the endogenous inhibitor, because the slope of the initial part of the rate curve becomes smaller if much more inhibitor is included. Rat liver homogenate evidently included a higher amount of inhibitor than other samples. In the case of cerebral super-

50

RAT0

AND SUZUKI TABLE 3

GALACTOSIDASEACTIVITIESOFHUMANSAMPLES MU-&Galactosidase (mmol of umbelliferone released/kg of protein/h) Sample

Control

Krabbe patients 17.6 29.9

Galactosylceramide galactosidase (mmol of galactose released/kg of protein/h) Control 2.85 2 0.30

Krabbe patients

Cerebral cortex (8)”

21.3 + 1.2

0.89 (0.74) 0.39 (0.30)

Cerebral white matter (3)

13.7 * 1.9

Liver (10)

138 f 17

64 175 189

1.42 f 0.28

0.14 (0.16) 0.14 (0.13) 0 (0.02)

Kidney (5)

221 + 19

409 302

3.03 + 0.33

0.32 (0.29) 0.49 (0.34)

7.77 * 0.91

Note. Both galactosidase activities (average + SEM) are statistically different ( p i 0.01) between any two of the control organs. The apparently linear range for the reaction rates of galactosylceramide galactosidase was 50-370 pg of protein/ml with the control organs and all assaysof this enzyme were carried out in this range. The radiometric activities of the control samples (not shown) had values similar to those obtained from the present assay. ,JRadiometric activities of patient organs. b Number of samples.

natant, a large part of inhibitor was eliminated by centrifugation from cerebral homogenate. DISCUSSION

The galactosylceramide galactosidase assay by using enzymatic measurement of galactose was once undertaken by Handa and Yamakawa (25) and the activity in cat cerebral supematant was determined by directly measuring NADI-I fluorescence. However, with crude enzyme samples, like homogenates of tissues, the blank was too high to measure the enzyme activity accurately, because a large amount sample was required due to the very low activities in the tissues (Table 2). In the present method, the high blank was overcome by specific amplification of NADH. The specific activity of cerebral homogenate (Table 1) was similar to the value for the same organ estimated by analyzing the activity of a partially purified enzyme preparation (Table 1 of Ref. (9)). The amplification mechanism provided a sensi-

tivity more than 40-fold higher than that of the radiometric method in routine assays. A few chromogenic synthetic substrates have recently been prepared for the galactosidase assay (26,27). One of them (2-hexadecanoylamino-4-nitrophenyl&D-galactosylpyranoside) was shown to be nonspecific for the enzyme and the sensitivity of the assay with this substrate was low (28,29). The natural substrate still seems to be necessary for the specific determination method. Nonproportionality was found between the reaction rates and the concentrations of samples, but the approximation, which regards as linear the initial part of the rate curves (Fig. 2a), is useful for diagnostic purposes (Table 3). However, a quantitative comparison was made feasible based on the double-reciprocal plot (Fig. 2b). In the radiametric method, the effect of dilution of radiolabeled substrate can also be eliminated by using the double-reciprocal plot since the endogenous substrate is thought to be a competitive substrate. In practice, the radiometric rate curve for rat cerebral homogenate

GALACTOSYLCERAMIDE

(Fig. 5 in Ref. (30)) gave a straight line if replotted according to the above method. This double-reciprocal plot is a by-product during development of the present method but is believed to be applicable to general analyses of impure enzyme samples. ACKNOWLEDGMENTS The authors thank Dr. K. Inoue and Professor T. Yamakawa for their discussions and suggestions.

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