Genetic Analysis of Nucleotide Triphosphatase Activity in ...Copyright 0 1994 by the Genetics Society of America Genetic Analysis of Nucleotide Triphosphatase Activity in the Mouse

Copyright 0 1994 by the Genetics Society of America

Genetic Analysis of Nucleotide Triphosphatase Activity in the Mouse Brain

Kristina M. Men' and Thomas N. Seyfried

Biology Department, Boston College, Chestnut Hill, Massachusetts 021 67 Manuscript received August 16, 1993

Accepted for publication February 1, 1994

ABSTRACT A Ca2'- or Mg2+-stimulated ecto-ATPase is thought to regulate the hydrolysis of extracellular ATP in

nervous tissues. The hydrolysis of nucleotide triphosphates (NTPs) was analyzed in brain microsomal fractions from crosses of DBA/2J (D2) and C57BL/6J (B6) mice. The nucleotide triphosphatase (NTPase) activity was significantly reduced in D2 mice as compared to B6 mice, and B6D2FI hybrids had activities intermediate to the parentals. A significant positive correlation was found between the hydrolysis of four NTPs (ATP, CTP, GTP and UTP) in 24 B6 X D2 (BXD) recombinant inbred (RI) strains of mice and in 80 B6D2FI X D2 backcross mice. The RI strains and backcross mice fell into two distinct groupswith respect to the NTPase activity. Linkage of NTPase activity was suggested with the chromosome 2 markers, DZMit6 and Ass-I, in the RI strains, and was confirmed by analysis of other markers in the backcross population. These data suggest that the Ca2+- or Mg*+-stimulated hydrolysis of NTPs, designated Ntp, is regulated by a single gene located on proximal chromosome 2. Although an association was observed previously between Ca2+-ATPase activity and susceptibility to audiogenic seizures (AGS), no significant association was observed for the expression of Ntp and AGS susceptibility.

T HE brain Ca2+- or Mg2+-stimulated ATPase is thought to reside on the outer surface of neuronal

and glial membranes (TRAMS and LAUTER 1974; STEFANOVIC et al. 1974a,b, 1976; SORENSEN and MAHLER

1982; NAGY et al. 1983,1986; NAGY 1986). Although the function of this ATPase is unclear, it may regulate hydrolysis of extracellular ATP, controlling ATP and adenosine levels in the synaptic cleft (ROSENBLATT et al. 1976; TRAMS and LAUTER 1978; ZIMMERMANN et al. 1979, 1986; NAGY et al. 1983, 1986; NAGY 1986; RICHARDSON and BROWN 1987; RICHARDSON et al. 1987; TERRAIN et al. 1989; WIERASZKO and SEYFRIED 1989; LAI and WONG 1991; JAMES

and RICHARDSON 1993). Most studies suggest that the brain Ca2+- or Mg2+-stimulated ATPases represent two separate enzymes (LIN 1983; LIN and WAY 1984; NAGY 1986; PALAYOOR and SEYFRIED 1984a,b; STAUDERMAN et al. 1985; GHANDI and Ross 1988), but some evidence sug- gests that they represent a single enzyme (STEFANOVIC et al. 1976; LAFFERTY et al. 1985; DING and SEWED 1992; DINGALLEN and SEYFRIED 1992, 1993). Support for the single or multiple enzyme hypothesis was based mainly on biochemical and pharmacological evidence.

ROSENBLATT et al. (1976), found that the Ca2+-ATPase activity in brain membrane enriched preparations was significantly lower in audiogenic seizures (AGS) suscep tible D2 mice than in seizure resistant B6 and C3H/HeN (C3H) mice. This reduction was also observed in cultured glial cells from AGS susceptible mice (TRAMS and LAUTER 1978). Furthermore, a correlation was found between reduced brain Ca2+-ATPase activity and AGS

Medical School, Warren Alpert Building 347, 200 Longwood Avenue, Boston, I Present address: Neurology Department, Beth Israel Hospital, Harvard

Massachusetts 021 15.

Genetics 137: 257-265 (May, 1994)

susceptibility in the BXD RI strains (PALAYOOR and SEYFRIED 1984a). Thus, reduced Ca2+-ATPase was thought to play a role in the mechanism of AGS sus ceptibility (ABOOD and GERARD 1955; ROSENBLATT et al. 1976, 1977; TRAMS and LAUTER 1978; PALAYOOR and SEYFRIED 1984a; PALAYOOR et al. 1986; WIERA~ZKO and SEYFRIED 1989; NAGY et al. 1990a). The chromosomal location for the Ca2+-ATPase ( Caa) locus was predicted on mouse chromosome 12 because of the association between the major AGS gene (Asp-1 ), Ca2+-ATPase activity, and the Ah locus (SEWED and GWER 1981; PALAYOOR and SEYFRIED 1984a; NEUMANN and SEYFRIED 1990). Our goal was to reevaluate the association between AGS, Ca2+- and Mg2+-stimulated ATPase activity, and other NTPase activities in brain microsomes. Portions of this research have been presented previously (DING and SEWED 1992; DINGALLEN and SEWED 1992, 1993).

MATERIALS AND METHODS

Mice: The B6, D2 and BXD RI strains of mice were purchased from The Jackson Laboratory, Bar Harbor, Maine. The production and use of RI strains was described previously (TAYLOR 1978; BAILEY 1981). The backcross mice were generated from the mating of B6D2F, X D2 mice. Female and male F, mice were used to produce the backcross offspring. The mice were housed in plastic cages and food ( w a y Prolab Rodent Diet Formula) and water were provided ad lib. Hard- wood sanichips (changed weekly) were used as the bedding material and the mice were kept on a 12-hr light-12-hr dark cycle. The mice used for these experiments were propagated in the Animal Care Facility of Boston College and were 18-28 days of age, except BXD-8, -13 and -30. These mice were tested for NTPase activities at 7-1 1 months of age because younger mice were not available. To account for any changes in NTPase

258 K M. Allen and T. N. Seyfned

TABLE 1

Analysis of Ca*+- or M$+-stimulated hydrolysis of nucleotide triphosphates in brain microsomes from B6, D2 and B6D2F, mice

Enzyme activity (pmol P,/mg protein/hr) ~~~ -

Ca*+-NTPase Mg2'-NTPase

Substrate B6 Fl D2 B6 F, D2

ATP CTP GTP UTP

8.1 2 0.3 6.2 2 0.1 4.1 ? 0.2 10.1 & 0.3 7.4 ? 0.1 13.2 ? 0.7

4.6 -C 0.1 10.6 2 0.1 7.4 -C 0.3 10.4 2 0.4 8.5 -C 0.2 5.5 -C 0.3

8.8 2 0.3 6.5 2 0.2 4.9 * 0.2 11.4 2 0.2 8.7 -C 0.2 13.0 2 0.3

5.8 2 0.2 9.7 i 0.2 7.2 2 0.2 11.9 2 0.3 8.9 2 0.2 6.0 -C 0.2

Activities are expressed as mean 2 SEM and 7-14 independent microsome preparations were used for each value. ' D2 values are significantly lower than the B6 or F, values at P < 0.001 and P < 0.01, respectively.

activity with age, the NTPase activities in these strains were compared to age-matched B6 and D2 control mice.

Coat color and AGS typing: The B6D2F, X D2 backcross mice were typed for coat color and AGS at 18-28 days of age. The backcross mice segregated for four coat color phenotypes; black ( + /b ; + / d ) , dilute ( + /b ; d / d ) , brown ( b / b ; + / d ) , and dilute brown ( b / b ; d / d ) . The mice tested for AGS received only one exposure to a pure tone sound of 120 dB at 11 kHz for no more than 45 sec and none were exposed to loud, high frequency sounds prior to testing. The AGS response pheno- type consists of a progressive sequence of phases (SEYFRIED 1982a,b). The initial response in seizure susceptible mice was wild running, which progressed to a clonic seizure, followed by a tonic seizure. A mean seizure severity score (SSS) was given to each mouse based on the severity of its response to sound (ie., no response = 0, wild running = 1, and clonic or tonic seizures = 2).

Preparation of microsomes: The microsomes were isolated according to PUYOOR et al. (1986). Briefly, mice were killed by cervical dislocation and the brains collected and placed in 3.0 ml ice-cold 0.32 M sucrose. The brains were homogenized with 10 strokes of a motor-driven Teflon pestle. The homc- genate was centrifuged at 900 X gfor 3 min. The nuclear pellet (Pl) was discarded and the supernatant centrifuged at 17,000 X gfor 10 min to obtain a crude mitochondrial (P2) pellet and a supernatant containing microsomes. The supernatant was centrifuged at 90,000 X gfor 40 min to obtain the microsomal pellet. This pellet was resuspended in 2.0 ml 0.32 M sucrose.

NTPase anah/i: A modification of the PALAYOOR and SEY- FRIED (1984a) method was used to determine the NTPase activity. Briefly, the NTPase activity was measured in an incubation medium containing 25 mM TrisHC1, pH 7.4, 2 mM CaCl, or MgCl,, 0.2 M sucrose, and 0.63 mM Tris-NTP (ATP, CTP, GTP or UTP) . The final volume of the reaction medium was 1.5 ml, which included about 100 pg of microsomal protein. After 5 min of preincubation, the reaction was started by adding the Tris-NTP. After 15 min of incubation, the reaction was stopped by immersing the tubes in ice and adding 0.5 ml of cold 10% trichloracetic acid. There was a significant difference in NTPase activity between the B6 and D2 mice over the tem- perature range of 10" to 40" (DING et al. 1991). Thus, the standard assay was carried out at 20".

The amount of inorganic phosphate liberated was determined by the method of MUSZBEK et al. (1977). The method of Lomv et al. (1951) was used to determine the total protein content of the enzyme preparations. The relative specific activity of the NTPase was expressed as pmol P,/mg protein/hr.

Polymerase chain reaction (PCR): Mouse genomic DNA was isolated from mouse spleen by the method of FRANKEL et al. (1989). Oligonucleotide primers were purchased from Re- search Genetics (Huntsville, Alabama). A modification of the DIETRICH et al. (1992) method was used for PCR analysis. Prim-

ers were end-labeled with [y32P]ATP (specific activity, 6000 Ci/mmol, DuPont-NEN) using T4 polynucleotide kinase (Pro- mega). A 20-ng aliquot of genomic DNA was amplified with both labeled and unlabeled primers in a 10-pl PCR reaction using Taq DNA polymerase (Perkin-Elmer Cetus) according to manufacturer's specifications. The samples were overlaid with 20 pl of light mineral oil. In a thermal cycler (MJ Research PTC 100 Programable Thermal Controller), reaction mixtures were initially denatured for 4 min at 94" and then amplified for 30 cycles using the following protocol: 94" for 1 min, 60" for 2 min, and 72" for 2.5 min. The PCRproducts were diluted twofold with a loading buffer consisting of xylene cyano1 and bromphenol blue in 100% formamide. The products were denatured for 5 min on a 95" heating block and electrophoresed on 6% polyacrylamide denaturing gels for 2 hr at 20 V/cm (120 W). Autoradiography was carried out using Kodak X-Omat ARX-ray film and Dupont Hi-Plus intensifymg screens for 4-16 hr at -70".

Linkage analysis Linkage analysis in the RI strains and backcross generation was facilitated using the RI Manager program (Version 2.3) (MANLY and ELLIOTT 1991).

RESULTS

We found that the Ca2+- or Mg2+-stimulated hydrolysis of several NTPs (ATP, CTP, GTP and UTP) was significantly lower in brain microsomes of D2 mice than B6 mice (Table 1). The NTPase activities in the D2 mice were about one-half of those in the B6 mice and activities in the B6D2F, hybrids were intermediate to those of the parental strains (Table 1). In the presence of Ca", the substrate specificity was in the order of CTP = UTP >. GTP = ATP. The pyrimidines (CTP and UTP) were a better substrate than the purines (ATP and GTP) for the Ca2+-stimulated enzyme activity. In the presence of Mg2+ on the other hand, the enzyme did not discriminate between purines and pyrimidines (Table l ) .

The BXD RI strains were divided into two distinct classes with respect to Ca2'- or Mg2+-stimulated ATPase activity (Figure 1). Fourteen of the strains (BXD-1, -5, -8, -9, -12, -13, -14, -21, -22, -24, -25, -30, -31 and -32) had low activities resembling the D2 progenitor strain, whereas the remaining 10 strains (BXD-2, -6, -11, -15, -16, -18, -19, -27, -28 and -29) had high activities resembling the B6 progenitor strain. The NTPase activities in these groups were typed D and B, respectively. The mean activity of the 14 D2-like strains (4.2 2 0.1 for Ca2+-ATPase; 4.7 2

A '1

Genetics of Brain NTPase in Mice

Ca2+-ATPase

259

T

., B6 28 16 18 19 29 27 11 6 15 2 8 30 14 24 12 31 22 1 13 5 21 9 25 32 02

FIGURE 1 .-Ca2+- and Mg2+-stimulated hydrolysis of ATP in brain microsomes of B6 and D2 mice and in 24 BXD recombinant inbred (RI) strain. Each value represents the mean 2 SEM. Three to 13 independent microsomal preparations were used for

RI Strains each strain except for BXD-13 ( n = 1) and BXD-8 ( n = 2) , because of the unavailability of these strains. The RI strains are arranged from highest to lowest ATPase activities.

ATPase; >5.1 for Mg2+-ATPase) had BfYike

(<4.7 for Ca2+-ATPase; <5.1 for Mg2+-

B 11

10 Mg2+-ATPase The high activity strains (>4.7 for Ca2'-

9 activity, whereas the lower activity strains

8 ATPase) had D2-like activity. c C I

$5 c 7

SF o n 6 *In 3: 5 Ecr "'0 4

E, 3

2

1

0 B6 19 16 28 18 29 27 11 2 15 6 22 8 24 30 31 14 1 12 9 5 25 13 21 32 D2

RI Strains

0.1 for Mg'+-ATPase) was significantly lower than that of the 10 B6-like strains (7.3 2 0.2 for Ca'+-ATPase; 9.1 2 0.2 for Mg'+-ATPase) ( P < 0.01; determined by the t-test). Two NTPase phenotypes were also found among the B6D2F1 X D2 backcross progeny, i. e . , low like the D2 parentals and intermediate like the F, hybrids.

The overall patterns of inheritance for the Ca"- or Mg2+-stimulated ATPases among the RI strains (Figure 1) were similar to those for Ca2+- or Mg'+-stimulated CTPase, GTPase and UTPase (DINGALLEN and SEYFRIED 1993). Highly significant positive correlations (Pearson product moment correlation, r ) were found between the SDPs for the Ca2+- or Mg'+-stimulated hydrolysis of

the nucleotide triphosphates (ATP, CTP, GTP and UTP) in both the RI strains and backcross mice. In the RI strains, the values for the correlation coefficients ranged between r = 0.94 (Ca'+-CTPase compared to Mg'+-ATPase, P < 0.01) and r = 0.99 (Ca'+-ATPase compared to Mg2+-ATPase, P > 0.01). There were no dis- cordancies for any of the NTPase activities among the RI strains analyzed. In the backcross mice, the correlation coefficients ranged from r = 0.87 (Ca"-CTPase compared to Ca'+-UTPase; P < 0.01) to r = 0.94 (Ca2+- CTPase compared to Mg'+-GTPase; P < 0.01).

To map the gene for brain NTPase activity, designated Ntp, the SDP for NTPase activity was compared to over

260 K M. Allen and T. N. Seyfried

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n n n n n

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m 3 n x m m

n n n n n

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m m x n n n

n x m m m m

m m x n n x m

m m m m m

n c a n n n

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n n n n n

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3 n x m m D

% D

E 3 ‘.u 3 Q $ h Q

5 1

hl z 675 SDPs for loci previously mapped in the BXD RI

Y strains ( ~ L Y and ELLIOTT 1991). The pattern of seg- B regation for NTPase activity among the 24 RI strains B suggested linkage with loci located on proximal c h r e 1 mosome 2, i.e., D2Mit6 (microsatellite marker, DIET- 8 s RICH et al. 1992), and Ass-1 (arginosuccinate synthetase, s m JACKSON et al. 1990). Table 2 shows the SDPs for the $ 5 chromosome 2 markers and NTPase activity. One re-

“0 not analyzed for NTPase activity). The mean recombi- $ 8

% $ 2 $ 2 3 j g In addition to typing the RI strains for NTPase activity, d % s several RI strains were typed for previously untyped 5 - 9 .- g 2 c markers. Strains BXD-5, -31 and -32 were typed for t p D2Mit6 and strains BXD-31 and -32 were typed for 3 w 4 s ji 2 5 : and D2Mit 7. The revised distance between D2Mit6 and 4 @ D2Mit 7 is approximately 10.1 cM. This is less than the $ E: approximate 15.0cM distance presented in the compos- 9 gq 2 ite linkage map based on the incomplete RI patterns

5 s 5 3 54.c;;i

5 .g $ lished tight linkage of D2Mit 7 to Hc (GBASE 1993). p.%e From the RI strain data, the following map distances m x gb.2 6 5 2 % y . 2 d l $ .“&%g cM)-NtP5.6cM (1.3-21.3 cM)-Ass-1-2.2 CM (0.2-10.1 $ 2 p z cM)-Hc-1.1 cM (0.0-7.7 cM)-D2Mit7. .z bng ;$ 5:s - To confirm the chromosome 2 assignment of NTPase

g z a m 0 - k& B 8

- 4 3 D2 backcross population. The backcross mice were 2=.:$ 3 3 , g 5 typed for Ca2+- and Mg2+-NTPase activity, and the mic-

& E ‘ 3 G E . a n -der 3 1 h g were not separated according to sex as there was no ‘6 5” !iW& affect of sex on the segregation of the markers analyzed. c c %n 2 . 9

5 . 5 E g g QJ, The published gene order of the microsatellite markers ci; c - . ~ & Bn on chromosome 2 is D2Mitl-D2Mit6-D2Mit7 8 3 z g . a q ,z -;a .: (DIETRICH et al. 1992) (Figure 2). The frequency of re- - e, d 6-8 Pg .9 2 ,s c VI

combination observed between each pair of markers is u ‘c1 E =.z a E y.:n sh 3 e, shown in Table 3. In these painvise comparisons, ani- 0 .e 0 QJ 2.8 $ - u L b h l u u mals carrying a B6 allele for one of the pairs of markers

and a D2 allele for the other are scored as recombinant.

k m o . ~ \ c ’ f g Animals that were doubly recombinant in the interval

a, &n - between a given pair of markers were also scored as re- 5 i 2 gig?$- E i & @ a combinant. Only those mice that could be scored for

2 2 : II sets Q u e , each marker were shown in Table 3 (80/82 mice). ?? .: ::ye Two crossovers were found between NTPase activity

3 and D2Mit6 and 3 crossovers were found between

d

- “ 9

% ” E B

.$ 2

combinant was detected between the NTPase activity m and D2Mit6 among 22 FU strains (BXD-20 and -23 were a

nation frequency (95% confidence limits; SILVER 1985; SILVER and BUCKLER 1986) was estimated at 1.2 cM (0.0- 8.7 cM) . Four recombinants in 24 strains were detected between the NTPase activity and Ass-1, indicating an estimated map distance of 5.6 cM (1.3-21.0 cM).

-

5 a

D2Mit7 (Table 2). Using these additional RI strains we revised the SDPs for more precise mapping of D2Mit6

2 2 s Le, (GBASE 1993). D2Mit 7 maps approximately 1.1 cM

(0.0-7.7 cM) distal to H c (hemolytic complement, D’EUSTACHIO et al. 1986), in agreement with the pub- n 8 2

c,

k c (95% confidence intervals) were derived for the loci on proximal chromosome 2: D2Mit6-1.2 cM (0.0-8.7

c c c 0 u. j : ,z u

activity and to more precisely map this gene, a multi- point linkage analysis was carried out in the B6D2F, X

- & p rosatellite markers D2Mit1, D2Mit6 and D2Mit7. Data

i; @ I $. 2 c Q 2

c

Genetics of Brain NTPase in Mice 261

Chromosome 2

D2Mit6

15

D2Mit7

20

FIGURE 2.-Linkage map of proximal mouse chromosome 2 deduced from segregation of NTPase activity with other markers in B6D2F, X D2 backcross mice. The loci mapped are listed to the right of the chromosome and the distance from the centromere is indicated to the left.

TABLE 3

Recombination frequencies between pairs of scored markers on chromosome 2 in the B6D2F, X D2 backcross mice

Interval of Recombinations/80 Recombination recombination" total mice analyzed frequencyb

DZMitI-D2Mit6 8 10.0 2 3.4 DPMitl-Ntp 10 DPMitl-D2Mit7

12.5 -t 3.7 12c 13.8 -t 3.9

DPMit6-Ntp 2 DZMit6-DPMit7

2.5 -t 1.8 4c 5.0 -+ 2.4

NtP-DPMit7 3 3.8 -t 2.2

a The most proximal locus is listed first. bThe recombination distance is expressed in cM 2 the standard

error. Includes double recombinants between each pair of loci.

NTPase activity and D2Mit 7 (Table 3). These data place the gene for NTPase activity approximately 2.5 5 1.8 cM ( +SEM) distal to D2Mit6 and 3.8 t 2.1 cM proximal to D2Mit7. This order is also supported by the recombination distances with the most proximal marker tested, D2Mitl. Recombination (including double crossovers) between D2Mitl and D2Mit6 occurred in 8/80 mice, between D2Mitl and Ntp in 10/80 mice, and between D2Mit 1 and D2Mit 7 in 12/80 mice These data are com- patible with the gene order and map distances ( ~ S E M ) : D2Mitl-10.0 t 3.4 cM-D2Mit6-2.5 t 1.8 cM-Ntp-3.8 t 2.1 cM-DBMit7 (Figure 2) . The distance between D2Mitl and D2Mit6 (10.0 t 3.4 cM) is in agreement with the approximate 10.0 cM distance presented in the composite genetic map (GBASE 1993). The distance between D2Mit6 and D2Mit7 based on backcross data is approximately 6.3 cM, compared to approximately 10.1 cM based on our RI strain data. Our results from RI

strain data and backcross data place the gene for NTPase activity (Ntp) on the proximal region of mouse chromosome 2 between D2Mit6 and Ass-1, near the gene for CY brain spectrin-2, Spna-2 (GBASE 1993).

No significant associations were found between the Ca2+- or Mg2+-stimulated ATPase activities and mean seizure severity scores (SSS) in the 24 RI strains ( T = -0.11 and T = -0.06, repectively). The mean SSS of the 14 RI strains with low NTPase activity (0.77 2 0.20) was not significantly different from the mean SSS of the 10 RI strains with high NTPase activity (0.83 t 0.23) ( P < 0.4; as determined by the t-test). Previous studies reported the mean SSS for 22 of the BXD RI strains analyzed (SEYFRIED et al. 1980; PALAYOOR and SEYFRIED 1984a). We typed two additional strains, BXD-22, and BXD-31. A total of 30 BXD-22 mice were tested; 20 had no response, 6 had wild running, and 4 had clonic or tonic seizures. A total of 44 BXD-31 mice were tested and none had a seizure response. The mean SSS of the BXD-22 and the BXD-31 mice, were computed as 0.47 and 0.0, respectively.

The backcross mice were typed for NTPase activity and the microsatellite markers, as well as for AGS susceptibility, and the brown ( b ) and dilute ( d ) coat color genes. Chi-square analysis indicated no significant association between Ca2+-stimulated NTPase activity and AGS susceptibility (x2 = 0.054, P 9 0.9), the dilute coat color ( d ) locus on chromosome 9 (2 = 0.045, P 5 0.9), or the brown coat color ( b ) locus on chromosome 4 (x' = 0.195, P 5 0.7). The incidence of AGS was higher in brown mice (80%) than non-brown mice (49%). The mean SSS ( I~IsEM) of the 39 brown mice (1.8 t 0.1) was significantly higher than that of the 41 non-brown mice (1.2 5 0.1) ( P < 0.01; as determined by the t-test). Chi- square analysis indicated that this association was significant (x' = 8.2, P 5 0.01). There was no association between AGS and the d locus.

DISCUSSION

Our previous studies showed that the Ca2+-ATPase reduction in D2 mice was expressed in brain homogenates as well as in microsomes, synaptosomes, and synaptic plasma membranes prepared from the whole brain (PALAYOOR et al. 1986). We analyzed Ca'+- and Mg2+- stimulated NTPase activities in brain microsomes because this preparation had the greatest difference in activity between the B6 and D2 strains and was easily isolated (PALAYOOR et al. 1986).

ATPases in mammalian brain fractions and cultured neural cells can be activated by Ca" or Mg2+ (HOSIE 1965; GERMAIN and PROULX 1965; KADOTA et al. 1967; STEFANOVIC et al. 1974a, 1976; ROSENBLATT et al. 1976, 1977; SORENSEN and MAHLER 1981, 1982; LIN and WAY 1982,1984; NAGY 1986; NAGY et al. 1986). We found that both Ca2+ and Mg" could activate ATP hydrolysis in mouse brain microsomes. ATPases from brain have a

262 K. M. Allen and T. N. Seyfried

relatively broad nucleotide triphosphate specificity (GINSBURG et al. 1967; STEFANOVIC et al. 1974b, 1976; TRAMS and LAUTER 1974; LIN and WAY 1982, 1984; NAGY 1986; NAGY et dl . 1986). We also found that several NTPs (ATP, CTP, GTP or UTP) could serve as substrates for brain Ca2+- or Mg2+-ATPase activity. The similar patterns of inheritance and correlation coefflcients among the NTPase activities suggested that the brain Ca2+- or Mg2+- ATPase activities represent a single enzyme.

Presently, there is controversy over whether the Ca2+- and Mg2+-ATPases represent a single enzyme or separate enzymes. Several investigators have concluded that a single enzyme, stimulated by Ca" or Mg2+ could hydro- lyze NTPs. STEFANOVIC et al. (1976) suggested that one enzyme was involved since the Ca2+ or Mg2+ stimulated ATPase in cultured neuronal and glial cells cleaved several NTPs and was equally affected by several inhibitors. Furthermore, Ca'+- or Mg'+-stimulated competition by ATP and ITP for the same binding site suggested that the enzyme represented a divalent cation stimulated nucleotide triphosphatase. The thermodynamic properties of the Ca2+- and Mg2+-stirnulated activity were similar to each other suggesting that only one enzyme moiety was present (LAFFERTY et al. 1985). Additionally, both the Ca2+ and Mg"-ATPases had similar maximal stimulation and responses to most inhibitors and drugs. The similar responses to divalent cations, NTPs, tempera- ture, inhibitors and drugs suggested that Ca2+- and Mg2+-stimulated NTPase activities represent the same enzyme.

Although the above studies demonstrated similarities between Ca2+- and Mg2+-stimulated ATPase activities, others have presented data that differentiate between the two enzymes. In synaptic plasma membranes, Ca2+- and Mg2+-ATPase have been shown to respond differ- ently to several inhibitors, and activators (NAGY et al. 1986; GHANDI and ROSS 1988). Functional differences between the enzymes were suggested by a selective increase in Ca2+-ATPase activity in response to dibutyryl cyclic AMP (STAUDERMAN et al. 1985). Kinetic analysis and the effects of monovalent cations differentiated Ca2+- from Mg2+-stimulated ATPase activity (LIN 1983; LIN and WAY 1984). Mg2+-ATPase was also differentiated from Ca2+-ATPase due to the association between AGS susceptibility and Ca2+-ATPase activity in the BXD RI strains (PALAYOOR and SEYFRIED 1984a,b),

In many ways, the behavior of the Ca2+- and Mg2+- ATPase activities are similar but it was argued that se- lectivity to different conditions supported the two enzyme concept. Rather than separate enzymes, in the presence of different divalent metal ions the enzyme may assume different conformations (SORENSEN and MAHLER 1981; LAFTERTY et al. 1985). Thus, the enzyme varies its accessibility to inhibitors, activators, monovalent cations and NTPs. The difference in the pattern of substrate specificity between Ca'+- and Mg2+-stimulated

NTPase activities may result from differential binding of the cations to the same enzyme. LIN and WAY (1984) suggested that Ca2+ and Mg" bind to different sites on the enzyme, but may share the same ATP site. However, they concluded that the Ca2'- and Mg2+-ATPases represent separate enzymes based on kinetic studies.

Our results show that the Ca2+- or Mg2+-stimulated hydrolysis of NTPs in mouse brain is regulated through the action of a single gene. The bimodal pattern of inheritance of Ca2+- or Mg2+-stimulated NTPase activities among the 24 RI strains suggested a single gene. In the backcross mice, the 1:l phenotypic ratio of DP-like to F,-like NTPase activities was also consistent with the mo- nogenic inheritance of NTPase activity. The results of our genetic analysis therefore support the single enzyme hypothesis of the Ca2+- and Mg2+-ATPase.

A strong correlation was found between our patterns for microsomal Ca2+- or Mg2+-ATPase activities and those previously found by PALAYOOR and SEYFRIED (1984a,b) using brain homogenates from the RI strains ( r = 0.75 and r =0.69, respectively). The previous pattern for activity in brain homogenates, however, was continuous and could not be clearly divided into two distinct groups. Multiple factors in the crude brain homogenate, including several other ATPase activities ( e . g . , Na,K- ATPase, mitochondrial ATPase, Ca2+ t Mg2+-ATPase) may have obscured the bimodal distribution. A SDP was generated from the continuous distribution of brain homogenate Ca2+-ATPase activities by dividing the strains into those with higher activity (>2.4) and those with lower activity (<2.4) (PALAYOOR and SEYFRIED 1984a). Based on this SDP, a gene for Ca2+-ATPase activity ( Caa) was predicted on chromosome 12 near the Ah locus and a gene influencing AGS susceptibility (SEYFRIED et al. 1980; SEYFRIED and GLASER 1981; PALAYOOR and SEYFRIED 1984a; NEUMANN and SEYFRIED 1990). Reanalysis of the SDP for Caa compared with other previously mapped loci showed linkage to proximal chromosome 2 between Neb and Pmu. Our pattern of segregation in the RI strains and backcross progeny indicated that N t p was located on proximal mouse chromosome 2 between D2Mit6 and Ass-1. The predicted location of Caa, based on data reanalysis, was aproximately 10 cM distal to Ntp. This provides further evidence that NTPase activities map to proximal chromosome 2, rather than chromosome 12. The linkage to chromosome 2 was not recog- nized previously because of the lack of sufficient markers in this region of the genome.

Reduced brain Ca2+-ATPase activity was previously suggested as a possible mechanism of AGS since those strains with a lower Ca2+-ATPase activity had higher AGS susceptibility (PALAYOOR and SEYFRIED 1984a; PALAYOOR et al. 1986). A negative correlation ( r = -0.66; P< 0.01) was found between brain homogenate Ca'+-ATPase activity and AGS susceptibility among 21 BXD RI strains


(PALAYOOR and SEYFR~ED 1984a). The correlation coefficient included data for the B6 and D2 progenitor strains. The inclusion of progenitor strain data tends to increase the random association between linked loci, shifting the correlation in the direction of the parental difference (NEUMANN 1992). Eliminating the parental values would result in a lower correlation coefficient. Four of the strains, which were not used in the original correlation analysis between brain homogenate Ca*’-ATPase activities and AGS susceptibility (BXD-22, -25, -30 and - 31) had low ATPase activity and low AGS susceptibility. In- clusion of these new RI strains further weakens the association between low Ca2+-ATPase activity and high AGS susceptibility.

Furthermore, linkage was not suggested between NTPase activity and a n y of the known AGS genes. The major gene for AGS, Asp-1, maps to chromosome 12, whereas the minor modifiers Asp-2 and Asp-3 map to chromosomes 4 and 7, respectively (COLLINS and FULLER 1968; COLLINS 1970; SEYFRIED et al. 1980; SEYFRIED and GLASER 1981 ; NEUMANN and SEYFRIED 1990; NEUMANN and COLLINS 1991, 1992). The RT strain pattern for NTPase activity shows no association with markers on chromosomes 4, 7 or 12. Although our backcross data showed linkage between the b locus and AGS, supporting the chromosome 4 assignment of Asp-2 (COLLINS and FULLER 1968; COLLINS 1970; SEYFRIED et al. 1980; NEUMANN and SEYFRIED 1990; NEUMANN and COLLINS 1991), no association was found between NTPase activity and AGS susceptibility or the b locus on chromosome 4. Our data also support the conclusion that the d locus was not associated with AGS in crosses involving the B6 and D2 strains (COLLINS 1974). Although linkage was previously suggested between the d locus and a brain NTPase activity, having similar properties to our microsomal enzyme (GINSBURG et al. 1967), we found no association between the d locus and NTPase activity.

The role of brain ecto-NTPase activity in mechanisms of epilepsy is not clear. No consistent differences in brain ecto-ATPase activities were found among various genetic animal models of epilepsy including, parox- ysmal chicks, epi chicks, hamsters, gerbils, grass- hopper mice or the mouse mutants tottering and EL (ROSENBLATT et al. 1977; PALAYOOR and SEYFRIED 1988; NAGY et al. 1990b). The results from this study show no association with AGS susceptibility in D2 mice. Although enhanced brain ecto-ATPase activity was found in the posterior hippocampus of humans with temporal lobe epilepsy (NAGY et al. 1990a), it was not clear whether the enzyme change was associated with the seizure-induced neuronal loss that occurs in this tissue. Elevated NTPase activities were previously found in association with neuronal loss in various cerebellar mutants i . e . , weaver, stag- gerer and Purkinje cell degeneration (S. T. PALAYOOR and T. N. SEYFRIED, unpublished observations) and with progressive sciatic nerve degeneration in Trembler mice

(B~RGISSER et al. 1987). Hence, altered NTPase activity may be associated with the effect, Le. , neuronal loss or damage, rather than the cause of epileptic seizure activity. Thus the role of NTPase activity in epilepsy should be reevaluated.

Linkage was not suggested between N t p and other previously mapped genes with NTPase activity. Ntp does not map near either the erythrocyte inosine triphosphatase locus, Itp, on distal chromosome 2 (SICILIANO et al. 1984; TAYLOR et al. 1987) or the Na,K-ATPase s u b units on chromosomes 1,3, 7 and 11 (KENT et al. 1987; MALO et al. 1990). The gene for NTPase activity maps near Spna-2, a brain spectrin-2, on chromosome 2. Al- though the role of brain spectrin is unclear, it can be deduced from the role of erythrocyte spectrin. In the erythrocyte cytoskeleton, actin ATPase activity may be stimulated by spectrin and the erythrocyte ATPase activity may be involved with the maintenance of erythrocyte plasticity and shape (KIRKPATRICK et al. 1975; BASKIN and LANGDON 1981; MIRCEVOVA et al. 1983; SATO et al. 1986; Xu et al. 1991). Thus, a brain spectrin-2 may be associated with NTPase activity and together they may be involved with the release of synaptic vesicles or the regu- lation of the synaptic cleft in the brain. Cosegregation of the a1 catalytic subunit of the Na,K-ATPase gene, Atpa-3, with an a-spectrin gene on chromosome 1, s u p ports the possible association between NTPase activity and spectrin (KENT et al. 1987). Further study is needed to establish the relationship between NTPase activity and spectrin genes.

While NTPase activity has not yet been mapped on the human genome, we can predict its location on the basis of known human and mouse linkage homologies (NADEAU 1989; O’BRIEN et al. 1993). The N t p locus maps within the region of proximal chromosome 2 syntenic with human chromosome 99, suggesting that the gene for NTPase activity in humans may also map to chromosome 9 (GBASE 1993). Although there are several genes in this region of human chromosome 9, none appear to be candidate genes for NTPase activity.

We thank PAUL NEUMAN for valuable comments. The research was submitted to Boston College in partial fulfillment of the Ph.D. degree (K.M.A.) and was supported by National Institutes of Health grants NS23355, and NRSA MH10138, and by a Boston College REG.

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