Magnesium-dependent NAD reductase. 
Protocol: Tris buffer, 
0.1 M, pH 9.0 = 1.80 ml 
NAD, 13 mM, 
10 mg/ml H,O = 0.10 ml 
MgCl.°6 H,O, 0.06 M 
(concn varied) 
H,O 
E to start reaction 
X ml (0, 0.10, 1.00) 
(1.00-X) ml 
0.10 ml 
The rate of reduction of NAD was followed 
spectrophotometrically at 340 nm. The control 
cuvette contained everything but the enzyme solu- 
tion. 
Malic enzyme.—The assay for ME, a measure of 
the rate of NADP reduction in the presence of malate, 
is based on the work of Ochoa et al. (1948), and has 
been described elsewhere (Gould, 1965). The buffer 
used here was Tris, 0.1 M, pH 8.0. 
a-glycerophosphate dehydrogenase.—This 
assay, with a discussion of the cation-dilution 
technique, is published in detail elsewhere (Gould, 
1969). 
Electrophoretic Procedures 
Electrophoresis.—Electrophoresis of cunner serum 
(ca 3 ul/column) was performed at 4°C using a dis- 
continuous buffer system, on 7% polyacrylamide gel 
columns, pH 9.1, with sample and stacker gels of 3% 
polyacrylamide, pH 5.2. The electrode buffer was Tris 
(0.005 M)-Glycine (0.038 M), pH 8.3. Running time 
was 60 min at 1mA/column followed by 105 min at 
3mA/column, using constant current. Both the gel for- 
mularies and the electrophoretic procedure are based 
on the work of Davis (1964) and have been fully 
described elsewhere (Gould and Medler, 1970). 
Stains.—For total-protein patterns, the gels were 
stained with amido schwartz 10B, 1% in 7% acetic 
acid. They were destained by passive diffusion in 
several changes of methanol-glacial acetic acid- 
distilled water (5:1:5), for a total of about 20 hr. 
For visualization of esterase sites, the gels were 
stained with a medium containing «a-naphthyl 
butyrate (50 mg in 2 ml acetone to dissolve, then 2 ml 
H,0), coupled with Fast Garnet GBC in 46 ml 
phosphate buffer, 0.1 M, pH 7.0. Polyvinylpyrrolidone 
(PVP) (ca 500 mg) was added to the buffer to aid 
solubilization of the dye. The substrate was added to 
the dye-PVP-buffer solution immediately prior to use, 
and the whole filtered through glass wool. Incubation 
was in the dark at room temperature for 45 min. 
RESULTS AND DISCUSSION 
In homogenates of fresh-frozen livers from cunners 
exposed to cadmium, AAT activity was significantly 
lower than in the controls (Table 1). Livers of fish ex- 
posed to 3 ppm Cd had only 71% of the AAT activity 
observed in livers of control fish and in fish exposed to 
24 ppm Cd, activity dropped to 59% of the control. 
Whether this cadmium-induced drop in activity 
represents a simple enzyme block, a depression of 
microsomal biosynthesis, or a more involved 
mechanism of inhibition cannot be speculated from 
these few data; the observations here serve only to in- 
dicate possibly profitable areas for further work. 
Parenthetically, activity of this transaminase in fresh- 
frozen livers had roughly 10 times the activity of livers 
frozen-stored for longer than 1 wk. Livers frozen for 2- 
8 wk (at ca +5°C) not only had much lower AAT ac- 
tivity than the fresh livers, but also had widely 
variable rates of loss of activity. 
Another fresh cunner-cadmium experimental 
series, with 14 fish at 0 ppm Cd and 14 at 24 ppm Cad, 
provided livers for a study of what appears to be a 
soluble, magnesium-linked NAD reductase (NADR- 
Mg). Initially, the assay was intended to be for a- 
GPdH, but it was discovered that the magnesium 
effect was stronger without the aGP substrate: in the 
presence of @GP(10 mM), 2 mM Mg produced only a 
1.2% and 20 mM only a 1.8% increase in reductase ac- 
tivity over activity with no added magnesium; 
whereas with no added substrate, 2 mM Mg produced 
58% and 20 mM produced 130% increase in reductase 
activity over that with no added magnesium (Table 
2). The endogenous substrate pool might be expected 
to contribute a strong variable to NADR activity 
(although not so much in teleosts as in marine in- 
Table 1.—Aspartate aminotransferase activity in liver of cunner, 
Tautogolabrus adspersus, exposed for 96 hr to varying concen- 
trations of cadmium, 25 ppt salinity. Each value is the change in 
absorbance at 340 nm for 1 min under assay conditions and 
represents the average of 2 tests. Each sample is a pool of livers 
from three fish; the enzyme preparation (E) has a 100X dilution 
factor. 
AAT activity 
Test concen (A A*°X10°/min/ 
Cd (ppm) 0.10 ml E) 
0 ppm Cd 151.5 
170.0 
152.5 
3 ppm Cd 110.0 
115.0 
24 ppm Cd 84.0 
93.5 
101.5 
