Professional Documents
Culture Documents
J. Biol. Chem.-1951-Swanson-577-90 PDF
J. Biol. Chem.-1951-Swanson-577-90 PDF
BY MARJORIE A. SWANSON
(From the Department of Biochemistry, Bowman Gray School of Medicine, Wake Forest
College, Winston-Salem, North Carolina)
with a “Pyre-Pase” which they have isolated from brain.2 This Pyro-
Pase splits only Pyro-P and ATP, and is completely dependent on the
presence of magnesium ions for activity. Pyro-Pases requiring magne-
sium have also been described in liver, erythrocytes, yeasts, and other
tissues. Since the Pyro-Pases occur along with “ATPase,” it is not easy
to decide whether or not the two activities are due to the same protein.
However, yeast Pyro-Pase, which has been obtained by Bailey and Webb
(5), did not split ATP, so that at least in this case the two activities are
not inseparable.
There has been a tendency to think of ATPases as requiring calcium
ions, probably because the calcium activation of myosin is so well known.
However, Bailey (6) has found that liver ATPase was much more strongly
HDP, Schwarz, purified through the acid barium salt according to Neu-
berg et al. (8).
Rib-5-P, prepared by the acid hydrolysis of ATP, followed by the iso-
lation and purification of the ethanol-insoluble barium salt.
The animals used were healthy, adult albino rats maintained on the
Rockland rat diet (complete). They were not fasted, and were sacrificed
by decapitation. Homogenates were prepared in a Potter-Elvehjem all-
glass homogenizer.
In the earlier experiments, 0.1 M maleate buffer was used at pH 7.4,
and a series of lactate, acetate, maleate, and barbiturate buffers to cover
the range between pH 3 to 10. Later, an equimolecular mixture of ace-
tate, maleate, and glycine (to be referred to as AMG buffer), 0.1 M with
Results
Action of Fresh Homogenate on Various Substrates-A typical experiment
of the survey series is shown in Table I. It can be seen that there is
significant activity against all the substrates used except A-3-MP. Gua-
nylic and cytidylic acids and cu- Bnd /3-Gly-P were omitted for the sake of
brevity. They are split at an even lower rate than A-3-MP. Glucose-
6-phosphatase is not very active3 at this pH. Ca++ stimulates the splitting
of only ADP and ATP, while Mg++ increases the rate of splitting these
substrates and also that of the inorganic polyphosphates. There is little
evidence of “non-specific” phosphatase at this pH, even in the presence
TABLE I
M
Ad-MP ............... 0.005 4.3 4.0 5.1
ADP .................. 0.005 2.0 3.9 6.7
ATP (b) .............. 0.004 3.8 11.6 27.9
Pyro-P . .............. 0.006 2.1 3.1 20.0
‘I ................ 0.0012 0.9 0.9 24.1
Tri-P-P ............... 0.005 3.7 3.4 6.4
‘I ............... 0.001 0.9 0.5 5.7
Phenyl-P ............. 0.006 4.1 5.4 4.5
A-3-MP . .............. 0.006 0.9 0.9 0.9
Rib-5-P. .............. 0.007 2.7
HDP .................. 0.012 3.1
was removed, diluted 1: 10 with water, and tested immediately. The re-
sults are shown in Fig. 1, from which it appears that about one-third of
the Mg++-activated ATPase is lost in the first 4 hours. There is appar-
ently no loss of A-5-MPase (5-nucleotidase of Reis (12)) or of the Ca++-
activated ATPase during the 1st day. They were not followed any longer
than this.
TABLE II
Rate of Splitting Various Phosphate Compounds by Fresh Rat Kidney Homogenate
Conditions as in Table I.
Substrate Finalconcentration Control With Ca++ With MpH
-
cent either in 0.15 M KC1 or in distilled water, were made and stored in
ice. Immediately before testing, a portion was diluted 1: 10 with water.
Some of the experiments were carried on consecutively with the same
homogenate. Because of the large number of samples required for one
curve, this occasioned a delay of as much as 4 hours between the death of
the rat and the commencement of an experiment. For this reason, the ab-
solute values were not always the same, but the several individual curves
all had similar characteristics. In Figs. 2 and 3 are presented the curves
which are averages of several experiments comparable with respect to the
time after the death of the animal. It can be seen that all the other ions
tested gave a greater activation than Ca++, but that, at higher concen-
trations, the extent of activation decreased. This confirms the results
reported by DuBois and Potter (7). *However, it should be noted that,
if 0.1 M Mg++ had been the only concentration tested, one would still have
observed a doubling of the activity, as compared to the control with no
added Mg++. Therefore, it seems incorrect to say that Mg++ inhibits
ATPase, as DuBois and Potter have said.
On the basis of these results a standard assay system was adopted, which
contained ATP (b) 0.004 M and Mg++ 0.005 M, final concentrations. With
this system, we assayed numerous fresh homogenates over the course of
a year and found excellent agreement between the levels of ATPase ac-
tivity. With Mg++, direct proportionality between activity and tissue
concentration is obtained only for the smaller amounts of tissue, as shown
in Fig. 4.
584 PHOSPHATASES OF LIVER. II
60
20
IO
I 2 3 4 5
MGM TISSUE
FIG. 4. Influence of tissue concentration on rate of splitting ATP by fresh homo-
genate. AMG buffer, pH 7.4, 15 minutes, 37’; w 0.005 Mg* final concentration; l
no added ions.
activating ions used. It is also possible that changes occur in the distri-
bution of some enzymes by the presence of salts in the homogenizing
medium. At the present time, it is apparent that the whole problem
could bear thorough reinvestigation, with determination of the optimal
conditions for measuring the activity of each fraction.
Chemical Fractionation-An ATPase splitting off only one phosphate
could be obtained from homogenates of rat liver in 0.88 M sucrose after
removal of cells and nuclei, followed by dialysis overnight in order to
remove the sucrose. A quicker procedure involved starting with a 0.15 M
KC1 homogenate and removing cells, nuclei, and mitochondria in one cen-
trifugation. 0.1 volume of acetate buffer, 0.1 M, pH 5.2, was added to
the supernatant solution. The pH of the solution becomesabout 5.4 and
M. A. SWANSON 585
varied with the extent to which the enzyme is in true solution, the more
soluble enzyme being the less activated. Here the situation is reversed,
the more soluble enzyme being markedly activated by Mg++. Further
study is obviously required, with each series of experiments including sep-
arate assays with Ca++ and Mg++. Apparently for technical reasons, most
authors who have studied the problem have omitted one or the other of
the ions.
Fraction 4 is usually red, giving a solution resembling dilute hemo-
globin. However, the Pyro-Pase of this fraction is very much more active
than that of the red cells, so that this activity cannot all be due to ery-
throcyte Pyro-Pase. The ATPase of Fraction 4 slowly loses its activity
when stored in ice, so that the solution becomes unusable after about 3
Time
I Inorganic P Remaining easily hydrolyzable P
min. Y Y
15 8.5 96.5
30 11.8 93.2
45 17.2 87.8
60 17.0 88.0
90 16.0 89.0
results obtained with whole homogenate, and instead rather resembles the
behavior of the purified brain Pyro-Pase of Binkley and Olson (4). Not
only does Ca++ not activate the ATPase, but, when it is added to the
z
ITI
l-
g 80 40r
a
5 30 4
I 60
oc
k!
0. 40
E
0.006 M 0.006 M
DISCUSSION
Several years ago the idea that there were only a few phosphatases of
rather low specificity was seemingly well established. However, more re-
cent evidence, with which the present results agree, indicates not only
that there may be a distinct enzyme catalyzing each reaction, but also
that one tissue may have more than one enzyme catalyzing what is ap-
parently the same reaction. Thus muscle has been shown to have two
ATPases, and indications of a similar situation have now been obtained
for rat liver. All of the enzymes of this kind which have been studied up
to date require a divalent metallic ion as cofactor, magnesium and calcium
being the most effective. When both these ions are present, they appear
The author wishes to thank Dr. Camillo Artom for his constant interest
SUMMARY
BIBLIOGRAPHY
1. de Duve, C., Berthet, J., Hers, H. G., and Dupret, L., Bull. sot. chim. biol., 31,
1242 (1949).
2. Swanson, M. A., J. Biol. Chem., 184, 647 (1950).
3. Kielley, W. W., and Meyerhof, O., J. BioZ. Chem., 176, 591 (1948).
4. Binkley, F., and Olson, C. K., J. BioZ. Chem., 186, 725 (1950).
5. Bailey, K., and Webb, E. C., Biochem. J., 38,394 (1944).
6. Bailey, K., Biochem. J., 36, 121 (1942).
7. DuBois, K. P., and Potter, V. R., J. BioZ. Chem., 160, 185 (1943).
8. Neuberg, C., Lustig, H., and Rothenberg, M. A., Arch. Biochem., 3,33 (194344).
9. Fiske, C. H., and Subbarow, Y., J. BioZ. Chem., 66, 375 (1925).
10. Naganna, B., and Narayana menon, V. K., J. BioZ. Chem., 174,501 (1948).
11. Margaria, R., Arch. SC. biol., 9, 305 (1927).
12. Reis, J., Enzymologia, 2, 110, 180 (1937).
590 PHOSPHATASES OF LIVER. II
13. Rosenthal, O., Rogers, C. S., Vars, H. M., and Ferguson, C. C., J. Biol. Chem.,
186,669 (1950).
14. Rowles, S. L., and Stocken, L. A., Biochem. J., 47,489 (1950).
15. Michelson, A. M., and Todd, A. R., J. Chem. Xoc., 2487 (1949).
16. Jones, L. J., Ind. and Eng. Chem., Anal. Ed., 14, 536 (1942).
17. Lehninger, A. L., and Smith, S. W., J. Biol. Chem., 181, 415 (1949).
18. Hogeboom, G. H., Schneider, W. C., and Pallade, G. E., J. Biol. Chem., 173,
619 (1948).
19. Kennedy, E. P., and Lehninger, A. L., J. Biol. Chem., 179, 957 (1949).
20. Swanson, M. A., Federation Proc., 9,236 (1950).
21. Schneider, W. C., J. Biol. Chem., 186, 585 (1946).
22. Steinbach, H. B., and Moog, F., J. Cell. and Comp. Physiol., 26, 175 (1945).
23. Kornberg, A., J. BioZ. Chem., 182,779 (1950).
24. Gordon, J. J., Biochem. J., 46, 121 (1950).