JBC, Vol. 250, Issue 11, 4178-4184, Jun, 1975
Molecular properties of purified (sodium + potassium)-activated adenosine triphosphatases and their subunits from the rectal gland of Squalus acanthias and the electric organ of Electrophorus electricus
J. R. Perrone, J. F. Hackney, J. F. Dixon and L. E. Hokin
The chemical properties of two highly purified preparations of (sodium +
potassium)-activated adenosine triphosphatase (NaK ATPase) and their
subunits have been compared. One preparation is derived from the rectal
gland of the spiny dogfish shark, Squalus acanthias and the other
preparation is derived from the electric organ of the electric eel,
Electrophorus electricus. Ouabain binding and phosphorylation from
[gamma-32-P]ATP for both enzymes ranged from 4000 to 4300 pmol per mg of
protein. This gives a stoichiometry for ouabain binding and phosphorylation
of 1:1 for both enzymes. The molar ratios of catalytic subunit to
glycoprotein was 2:1 for both enzymes, suggesting a minimum molecular
weight of 250, 000, which agrees with the molecular weight obtained by
radiation inactivation. Assuming that only one of the two catalytic
subunits is phosphorylated and binds ouabain per (sodium +
potassium)-activated adenosine triphosphatase molecule the data on
phosphorylation and ouabain binding also give a molecular weight of 250,
000. The data on phosphorylatiion, ouabain binding, subunit composition,
and molecular weight based on radiaion inactivation are thus all internally
consistent. A technique has been developed for isolation of pure catalytic
subunit and glycoprotein in good yields by preparative sodium dodecyl
sulfate-polyacrylamide gel electrophoresis. A variety of chemical studies
have been carried out with the purified subunits. The amino acid
composition of the catalytic subunit was different from that of the
glycoprotein, but the amino acid composition of each of the two subunits
was essentially the same for both species. However, the NH2-terminal amino
acid for the catalytic subunit was alanine for the rectal gland enzyme and
serine for the electric organ enzyme, suggesting some differencesin amino
acid sequences for the two species. The NH2-terminal amino acid for the
glycoprotein was alanine for the two species. The glycoproteins from both
species contained the same carbohydrates but in quite differing amounts.
The carbohydrates were glucosamine, sialic acid, fucose, galactose,
mannose, and glucose. The release of all the sialic acid from the electric
organ enzyme and the release of 40% of the sialic acid from the rectal
gland enzyme did not affect (sodium + potassium)-activated adenosine
triphosphatase activity. Both enzymes contained the following
phospholipids, which accounted for 98 to 100% of the total phospholipid
phosphorus: sphingomyelin, lecithin, phosphatidylserine,
phosphatidylethanolamine, and phosphatidylinositol. With the exception of
phosphatidylethanolamine, and phosphatidylinositol. With the exception of
phosphatidylserine, the amount of any phospholipid per mg of enzyme as well
as the total phospholipid content were quite different for the two enzymes.
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