J. Biol. Chem., Vol. 258, Issue 17, 10341-10347, 09, 1983
Tyrosine protein kinase activity of rat spleen and other tissues
G Swarup, JD Dasgupta and DL Garbers
Using a synthetic peptide (Glu-Asp-Ala-Glu-Tyr-Ala-Ala-Arg-Arg-Arg-Gly) as
a substrate, various normal tissues from the rat were probed for tyrosine
protein kinase activity. Spleen was shown to contain much higher tyrosine
protein kinase activity than other rat tissues (lung, brain, testes, liver,
kidney, heart, and thymus, in decreasing order of specific activity). Most
of the tyrosine protein kinase activity of the various rat tissues (greater
than 80%) was present in the particulate fraction, and Nonidet P-40, a
nonionic detergent, could activate the particulate form of the enzyme
2-20-fold in many of the tissues. Epidermal growth factor (1 microgram/ml),
cyclic AMP, cyclic GMP, or Ca2+ did not increase spleen tyrosine protein
kinase activity. Half- maximal enzyme activity was observed at 60-80 microM
MgATP and at 2.2 mM peptide, and both Mg2+ (10 mM) and Mn2+ (0.5-1.0 mM)
were effective divalent metal ions for the expression of activity. When the
particulate fraction of spleen was incubated with [gamma-32P]ATP followed
by polyacrylamide gel electrophoresis in the presence of Na dodecyl SO4, a
number of alkali-stable bands were identified by autoradiography. Two major
bands at Mr = 53,000 and 56,000 were shown to contain phosphotyrosine. Two
similar alkali-stable bands containing phosphotyrosine but with lower
amounts of 32P labeling were also observed in the particulate fractions of
various other tissues (lung, brain, kidney, and testes). The particulate
form of tyrosine protein kinase of rat spleen could be solubilized by using
high concentrations of Nonidet P-40 (5%) at an alkaline pH (pH 9.0).
Partial purification and subsequent filtration on Sephacryl S-200 yielded a
peak of tyrosine protein kinase activity with an apparent molecular weight
of 55,000. The two major phosphorylated bands of Mr = 53,000 and 56,000 co-
migrated with the peak of enzyme activity. The solubilized and partially
purified enzyme preparation phosphorylated only tyrosine residues when
either endogenous proteins or casein were used as substrates. These results
suggest that relatively high activities of tyrosine protein kinase exist in
a normal tissue (rat spleen). Major endogenous substrates of the enzyme(s)
appear to be represented by two proteins of Mr = 53,000 and 56,000; one or
both of these substrates may be the tyrosine protein kinase itself.
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