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???displayArticle.abstract??? Titin and nebulin are two major protein components of the sarcomere matrix in striated muscles. Purified titin and nebulin from frog (Xenopus laevis) skeletal muscle are similar in size and in amino acid composition to their mammalian or avian counterparts. Both proteins contain substantial amounts of protein-bound phosphate: about 5 to 6 per titin subunit and 8 to 9 per nebulin subunit. Injection of radioactive inorganic phosphate into the dorsal lymph sacs of Xenopus laevis resulted in the significant incorporation of radioactivity into titin and nebulin within three days of incubation. Purified titin from in vivo labeled frog gastrocnemius muscle contains one mole of radioactive phosphoserine per mole of titin subunit. These data indicate that phosphorylation of frog titin and nebulin occurs in vivo.
Fig~ 1. Myofibrillar proteins of rabbit and frog skeletal muscles. SDS gel patterns of
rabbit and frog myofibril preparations are compared (2 to 12% gradient gels). Note that titin
and nebulin have similar inabilities, whereas the low molecular weight bands below actin differ
significantly.
Fig. 2. In vivo phosphorylation of titin and nebulin in live frog muscle. Frog
gastrocnemius muscle was isolated from [32p]-phosphate-injected Xenopus laevis, solubilized
and analyzed on 2 to 12% gradient polyacrylamide gels. Pairs of coomassie blue staining
patterns of wet gels (CB) and corresponding autoradiograms of dried gels (32p) are shown. (A)
Guanidinium chloride-solubilized frozen tissue (9 ug protein). Four radioactive bands are
detected in the titin/nebulin region (42-day exposure). Only two cornigrate with titin (T) and
nebulin (N), respectively. Bands Na and Nb have no corresponding coomassie blue bands. (B)
SDS-solubilized frog myofibrils that were washed without Triton X-100 (22 ug protein). Four
radioactive bands are detected in the titin/nebulin region (31-day exposure). (C) SDSsolubilized
frog myofibrils that were washed with Triton X-100 (16 ug protein). Note that
bands Na and Nb are absent. Only two radioactive bands, corresponding to titin (T) and nebulin
(N) are seen in this region (60-day exposure).
F!g. 3. Phosphoamino acid of labeled frog titin. Labeled titin purified (700 cpm, 400 ug
protein) was partially hydrolyzed and the acid-hydroiysate was analyzed by one-dimensional
paper electrophoresis. (A) Ninhydrin-stained paper electrophoretegram of titin hydrolysate
plus phosphoamino acid standards. (B) Phosphoamino acid standards. (C) Autoradiegram (27-
day exposure) corresponding to (A).
