A HIGH-MOLECULAR-MASS PROTEIN ISOLATED FROM CHICKEN GIZZARD - ITS LOCALIZATION AT THE DENSE PLAQUES AND DENSE BODIES OF SMOOTH-MUSCLE AND THE Z-DISKS OF SKELETAL-MUSCLE

We purified a 450 kDa protein from a low-salt alkaline extract of chic ken gizzard smooth muscle, This high molecular mass protein could be e xtracted with the low-salt alkaline solution at 37 degrees C but not a t 4 degrees C. The 450 kDa protein was isolated from the extract by am monium sulfate fractionation and following sequential column chromatog raphy using hydroxylapatite, DEAE-Cellulofine A-800m and phenyl-Sephar ose CL-4B resins, The partially purified protein molecule resembled a flexible rod with a globular head and an irregular-shaped tail. Its le ngth was approximately 300 nm, The nucleotide sequence of the partial cDNA encoding this protein was determined and analyzed with a data bas e, The analysis showed that the protein revealed significant homology with the rod region of chicken filamin (57% homology in amino acid seq uence). Immunoblot analysis showed that an affinity-purified antibody reacted exclusively with the 450 kDa protein band of smooth, skeletal and cardiac muscle tissues, By indirect immunofluorescence microscopy, we examined the localization of the 450 kDa protein in smooth and ske letal muscle cells, The affinity-purified antibody against the 450 kDa protein stained the dense plaques and dense bodies of smooth muscle, the peripheral region of Z-disks and the subsarcolemmal region of skel etal muscle. Immunoelectron microscopy confirmed the localization of t he 450 kDa protein at the peripheral regions of the actin anchoring st ructures mentioned above, Judging from its amino acid sequence, molecu lar size, molecular shape, immunological reactivity and localization i n muscle cells, the 450 kDa protein seemed to be a new component assoc iated with the actin-anchoring structures of muscle tissues, We tentat ively call this protein 'fulcin'.