Urea extraction of Z bands, intercalated disks, and desmosomes

Preliminary biochemical investigations of the intermediate filaments

“…× 24 000 and × 72 000. attempted, we have assumed a protein composition. In this report we have demonstrated limited biochemical evidence based on extraction experiments which support the physical data and which initially indicated the presence of the intermediate filaments (23).…”

“…The present investigation, initiated in 1967 and completed in 1968 (22, 23), was originally conceived as a series of experiments to identify and characterize the presarcomere subunits and to determine whether the "free cytoplasmic thin filaments" were composed of actin (20), myosin (11), or both (10). As a direct result of this investigation, a third class of filaments differing significantly in composition and reactivities from the thick and thin filaments of cardiac muscle was first recognized (22,23,25) and is the subject of an accompanying report (26).…”

“…In an effort to differentiate between chemically different but perhaps physically similar filaments, electron microscope examinations followed nonspecific extractions of actin (4,30,31) and of thin filaments (22,23,25) using one of several neutral salt extraction procedures. Embryonic hearts of stages 11 to 15 were excised and placed for 1 hour in the "actomyosin" extractant (2,8,9,15,(30)(31)(32) composed of 0.6 M potassium iodide, 5 mM sodium thiosulfate, and 0.01 M /%mercaptoethanol solution, removed, and fixed in phosphate-buffered glutaraldehyde (26).…”

“…Additional extractions were carried out with buffered KI, slowly diluted from 0.6 M to 0.1 M before fixation. In an attempt to reduce osmotic damage to the tissue and to increase the opacity of the almost transparent hearts, additional extractions 'were attempted following brief exposure (30 seconds) to buffered osmium tetroxide, a procedure reported (23,25) to allow removal of the thin filaments while partially stabilizing the thick filaments, Z bands, and various other cytoplasmic structures. Although only 30 seconds of exposure to OsO4 was found to stabilize the Z bands [possibly indicating the presence of unsaturated carbon-carbon bonds characteristic of lipids (3)], longer periods of prefixation were employed before extraction, yielding data on the relative "fixability" of the thick, intermediate, and thin filaments.…”

Localization of [alpha]-actinin and tropomyosin in muscle and nonmuscle systems

“…Moreover, Hagopian and Spiro (1970) have demonstrated continuity between both the zonulae and the fasciae adherentes and Z-disks in embryonic chick myocardium, and have therefore suggested that Z-disks are derived from these cell junctions. Although this suggestion has been disputed by others, these ultrastructural findings suggesting a close similarity between Z-disks of skeletal muscle and the fascia adherens of cardiac muscle have been strengthened by finding that treatment of chick myocardium with a 1 to 3 M urea solution results in complete extraction of Z-disks and partial extraction of the dense material of the fasciae adherentes, while the thin and the thick myofila ments remain unaffected (Rash et , 1968 (Kawamura and James, 1971) to produce the five-layered, fused membrane structure. This appearance of the nexus led to the use of terms such as tight junction, fused membrane, quintuple-layered mem brane junction, pentalaminar junction, macula or fascia occludens and close junction to describe this structure.…”