PARAMYOSIN POLARITY IN THE THICK FILAMENT OF MOLLUSCAN SMOOTH MUSCLES

Paramyosin is the main structural component of the thick filament of m olluscan smooth muscles. These filaments consist of a large paracrysta lline core of paramyosin with myosin arranged on its surface. The deta iled molecular packing of paramyosin in the core and the array of myos in on the surface of the paramyosin core remain unknown. An unsolved p roblem is the polarity of the paramyosin molecules within these thick filaments (i.e., it is not known whether the paramyosin molecules asse mble with their NH2-terminal ends pointing toward the center or toward the end of the thick filament). Here a method to distinguish between the NH2- and the COOH-terminal ends of the paramyosin molecule by elec tron microscopy is described and used to determine their polarity in s ynthetic paracrystalline arrays. This method consists of labeling the cysteine residues of paramyosin molecules with the avidin-biotin syste m developed by Sutoh et al. (1984). Accordingly, the sulfhydryl groups of paramyosin-isolated from the anterior byssus retractor muscle (ABR M) of Mytilus edulis-were modified with maleimide-biotin, and the biot inylated thiols were visualized in the electron microscope after glyce rol spraying/rotary rotary metal shadowing by attaching monomeric avid in to them. Avidin-biotin labeling of the native molecule and its carb oxypeptidase fragments revealed that ABRM paramyosin contains one pair of cysteine at its NH2-terminal end and one pair at similar to 30 nm from its COOH-terminal end. Synthetic paracrystalline arrays of paramy osin with known axial arrangement were also labeled with the avidin-bi otin system. The location of the bound avidin in these paracrystals in dicated the polarity of paramyosin in these arrays. The polarity was a lso determined by comparison of the transverse band-like staining patt ern of paracrystals of alpha-paramyosin (intact protein) and beta-para myosin (a proteolytically cleaved alpha-paramyosin that has lost a sma ll segment at its COOH-terminal end). Both methods revealed that param yosin assembles with its NH2-terminal end pointing toward the center o f the paracrystals. The implications of this result for the polarity o f paramyosin in the native filament core, and for the arrangement of m yosin on the surface of molluscan thick filaments, are discussed. (C) 1994 Academic Press, Inc.