Biopolymers and cell. Volume 20. № 3. 207-214.

M. T. Bobrovskaya, V. A. Kovalev, T. V. Shirina, A. V. Rudenko, E. A. Kozlov

Dynamics of cleavage of the Bombyx mori nuclear polyhedrosis virus polyhedrin by polyhedral protease and identification of cleaved bonds

Summary

It is shown that polyhedral protease (PP) cleaves the polyhedrin polypeptide chain bonds Tyr-Ile (208-209), Tyr-Thr (186-187), Ala-Asn (121-122), Trp-Ser (88-89) upon polyhedron dissolution under alkaline (pH 11.0) conditions at room temperature during 2 hours. According to the data obtained by the sodium dodecyl sulfate (SDS) polyacrylamide gel  electrophoresis (PAGE), none of the bonds is cleaved completely, that results in the formation of overlapping polypeptides with molecular weights of 28000 (a whole polypeptide chain), 25000, 22000, 14500, 10500, 8000 (polypeptide chain fragments). We calculated the molecular weights of all possible fragments which could be derived from the polyhedrin polypeptide chain (28 kDa) by PP cleavage. The 25, 22, 14.5, 10.5, and 88 kDa polypeptides correspond to the polypeptide chain fragments 1-208 (m. w. 24715), 1-186 (m. w.  21980), 1-121 (14362), 1-88 (10443), 122-186 (7633). The bond 208-209 is the first to show the cleavage in 15 min of dissolution, the bond 186-187 is the second (in 45 min), and the bonds 121-122, 88-89 are cleaved in 120 min. These results do not depend on the prolongation of the dissolution time at room temperture, while the bonds Tyr-Leu (118-119), Phe-Val (193-194) and Tyr-Lys (203-204) are cleaved in addition after 120 min of dissolution at 37 ⁰C. Under these conditions the bonds 88-89, 118-119, 203-204, 208-209 are cleaved completely contrary to the bonds 121-122, 186-187, 193-194 cleaved only partially. A series of fragments 1-88, 118/122-186/193/203 with the calculated molecular weights of 7633, 7916, 8445, 8728, 9765, 10048, 10443 respectively is released by the PP cleavage at 37 ⁰C. These data are in accord with a diffused band in the range of molecular weights of 7000-10000, obtained by SDS-PAGE for the PP cleaved polyhedrin at 37 ⁰C. The PP cleavage effect on the association/dissociation process of polyhedrin in solution as well as on the association of polyhedrin and its fragments with virion membranes is discussed considering the data obtained.