The Primary Structure of the Heavy Chain of Chicken Gizzard Myosin

Research Project

Project/Area Number	59580107
Research Category	Grant-in-Aid for General Scientific Research (C)
Allocation Type	Single-year Grants
Research Field	物質生物化学
Research Institution	Nagasaki University
Principal Investigator	MAITA Tetsuo Assistant professor of Nagasaki University, School of Medicine, 医学部, 助教授 (80039502)
Project Period (FY)	1984 – 1986
Project Status	Completed (Fiscal Year 1986)
Budget Amount *help	¥1,600,000 (Direct Cost: ¥1,600,000) Fiscal Year 1986: ¥300,000 (Direct Cost: ¥300,000) Fiscal Year 1985: ¥300,000 (Direct Cost: ¥300,000) Fiscal Year 1984: ¥1,000,000 (Direct Cost: ¥1,000,000)
Keywords	Myosin / Smooth Muscle / Chicken Gizzard Muscle / 一次構造
Research Abstract	It may be necessary to reveal the primary structure of myosin, a major component of the contractile apparatus, for studies of muscle contraction at the molecular level. In the present study, we determined the N-terminal 203-residue and C-terminal 203-residue sequences of the S-1 heavy chain of chicken gizzard myosin. 1. Gizzard myosin was modified with N-iodoacetyl-N'-(5-sulfo-1-naphtyl)ethylenediamine (abbreviated as IAEDANS) in the presence of ATP. From the tryptic digest of the modified myosin, a fluorescent fragment (24 kilodalton) was isolated by gel filtration on a Sephadex G-100 column in the presence of 5 M guanidine-HCl followed by chromatography on a CM 52 column in the presence of 8 M urea. The amino acid sequence of the fragment was determined by conventional methods. The fragment contained 203 amino acid residues and a blocked N-terminus, and was assigned as an N-termnal part of the heavy chain. An <epsilon> -N-trimethyllysine was recognized at the homologous position with … More skeletal myosin, but no <epsilon> -N-monomethyllysine. Trp-130 of rabbit skeletal myosin heavy chain which is considered to be one of ATP-binding residue was replaced by gultamine in gizzard myosin. Cys-93 of the fragment was the amino acid residue whose reaction with IAEDANS alters the ATPase activity of gizzard myosin. 2. Gizzard myosin was modified with IAEDANS in the absence of ATP, and digested with papain. From the digest, a fluorescent fragment of Mr=23,800 was isolated, and sequenced as described above. The papain-fragment contained 203 amino acid residues which could be assigned as C-terminal part of the S-1 heavy chain based on the homology with the sequence of skeletal myosin. The C-terminal 53 residues of this fragment, constituting the neck part of myosin, contained 13 positively charged residues but no negatively charged residue. 3. We are sequencing the 50 kilodalton fragment which lies between the above two fragments. The primary structure of the gizzard myosin head could be revealed in the near future. Less