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M Nishigai

Publications and source records attributed to M Nishigai.

24 records · Page 2Linked to original sources
PubMed1985

The quaternary structure and activity of newly purified fatty acid synthetase from the Harderian gland of guinea-pig.

Fatty acid synthetase was isolated from the Harderian gland of guinea-pig. The fatty acids synthesized by the purified enzyme were analyzed by mass fragmentography. The purified enzyme had an inherent capacity to utilize methylmalonyl-CoA and synthesize methyl-branched fatty acids. Physicochemical studies indicated that an active enzyme was a dimer, consisted of two subunits of Mr = 2.5 X 10(5). The negatively stained enzyme had an electron micrographic image of an ellipsoidal contour with a continuous middle cleft along the major axis. The major and minor axes were approximately equal to 220 and 150 A, respectively. In a dimer, the subunit had a rod-like structure about 220 A long and 50 A wide. The enzyme was inactivated and dissociated into subunits by incubation at 0 degree C. The inactivated enzyme was fully reactivated by raising the temperature of the solution. The relationship between the quaternary structure of the enzyme and the occurrence of enzymatic activity was studied by high-performance liquid chromatography. Neither active monomers nor inactive dimers were found in inactivation and reactivation processes. The initial velocity of reactivation was proportional to the enzyme concentration over a concentration range of 160-800 micrograms/ml, indicating that the rate-determining step in the reactivation reaction was unimolecular.

Amino Acids↗
PubMed1983

Alpha-2-macroglobulin-like protease inhibitor from the egg white of cuban crocodile (Crocodylus rhombifer).

A high molecular weight protease inhibitor was purified from the egg white of Cuban crocodile (Crocodylus rhombifer). It inhibited the casein hydrolyzing activity of trypsin, subtilisin and papain. Its native molecular weight was 730,000 and it consisted of four subunits of equal molecular weight, each pair of which were disulfide bonded. The amino acid composition, circular dichroic spectrum and electron micrographs of this protein are also presented. Upon incubation with trypsin this protein yielded a fragment of Mr = 80,000, similar in size to the one known to originate from alpha 2-macroglobulin under the same conditions. The molecular parameters of this protein and the broad inhibitory activity towards thiol and serine proteases with different substrate specificities suggest that it is a protein closely related to alpha 2-macroglobulin in mammalian serum. From its native molecular weight and amino acid composition we believe that this protein is also a reptilian counterpart of the avian ovomacroglobulin described by Miller and Feeney (3).

Alligators and Crocodiles↗
PubMed1979

Orientation of the rhodopsin sugar moiety in bovine disk membrane.

Rhodopsin from the bovine rod outer segment contains a covalently linked carbohydrate moiety (Heller, J. & Lawrence, M.A. (1973) Biochemistry 9, 864--868). We studied the location of this carbohydrate moiety on the disk membrane by using ferritin-conjugated concanavalin A and concanavalin A labelled with fluorescein isothiocyanate. Electron microscopic observation of sonicated disk membrane that was labelled with ferritin-concanavalin A revealed the electron-dense image of ferritin on the inner surface of the disk membrane and not on its outer surface. Intact disk membrane that was similarly treated with ferritin-concanavalin A showed a complete absence of ferritin molecules on its surface. In an independent series of experiments we confirmed that the sonicated disk membrane bound three to five times more fluorescein-labelled concanavalin A than the intact disk membrane did. From these experiments we conclude that the carbohydrate moiety of bovine rhodopsin is located on the inner surface of the disk membrane, in agreement with the report by Rohlich on the frog rod outer segment disk membrane (Rohlich, P. (1976) Nature 263, 789--791).

Animals↗
PubMedDate not supplied

Open quaternary structure of the hagfish proteinase inhibitor with similar properties to human alpha-2-macroglobulin.

A homologous protein to human plasma alpha-2-macroglobulin (alpha-2-M) was purified from the blood plasma of hagfish (Eptatretus buergeri) and its structure and function were studied. The hagfish protein inhibited several proteinases and its inhibitory activity was blocked with methylamine as in the case of human alpha-2-M. The molecular weight and sedimentation coefficient of the hagfish inhibitor were 390,000 +/- 20,000 and 11.0 S, respectively, as determined by sedimentation studies. The frictional ratio calculated from these parameters was 1.75. The Stokes radius estimated from HPLC gel chromatography was 8.8-8.9 nm, which was similar to that of human alpha-2-M despite the fact that the hagfish inhibitor was only one-half as large as human alpha-2-M in molecular weight. The hagfish inhibitor was expected to be more asymmetric and/or more hydrated than the human inhibitor. The electron micrographs of the negatively stained hagfish inhibitor showed that it had an open, rectangular quaternary structure of 15 +/- 1.5 X 19 +/- 2 nm in which two semiglobular units were located at the two shorter sides with a gap of 8 +/- 1 nm in width. Each semiglobular unit had an approximate width of 5 +/- 0.5 nm. The thickness of the unit was estimated to be 3 to 3.5 nm from the result of fixed-angle shadowing experiments. Although the two semiglobular units must be connected by some structure, very little material could be seen between them. Such an open quaternary structure may explain the high frictional ratio and large Stokes radius of this protein. The structural change of the inhibitor after reaction with proteinases or methylamine could be detected by electron microscopy and gel chromatography.

Amino Acids↗