Structure and function of muscle myosin

We have studied the primary structures of myosins from chicken muscles in order to clarify the relationship between structure and function of muscle myosin. The primary structures of the various kinds of light chains from chicken muscle myosins have been determined. We also report the primary structure of the 23K fragment of subfragment-1 (S-1) component from the heavy chain of chicken fast skeletal muscle myosin. In addition, antibody was prepared against the 23K fragment. The antibody was found to inhibit the Mg²⁺-ATPase activity and the initial P_i burst of the ATPase in the S-1 component. The antibody suppressed the ATP-induced fluorescence enhancement of S-1, though it did not suppress the binding of ATP to S-1. These results are also discussed.This article was presented during the proceedings of the International Conference on Macromolecular Structure and Function, held at the National Defence Medical College, Tokorozawa, Japan, December 1985.     

Recent gene conversion between genes encoding human red and green visual pigments

Nucleotide sequences of the human X-linked red and green pigment genes were compared, and the number of silent substitutions per site (KSc) between these genes was analysed in comparison with the corresponding values of primate genes. Taking the retarded mutation rate of X-linked genes into consideration (Miyata et al., 1987), the red and green pigment genes were shown to have undergone gene conversion at around the time of separation of African apes and orangutan. Thus the recent gene conversion and retarded mutation rate in these X-linked genes are probably responsible for the strong sequence similarity between these genes, which is likely to facilitate the occurrence of red-green color blindness in the human population. It was also shown that the red pigment gene evolved about five times more rapidly than the green pigment gene since the latest gene conversion.     

Cell Structure of Yeasts Grown Anaerobically in Aspergillus orpzae-Proteolipid-supplemented Media

Effects of Aspergillus oryzae-proteolipid (PL) on the formation of high concentrations of alcohol, more than 20% as in sake brewing, have been studied. Electron microscopy revealed that there were no vacuoles but many lipid deposits in cytoplasm of the alcoholtolerant cells of Saccharomyces sake Kyokai No. 7, which were obtained by the anaerobic culture supplemented with PL. Sphaeroplasts from the alcohol-tolerant cells were stable in 20% alcohol, whereas those from the untolerant cells grown anaerobically in the PL-unsupplemented media were ruptured. The cell membrane became alcohol-endurable in the anaerobic cultures with PL. Synthetic phospholipid promoted yeast growth and the fermentative activity, whereas a small amount of sterol ester enhanced the alcohoi-endurability. The supplementation of both lipids anaerobically induced physiological properties characteristic of the alcohol-tolerant cells.     

Antitumor Activity of an Alkali Extract of Clostridium saccharoperbutylacetonicum Cells

Bacillus thuringiensis var. israelensis produces 130-kDa proteins which are toxic to mosquito larvae. The ISRH4 gene encoding 1,180 amino acids of the 130-kDa insecticidal protein was fused with lac Z′ on a plasmid, pUC19, and sequentially deleted from the C-terminus to construct a series of deletion mutants. All the deletion mutant genes directed the production of truncated ISRH4 proteins fused with the α-complementing fragment of β-galactosidase in Escherichia coli cells in the presence of isopropyl β-d-thiogalactopyranoside. Analysis of the mosquito larvicidal activity of deletion mutant proteins revealed that the N-terminal 29 amino acids and the C-terminal 485 amino acids could be removed without loss of the activity.     

High Concentration—Ethanol Fermentation of Raw Ground Corn

1-Pyrroline-5-carboxylate dehydrogenase was purified and crystallized from Bacillus sphaericus. The crystalline preparation gave a single band on polyacrylamide slab gel electrophoresis. The molecular weight of the enzyme was determined to be about 100,000 by gel filtration. The enzyme consists of two subunits which are identical in molecular weight (50,000), as judged on SDS slab gel electrophoresis. The enzyme shows an optimum pH of 6.5 to 7.0. Its activity was 8.1 times higher with NADP⁺ than with NAD ⁺, and the enzyme was stabilized by NADP⁺. The apparent Km values for l-l-pyrroline-5-carboxylate, NADP⁺ and NAD⁺ are 4.2 × 10^–5m (with NADP⁺), 9.5 × 10~⁶m and 2.5 × IO^-3 m, respectively. The enzyme reaction is irreversible. A simple method for the determination of l-ornithine involving ornithine ¿-aminotransferase and 1- pyrroline-5-carboxylate dehydrogenase from B. sphaericus was developed. A linear relationship was found between the absorbance at 340 nm and the amount of l-ornithine (50 ~ 400 nmol), and between the fluorescence and the amount of l-ornithine (0.2 ~ 10 nmol).     

Effects of Phosphatidylcholine or Ergosteryl Oleate on Physiological Properties of Saccharomyces sake

Chromatographically homogeneous phosphatidylcholine identified by thin-layer chromatography on silica gel G was isolated from mycelia of Aspergillus oryzae or egg yolk by a combination of alumina and silicic acid column chromatography. Although the 2-position in both Asp. oryzae- and egg yolk-phosphatidylcholine was occupied nearly exclusively by unsaturated fatty acids, significant proportions of unsaturated fatty acids were also found in the 1 -position of Asp. oryzae-phosphatidylcholine. In nitrogen gas-sparging anaerobic culture of Saccharomvces sake Kyokai No. 7, supplementing the basal synthetic medium with phosphatidylcholine enhanced the yeast growth and fermentative activity, whereas adding ergosteryl oleate enhanced alcohol-endurability. Supplementation with both phosphatidylcholine and ergosteryl oleate promoted the yeast growth, fermentative activity and alcohol-endurability of cells.     

Chicken IL-6 is a heat-shock gene

The febrile response is elicited by pyrogenic cytokines including IL-6 in response to microorganism infections and diseases in vertebrates. Mammalian HSF1, which senses elevations in temperature, negatively regulates the response by suppressing pyrogenic cytokine expression. We here showed that HSF3, an avian ortholog of mammalian HSF1, directly binds to and activates IL-6 during heat shock in chicken cells. Other components of the febrile response mechanism, such as IL-1β and ATF3, were also differently regulated in mammalian and chicken cells. These results suggest that the febrile response is exacerbated by a feed-forward circuit composed of the HSF3-IL-6 pathway in birds.