Proteolysis of abnormal prion protein with a thermostable protease from Thermococcus kodakarensis KOD1

The abnormal prion protein (scrapie-associated prion protein, PrP^Sc) is considered to be included in the group of infectious agents of transmissible spongiform encephalopathies. Since PrP^Sc is highly resistant to normal sterilization procedures, the decontamination of PrP^Sc is a significant public health issue. In the present study, a hyperthermostable protease, Tk-subtilisin, was used to degrade PrP^Sc. Although PrP^Sc is known to be resistant toward proteolytic enzymes, Tk-subtilisin was able to degrade PrP^Sc under extreme conditions. The level of PrP^Sc in brain homogenates was found to decrease significantly in vitro following Tk-subtilisin treatment at 100 °C, whereas some protease-resistant fractions remain after proteinase K treatment. Rather small amounts of Tk-subtilisin (0.3 U) were required to degrade PrP^Sc at 100 °C and pH 8.0. In addition, Tk-subtilisin was observed to degrade PrP^Sc in the presence of sodium dodecyl sulfate or other industrial surfactants. Although several proteases degrading PrP^Sc have been reported, practical decontamination procedures using enzymes are not available. This report aims to provide basic information for the practical use of a proteolytic enzyme for PrP^Sc degradation.     

Seasonal changes in activity of the enzyme system producing cis-3-hexenal and n-hexanal from linolenic and linoleic acids in tea leaves

The enzyme system producing cis-3-hexenal, a precursor of cis-3-hexenol(leaf alcohol) and trans-2-hexenal (leaf aldehyde), from linolenicacid showed high activity in summer and no activity in winterin tea (Thea sinensis) leaves and isolated chloroplasts. Theenzyme system producing n-hexanal from linoleic acid also showedsimilar seasonal changes in activity. These changes were closelyrelated to temperature and solar radiation. Enzyme activitycould not be induced after the leaves had been cut and was notaccompanied by de novo protein synthesis. (Received July 9, 1976; )     

Blätteralkohol (VIII)

Es wurde aufgeklärt, daβ die im Tee-blättern weit verbreitet vorkommende natürliche Blätter-alcohol-fraction aus einern Gemisch der cis, trans-Isomere besteht, wobei bisher das cis-Isomere besteht, wobei bisher das cis-Isomere stark überwiegt.     

Blätteralkohol (XVI)Blätteralkohol (XVII)

2-Methyl-4, 6-cyclohexadienaldehyde and n-butyraldehyde were treated with sodium in p-xylene to yield the aromatized “leaf alcohol reaction” product, 2-methyl-benzylalcohol, in a better yield than that with the cyclohexadienaldehyde alone. n-Butyric acid isolated from the reaction mixture unequivocally showed the operation of the “crossed Cannizzaro disproportionation” in this reaction, aliphatic aldehyde serving as the hydride donor. 2-Propyl-5-ethyl-4, 6-cyclohexadienaldehyde was obtained by the NaOH/H₂O-EtOH Michael-Aldol condensation of leaf aldehyde, gave 2-propyl-5-ethyl-benzylalcohol along with caproic acid.

On the basis of “leaf alcohol-reaction” mechanism, it was obtained following benzyl-alkohols; 2-methyl-, 2-propyl-, 2-methyl-5-ethyl-, 2-propyl-5-ethyl-benzylalcohol, from leaf alcohol and crotylalcohol.     

A Tracer Study on the Leaf Alcohol Reaction

The pathway of the leaf alcohol reaction, in which n-hexen-l-ols were converted to 2-propyl-5-ethylbenzylalcohol by refluxing with sodium at 160°C has been confirmed by the tracer technique. First, 2-trans-hexen-l-ol-l-¹⁴C and -5-¹⁴C were synthesized, then the labeled alcohols were subjected to the leaf alcohol reaction. The 2-propyl-5-ethylbenzyl alcohol-¹⁴C obtained was led to suitable degradation compounds.

By the radioassay of the starting, condensed and degradative compounds, the ratios of radioactivity among these compounds were determined. Results demonstrated that the C-l and C-3 of one molecule of 2-hexen-l-ol respectively combined with the C-4 and C-2 of another molecule of the compound to be converted to 2-propyl-5-ethylbenzyl alcohol.     

NAD-Dependent and NADP-Dependent Alcohol Dehydrogenases in Escherichia coli

The isomeric ratio and level of natural xanthoxin (XAN) in tomato plants (Lycopersicon esculentum) were examined by a more reliable analytical method than has been reported before. Efforts were made to avoid artificial isomerization between c-XAN and t-XAN throughout the isolation, derivatization and GC-MS procedures. Natural XAN was separated from contaminating chlorophylls before rev. HPLC purification, derivatized to abscisic acid methyl ester (MeABA) in four chemical steps, and quantified with the deuterium-labeled internal standards on clear and reproducible full GC-EI-MS. It was revealed that the isomeric composition of natural XAN was exclusively shifted to c-XAN. The level of c-XAN was higher and more significantly induced by water stress in older plants. The significant role of c-XAN as an ABA biosynthetic precursor is suggested.     

Purification and properties of a lectin from ascomycete mushroom,Ciborinia camelliae

A lectin was isolated from an ascomycete mushroom, Ciborinia camelliae which was specific to N-acetyl-D-galactosamine. On SDS-polyacrylamide gel electrophoresis; this lectin gave a single band of approximately 17-kDa in the presence of 2-mercaptoethanol, but formed dimers, trimers and tetramers in its absence. Amino acid analysis revealed the lectin contained two cysteines and no methionine. The N-terminal sequence was determined up to residue 21, and no homologous proteins including other ascomycete lectins were found.     

Blätteralkohol (IX)

Blätteraldehyd wurde durch die einfachste, ergiebigste Synthese dargestel1t. Demnach wurde es aufgeklärt, daß die Konfiguration des natürlichen Blätteraldehyds trans-Form ist.     

Biosynthesis of Leaf Alcohol: Kinetic Study of Lipoxygenase Activation Process Caused by Hydroperoxylinoleic Acid

Quaternary structure of ribulose-1, 5-bisphosphate (RuP₂) carboxylase from the autotrophically grown cells of blue-green alga, Anabaena cylindrica, was studied. Sedimentation coefficient (s_{20, w}) of the enzyme was determined to be 18.3 S by the sucrose density gradient centrifugation. The molecular weight was estimated to be 5.0 × 10⁵ by the Sepharose 4B gel filtration technique. The purification of the enzyme from the algal cells was undertaken by means of sucrose density gradient centrifugation and DEAE-Sephadex A–50 ion-exchange column chromatography, and the structural make-up of the enzyme containing two subunits, A (M. W., 5.2 × 10⁴) and B (M. W., 1.2 × 10⁴) was established by the Na-dodecylsulfate polyacrylamide gel electrophoresis experiment. Structural similarity of the algal RuP₂carboxylase with the spinach enzyme was further demonstrated by the Ouchterlony double immunodiffusion experiment.