Studies on the Proteolytic Enzymes of Black Aspergilli

In order to determine the specificity of Aspergillus Saitoi protease, the hydrolyzate of B-chain of insulin oxidized by this enzyme was investigated on paperchromatography according to the 2,4-dinitrofluorobenzene technique. Specificity was compared with pepsin and other proteolytic enzymes.     

Modification of Proteinase Inhibitor II in Adzuki Beans during Germination

We investigated the effects of compounds isolated from a methanolic extract of rose hips on melanin biosynthesis in B16 mouse melanoma cells and the possible mechanisms responsible for the inhibition of melanin biosynthesis. We found that, among the isolated compounds, quercetin was a particularly potent melanogenesis inhibitor. To reveal the mechanism for this inhibition, the effects on tyrosinase of B16 mouse melanoma were measured. Quercetin decreased the intracellular tyrosinase activity as well as the tyrosinase activity in a cell culture-free system. We also examined the cellular level of tyrosinase protein and found that quercetin dose-dependently inhibited tyrosinase protein expression. We consider from these results that the inhibition of melanogenesis by quercetin was due to the inhibition of both tyrosinase activity and of the protein expression.     

Post-translational Modification of κ-Casein in the Golgi Apparatus of Mid-lactating Mammary Glands from Rat and Cow

Bovine κ-casein, a phosphoglycoprotein, has mucin-type carbohydrate chains. Subcellular distribution of enzymes that take part in the post-translational modification of κ-casein was examined. In lactating mammary glands from rats and cows, N-acetyl-galactosaminyl transferase, galactosyl transferase, sialyl transferase, and casein kinase were localized specifically in the Golgi apparatus.

The substrate specificities indicate that these enzymes are actually responsible for the processing of κ-casein.

The presence of a phosphate group attached to κ-casein did not affect the rate of glycosylation by N-acetyl-galactosaminyl transferase, while the presence of carbohydrate chains attached to κ- casein strongly reduced the rate of phosphorylation by casein kinase. These results suggest that in the Golgi apparatus, phosphorylation of κ-casein precedes glycosylation.     

Simple Synthesis of (R)-5-Hexadecanolide

Chemical modification of tryptophan residues in abrin-a with N-bromosuccinimide (NBS) was studied with regard to saccharide-binding. The number of tryptophan residues available for NBS oxidation increased with lowering pH, and 11 out of the 13 tryptophan residues in abrin-a were eventually modified with NBS at pH 4.0, while 6 tryptophan residues were modified at pH 6.0 in the absence of specific saccharides. Modification of tryptophan residues at pH 6.0 greatly decreased the saccharide-binding ability of abrin-a, and only 2% of the hemagglutinating activity was retained after modification of 3 residues/mol. When the modification was done in the presence of lactose or galactose, 1 out of 3 residues/mol remained unmodified with a retention of a fairly high hemagglutinating activity. However, GalNAc did not show such a protective effect. NBS-oxidation led to a great loss of the fluorescence of abrin-a, and after modification of 3 tryptophan residues/mol, the fluorescence intensity at 345 nm was only 38% of that of the unmodified abrin-a. The binding of lactose to abrin-a altered the environment of the tryptophan residue at the saccharide-binding site of abrin-a, leading to a blue shift of the fluorescence spectrum. The ability to generate such fluorescence spectroscopic changes induced by lactose-binding was retained in the derivative in which 2 tryptophan residues/mol were oxidized in the presence of lactose, but not in the derivative in which 3 tryptophan residues/mol were oxidized in the absence of lactose. Importance of the tryptophan residue(s) in the saccharide-binding of abrin-a is suggested.     

Studies on the Proteolytic Enzymes of Black Aspergilli

The specificity of crystalline Asp. Saitoi proteinase on oxidized lysozyme has been investigated by application of the Sanger DNP-method.

It was found that this proteinase has a much broader specificity as compared with pepsin and Bac. subtilis proteinase.     

Characterization of a Potato Starch-digesting Bacterium and Its Production of Amylase

A bacterium which can utilize potato starch granules as sole carbon source was isolated and identified as Bacillus circulans from its physiological and biochemical properties. Scanning electron microscopic observation of potato starch granules recovered from the culture broth revealed that granules were degraded gradually from their surface resulting in elongated granules with layered structures on their surface. This bacterium produced extracellular amylase which can digest potato starch granules in vitro. The amylase has a unique property in that it produces only maltohexaose from gelatinized starch in the early stage of the reaction. For the production of this amylase potato starch was found to be most effective while soluble sugars including gelatinized starch and maltose had little effect.     

Lipid Compositions of Photomixotrophic Green Calluses and Chlorophyll Deficient Leaves of Tobacco

Among photomixotrophic green calluses tested (N. rustica. N. tobacum L. cv. BY-4 and Samsun), the callus of Samsun had the highest contents of chlorophyll and chloroplast lipids, such as monogalactosyldiglyceride (MGDG), digalactosyldiglyceride (DGDG), sulfoquinovosyldigly-ceride (SQDG) and phosphatidylglycerol (PG). However, the chlorophyll and chloroplast lipids in the green callus of Samsun were still 1/6 and 1/3 of that in the parent leaves, respectively. The relative content of a-linolenate in MGDG, DGDG and SQDG of the green calluses were higher than that of the white calluses. The ratios of hexadecatrienoate in MGDG and hexadeceno-ate (Δ³-trans) in PG in the green calluses were trace or less compared with that of the parent leaves. The crude lipids and total fatty acid contents of the chlorophyll deficient leaves (N. taba-cum L. cv. Consolation 402 and Dominant Aurea Su/su) were almost the same as those of the normal leaves (cv. BY-4 and Samsun), although the chlorophyll contents of the chlorophyll deficient leaves were 1/3 ~ 1/4 of that of the normal leaves. The ratios of chloroplast lipids in the total polar lipids in the chlorophyll deficient leaves were a little lower than that in the normal green leaves, but the former had a slightly higher ratio of phospholipids such as phosphatidylcholine and phosphatidylethanolamine than the latter. There were few differences in the fatty acid compositions of each individual lipid betweeen both types of leaves.     

Effect of 1-Anilino-8-naphthalenesulfonate on Properties and Associations of αs-, β- and κ-Caseins

It was indicated from fluorescence spectra and fluorescence titration that a hydrophobic probe, 1-anilino-8-naphthalenesulfonate (ANS), binds to casein components (α_s-, β- and κ-caseins). Fluorescence intensity and affinity of ANS-κ-casein complex were larger than that of ANS-α_s- and ANS-β-casein complexes. Enhancements of fluorescence intensity of complexes of casein components were observed by the addition of KCI or CaCl₂. Reason for the enhancement was postulated to be the increase of the quantum yield of the ANS fluorescence caused by the environmental change of ANS binding region of the casein components.

Marked increase of sedimentation coefficient of β-casein in the presence of KCl or CaCl₂ at 10°C was caused by the addition of ANS. This may be responsible for the stimulation of the Ca-dependent precipitation of β-casein by the addition of ANS.

It was found that α_s · κ-association was prevented by ANS and that hydrophobic interaction have an important role for α_s · κ-association.     

Changes of Nucleic Acids in the Preparation Process of Cell Protein Isolates from Yeast (Saccharomyces cerevisiae)

Cell protein isolates were prepared from yeast (S. cerevisiae) by alkali-extraction followed by acid precipitation. The relationships between alkali-treatment and nucleic acid contents in cell protein isolates were examined.

The isolate which was precipitated at pH 4.5 following extraction with 0.20 n NaOH at 80°C contained small amounts (less than 1 % of the isolate) of nucleic acids. However, the content of nucleotides in the isolate which was precipitated at pH 4.5 following extraction with 0.20 n NaOH at 37°C was 9.13% of the product. Treatment by washing or dialysis of the isolate had little effect in removing the nucleotides in the isolate.

This finding was explained by the interaction of nucleotide to cell protein isolate. The binding energy was measured by Hummel’s method.     

Role of Tyrosine Residues in Interactions between Casein Components

It was indicated from ultraviolet difference spectra and ultracentrifugal experiments that associations occurred between two casein components (α_s- and κ-caseins, β- and κ-caseins and α_s- and β-caseins) at lower CaCl₂ concentrations (2~3 mm) and that aromatic amino acid residues participated in the associations. Chemical modification studies with 2-hydroxy-5-nitrobenzylbromide indicated that tryptophane residues of each casein component were not essential for these associations. It was also demonstrated by nitration of tyrosine residues with tetranitromethane that tyrosine residues of κ-casein were essential for α_s·κ-association and for β·κ-association and that tyrosine residues of α_s-casein were important to α_s·β-association.

Interactions between casein components were also studied at higher CaCl₂ concentration (10 mm) which is enough for micelle formation. It was found that tyrosine residues of κ- casein played an important role for the stabilization of α_s- and β-caseins. Properties of the nitrated-β-casein were almost the same as that of the native β-casein except the absorption spectrum. α_s·β-Interaction in the presence of 10 mm CaCl₂ was investigated by use of the nitrated-β-casein instead of the native β-casein. It was proved that α_s-casein was stabilized by the nitrated-β-casein and that precipitation of the nitrated-β-casein increased in the presence of α_s-casein.

The mechanism of interactions between casein components at higher CaCl₂ concentration (10 mm) are discussed in connection with the associations at lower CaCl₂ concentrations (2~3 mm).