Purification and Properties of Maltose Phosphorylase from Lactobacillus brevis

Maltose phosphorylase (EC 2.4.1.8) from Lactobacillus brevis was purified 29-fold over the crude extract. The final preparation was at least 80% pure and had a specific activity of 18 units/mg protein. The molecular weights of the native enzyme and of the component dissociated in sodium dodecyl sulfate were 150,000 and 80,000, respectively. The enzyme does not contain pyridoxal-5′-phosphate as a cofactor. It can not act on maltitol, malto-triitol, sucrose, lactose and trehalose, and essentially not on isomaltose, maltobionic acid, maltotriose and maltotetraose. Inhibitory effect was observed with CuSO₄, HgCl₂ and p-chloromercuribenzoate. Some other properties were also examined. A possibility of using this enzyme for the analysis of maltose was proposed.     

Metabolism of Glutamate in Purple Nonsulfur Bacteria Participation of Vitamin B12

Purple nonsulfur bacteria, Rhodospirillum rubrum and Rhodopseudomonas spheroides were found to possess coenzyme B₁₂-dependent glutamate mutase activity. Cell-free extracts of these bacteria grown on Co²⁺-containing media catalyzed the conversion of glutamate to β-methylaspartate and further to mesaconate. The activity of the cell-free extracts of these organisms cultivated on Co²⁺-deficient media was markedly lower than that of the normal cells. Addition of coenzyme B₁₂ to the former reaction mixture enhanced the mesaconate formation via β-methylaspartate. These results indicate the involvement of coenzyme Independent glutamate mutase of these bacteria in the dissimilation of glutamate to acetyl-CoA and pyruvate through the following pathway.

glutamate→β→methylaspartate→mesaconate→citramalate→→acetyl-CoA, pyruvate On the other hand, a greater part of glutamate was converted to α-hydroxyglutarate and succinate with the cell-free extracts of these photosynthetic bacteria. This fact, taking account of the presence of propionyl-CoA carboxylase in these bacteria, implies the participation of coenzyme B₁₂-dependent (R)-methylmalonyl-CoA mutase in the formation of succinate via the following route.

glutamate→α-ketoglutarate→α-hydroxyglutarate→propionate→propionyl-CoA→(S)-methylmalonyl-CoA→(R)-methylmalonyl-CoA→succinyl-CoA     

The Biosynthetic Pathway of (Z)-11-Hexadecenal,the Sex Pheromone Component of the Rice Stem Borer,Chilo sappressalis WALKER (Lepidoptera: Pyralidae)

Lipoxygenase-3, the major component of the enzyme in rice grain, was purified 2980-fold with a yield of 7% from embryos. The purified enzyme had a specific activity of 280 μmol O₂ formed/min per mg protein. This enzyme was inactivated by SH compounds, such as cysteine and glutathione. The inactivation was prevented by the addition of catalase or replacement of the air by N₂ gas. These two treatments were also effective for the stable storage of the purified enzyme. The molecular weights measured by sodium dodecyl sulfate gel and gradient gel electrophoresis were 93,000 and 89,000, respectively, indicating that the enzyme is a single polypeptide chain. The purified enzyme contained 0.73 Fe atom per molecule. The absorption spectrum suggested that the enzyme is a non-heme iron protein. Some similarities in amino-acid composition were observed between rice, soybean, and pea lipoxygenases. The purified enzyme specifically produced 9-d-hydroperoxy-10,12(E,Z)-octadecadienoic acid when linoleic acid was used as a substrate.     

Bitter Taste of Synthetic C-Terminal Tetradecapeptide of Bovine β-Casein,H-Pro196-Val-Leu-Gly-Pro-Val-Arg-Gly-Pro-Phe-Pro-Ile-Ile-Val209-OH,and Its Related Peptides

The primary structure of bovine β-casein contains the partial sequence of -Pro¹⁹⁶-Val-Leu-Gly-Pro-Val-Arg-Gly-Pro-Phe-Pro-Ile-Ile-Val²⁰⁹ in the C-terminal portion. We previously reported that the synthetic C-terminal octapeptide, Arg²⁰²-Val²⁰⁹, is extremely bitter with its threshold value 0.004 mm, 250 times as strong as that of caffeine. To further investigate the bitter taste of the C-terminal portion of β-casein, we synthesized the C-terminal tetradecapeptide, Pro¹⁹⁶-Val²⁰⁹, and some of its fragments. A hydrophobic hexapeptide, Pro¹⁹⁶-Val²⁰¹, was twice as bitter as caffeine. The bitter taste of the decapeptide, Pro²⁰⁰-Val²⁰⁹, was the same as that of Arg²⁰²-Val²⁰⁹. Although the tetradecapeptide, Pro¹⁹⁶-Val²⁰⁹, was composed of two bitter peptides, Pro¹⁹⁶-Val²⁰¹ and Arg²⁰²-Val²⁰⁹, its bitter taste was weaker than that of Arg²⁰²-Val²⁰⁹ and its threshold value was 0.015 mm. We suggested that the increase of bitterness in peptides through the introduction of hydrophobic amino acids depended on the number of hydrophobic amino acids added. In addition, the synthetic retro analog of Arg²⁰²-Val²⁰⁹ (H-Val-Ile-Ile-Pro-Phe-Pro-Gly-Arg-OH) was not as bitter as Arg²⁰²-Val²⁰⁹. This indicated that the sequence of Arg²⁰²-Val²⁰⁹ is important for extreme bitterness.     

A New Isoflavone with Antifungal Activity from Immature Fruits of Lupinus luteus

A new isoflavone having antifungal activity was isolated from immature fruits of Lupinus luteus (Leguminosae), and named luteone. The structure was shown to be 5,7,2′,4′-tetrahy-droxy-6-(3,3-dimethylallyl)-isoflavone by degradative and spectroscopic studies.     

Bitter Taste of H-Pro-Phe-Pro-Gly-Pro-Ile-Pro-OH Corresponding to the Partial Sequence (Positions 61 ~ 67) of Bovine β-Casein,and Related Peptides

The bitter components of cheese are hydrophobic peptides which are produced during the process of enzymatic digestion, and some of the isolated bitter peptides are derived from the middle portion of β-casein. However, quantitative examination of the bitter taste is seldom performed. We synthesized two hydrophobic peptides, H-Pro⁶¹-Phe-Pro-Gly-Pro-Ile-Pro⁶⁷-OH and H-Tyr⁶⁰-Pro-Phe-Pro-Gly-Pro-Ile⁶⁶-OH, which correspond to common portions among the isolated bitter peptides, in order to determine how bitter they were. From the results of sensory analysis, it was found that the synthetic peptides exhibited a bitter taste with threshold values 0.25 and 0.16mm, respectively. We also synthesized their fragments and analogs, and discussed the structure-bitterness relationship.     

Subunit Structure of Glucoamylase of Saccharomyces diastaticus

Extracellular glucoamylase produced by a starch-fermenting yeast, Saccharomyces diastaticus 5106-9A, was purified. The enzyme was found to be heterogeneous in molecular weight, ranging from approximately 80K to 66K as estimated by gel filtration, and consisted of two subunits, H and Y. The molecular weight of subunit H was heterogeneous and was determined to be approximately 68K, 59K, and 53K by acrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The molecular weight of subunit Y was 14K, estimated by the same gel. the molecular weight of the deglycosylated form of subunit H was 41K, suggesting that the heterogeneity of the enzyme was due to glycosyl moieties of subunit H. Subunits H and Y were separated by gel filtration in the presence of sodium dodecyl sulfate. Subunit Y seemed to be hydrophobic, since it was insoluble in an aqueous buffer without detergent.     

Structures of Gibberellins A39, A48, A49 and a New Kaurenolide in Cucurbita pepo L.

The structures of three new gibberellins A₃₀, A₄₈ and A₄₉ and a new kaurenolide, isolated from seeds of Cucurbita pepo L., were elucidated. The structures of GA₃₉, GA₄₈ and GA₄₉ were shown to be ent-3α,12β-dihydroxygibberell-16-ene-7,19,20-trioic acid (1), ent-2α,3α,10,12α-tetrahydroxy-20-norgibberell-16-ene-7,19-dioic acid 19,10-lactone (5) and the epimer at C–12 of GA₄₈ (8), respectively. The kaurenolide was shown to have the structure: ent-6β,7α,12β-trihydroxykaur-16-en-19-oic acid 19,6-lactone (14).     

Induction of Catalase Activity by Hydrocarbons in Candida tropicalis рK 233

1. The catalase activity of Candida tropicalis pK 233 was induced by hydrocarbons but not by glucose, galactose, ethanol, acetate or lauryl alcohol.

2. The induction of the catalase activity depending upon hydrocarbons was sensitive to cycloheximide but not to chloramphenicol.

3. Glucose repressed strongly the induction of the catalase activity by hydrocarbons but galactose did not affect seriously.

4. When C. tropicalis was incubated with hydrocarbons, the appearance of microbodies was observed electronmicroscopicaliy.