The mechanism of starch degradation by the yeast Lipomyces starkeyi was studied in strain CBS 1807. It was shown that the amylolytic system is associated with cell walls. It is equally well induced by starch or maltose. The enzyme exhibits considerable a amylase type activity and also liberates a small amount of glucose. Enzyme synthesis occurs during the exponential growth phase. The regulation of the synthesis of the enzyme is discussed.

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The ¹³C-NMR spectra of five phenolic glycosides, dimetbyl crenatin (2), cretanin (4), neocretanin (5), chesnatin (8), and chestanin (9) isolated from galls were compared and the structures 8 and 9 were confirmed as proposed in the previous papers.

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The Oxygen activating mechanism of Fusarium lipoxygenase, a heme-containing dioxyge-nase, was studied. The enzyme did not require any cofactors, such as H₂O₂, however, both superoxide disniutase and catalase inhibited linoleate peroxidation by Fusarium lipoxygenase. A low concentration of H₂O₂ caused a distinct acceleration in enzymatic peroxidation. These results indicate that both O₂^- and H₂O₂ are produced as essential intermediates of oxygen activation during formation of linoleate hydroperoxides by Fusarium lipoxygenase, This peroxidation reaction was also prevented by scavengers of singlet oxygen (¹O₂), but not by scavengers of hydroxyl radical (OH•). Generation of O₂ in the enzyme reaction was detected by its ability to oxidize epinephrine to adrenochrome. Moreover, the rate of peroxide forma-tion was greater in the D₂O than in the H₂O buffer system. These results suggest that the Haber-Weiss reaction (O₂^-+H₂O₂→OOH^-+OH•+¹O₂) is taking part in linoleate peroxida-tion by Fusarium lipoxygenase, and the ¹O₂ evolved could be responsible for the peroxidation of linoleate. H₂O₂ produced endogenously in the enzyme reaction might act as an activating factor for the enzyme. This possible mechanism of oxygen activation can explain the absence of a need for exogenous cofactors with Fusarium lipoxygenase in contrast to an other home-containing dioxygenase, tryptophan pyrrolase, which requires an exogenous activating factor, such as H₂O₂.

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Tunicaminyl uracil, C₁₅H₂₃N₃O₁₀, was obtained by the hydrolysis of tunicamycin with 3 _N HCl at 100° for 3hr. By a combination of chemical and spectrometric methods, its structure was determined to be 1-β-(10'-amino-6', 10'-dideoxy-L-galacto-D-allo-undecodialdo-7', 11'-pyranose-1', 4'-furanosyl)-uracil (I).

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A sarcosine dehydrogenase was purified to homogeneity from cell free extract of Pseudomanas putida aerobically grown in a medium containing creatinine or betaine as the carbon and nitrogen sources. The enzyme catalyzed dehydrogenation of N-methyl derivatives of some amino acids but was inert toward dimethylglycine, betaine and choline. Phenazine methosulfate, 2, 6-dichlorophenol indophenol, methylene blue, meldora blue, nile blue and potassium ferricyanide served as electron carriers. The maximal activity was observed at pH 8.0_??_9.0. The Km and V_max values for sarcosine were 29 m_M and 1.2 μmol/min/mg, respectively. The molecular weight was estimated to be about 170, 000, presumably composed of four sub-units. Spectrophotometric and fluorometric analyses indicated that the enzyme was a flavoprotein.

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Lyophilized photosystem I subchloroplast fragments prepared from spinach chloroplasts were extracted and reconstituted. Hexane extraction eliminated 26% of the photosystem I activity without removing chlorophylls, and the reconstitution with β-carotene, an unkown lipid or chlorophyll a restored almost all the activity. Extraction with hexane-acetone (2:1, v/v) eliminated 81% of the activity with removal of 78% of the chlorophylls. In this case, reconstitution with chlorophyll a showed complete restoration of the activity, but β-carotene, the unknown lipid, and plastoquinone A caused a rather inhibitory effect. A possible explana-tion is that photosystem I reaction centers are closely surrounded by chlorophyll a; and β-carotene, the unknown lipid, and plastoquinone A function only through chlorophyll a.

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Inorganic phosphate-repressible and -constitutive acid phosphatases of baker's yeast were examined to determine whether these two acid phosphatases are the same or different mole-cular species. In DEAE-cellulose chromatography, the repressible enzyme was eluted by 50mM malonate buffer (pH 5.5), whereas the constitutive one was eluted by a much higher buffer concentration. Both enzymes showed non-specific acid phosphatase activities but no phosphodiesterase activity. The repressible enzyme was more sensitive to heat treatment and to chemicals such as Zn²⁺, Hg²⁺, ICH₂CONH₂, PCMB, NaF, urea and Triton X-100 than the constitutive one. Optimum pH of the repressible enzyme was 4.3 whereas that of the consti-tutive one was 3.6, and Km of the repressible enzyme for p-nitrophenylphosphate was 0.74×10^-3 as and that of the constitutive one was 1.1×10^-3M; both of them were competitively inhibited by inorganic phosphate. These results suggest that both enzymes represent different molecular species.

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The digestibility of denatured glycinin with various denaturants such as urea, GuHCl and SDS, and heat treatment, was studied by a pH -stat method. It increases with increasing concentration of the denaturants and heating temperature. However, further addition of the denaturants and 120°C heating caused decrease of digestibility. In urea denaturation, optical measurements indicate that conformation of the protein appreciably refolds after the removal of the denaturant. Gel filtration analyses also indicate the partial refolding of the structure of the protein. Therefore, the refolding of the protein may play an important role in the decrease of the digestibility. However, the structural differences of denatured glycinin from native one and reasons of the decrease of the digestibility can not be revealed yet.

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An enzyme which catalyzes the oxidation of poly(vinyl alcohol) has been purified to a homogeneous state from an enzyme preparation obtained from a culture broth of a strain of Pseudomonas and described previously as a polyvinyl alcohol)-degrading enzyme (Y. Wata-nabe et al., Agric. Biol. Chem., 39, 2447 (1975)). When the purified enzyme reacts on poly (vinyl alcohol), hydroxyl groups seem to be oxidized to keto groups and 1 mol of hydrogen peroxide is produced as 1 mol of molecular oxygen is consumed. Essentially no decrease is observed in the viscosity of poly(vinyl alcohol) solution. The enzyme is active not only to poly(vinyl alcohol) but also to a variety of low molecular weight secondary alcohols and 4-heptanone is formed from 4-heptanol. A name of secondary alcohol oxidase has been pro-posed to the enzyme. The enzyme is a single polypeptide with a molecular weight of 50, 000 with an isoelectric point at pH 10.3. Alanine and valine has been identified as N- and C-terminal residues, respectively. The enzyme is pink, has absorption maxima at 277, 364 and 466nm, and contains 1g atom of non-heme iron per molecule. No flavin has been detected. The enzyme is most active at pH 7 and at 50°C, and is stable between pH 4.5 and 9 and below 50°C. Among the compounds examined, Hg²⁺, Pb²⁺ Zn²⁺, o-phenanthroline and ethylene-diaminetetraacetate have shown weak inhibitions of the activity. The following two succes-sive reactions, the first is catalyzed by the secondary alcohol oxidase and the second by a pro-bable hydrolase have been postulated as a mechanism of bacterial degradation of poly (vinyl alcohol):
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A new cytotoxic substance, fusariocin C, was isolated from the culture filtrate of Fasarium moniliforme A-130. Fusariocin C, C₂₇H₂₈O₈, forms pale yellow prisms, melting at 242_??_244°C and has an optical rotation of [a]²⁰_D-20.6° (c=0.25, CHCl₃). The ultraviolet absorption spectrum was observed at 258 and 365nm.
It was cytotoxic to HeLa cell culture and leukemia L 1210 cells, and showed some anti-tumor activity in vivo for ascitic form of Ehrlich carcinoma. The LD₅₀ by intraperitoneal in-jection in mice was determined to be 3.97mg/kg. This compound was almost devoid of anti-microbial activity.

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β-Chloro-L-alanine was catalytically converted to pyruvate, ammonia and chloride by α-aminoisobutyrate (AIB) decomposing enzyme (α, β elimination), which was synchronously inactivated. There was a linear relationship between α, β elimination and inactivation. With apoenzyme, neither α, β elimination nor inactivation occurred. These facts suggest that α, β elimination is dependent on pyridoxal 5'-phosphate, and inactivation cooperates with α, β elimination (syncatalytic inactivation). But it seemed that D-form of β-chloroalanine was not a substrate for AM decomposing enzyme, because just half amount of β-chloro-DL-alanine was decomposed to pyruvate by the enzyme.
An identical active site for each of following three reactions were shown by the fact that AIB decomposing activity, transamination activity and α, β elimination activity were lost in parallel. From a kinetic study, the affinity of the enzyme toward β-chloro-L-alanine was shown to be higher than that toward AM or L-alanine. The turnover number, about 8, 000, of α, β elimination during the inactivation of one mol of the enzyme was much larger than that of n-amino acid transalninase or alanine racemase.

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Peroxidation of linoleic acid (LA) was promoted by ultraviolet (UV) light of main wave-length 254 nm in a 40% aqueous ethanol solution at pH 7.0 and 25°C. The reduction of nitroblue tetrazolium (NBT), an indicator of superoxide anion (O₂^-), was observed in the photooxidation of LA, and was dependent on the photolysis of linoleic acid hydroperoxides (LAHPO). The reduction of NBT in the photolysis of LAHPO required the presence of oxygen, and was inhibited by the addition of Cu (II)-Gly complex, which is known to have super-oxide dismutase-like activity. The photooxidation of LA was inhibited by the addition of NBT or Cu (II)-Gly complex, and these inhibitory effects depended on the O₂^- quenching activities of these additions rather than on the decrease in the transmittance of UV light absorbed by the additions. These facts suggest that Cu (II)-Gly complex inhibits the photooxi-dation of LA by quenching O₂^- or an O₂^-like factor derived from the photolysis of LAHPO.

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Order of the toxicities of the various cobalt complexes to E. coli B were as follows: Cot²⁺_??_ [Co(C₂O₄)₃]^3->[Co(NH₃)₆]³⁺>[Co(en)₃]³⁺, [Co(edta)]-, [Co(CN)₆]^3-=control. The toxici-ties of the complexes are enhanced under a magnesium-free condition. On the other hand, the uptake of cobalt by cells are ordered as follows: [Co(NH₃)₆]³⁺>[Co(en)₃]₃+>Co²⁺, Co(acac)₃, [Co(dip)₃]³⁺>[Co(edta)]^-, [Co(C₂O₄)₃]^3->[Co(CN)₆]^3-. In this system, the uptake of Co²⁺ is energy-linked, and dependent on the magnesium existent, while that of [Co(NH₃)₆]³⁺ does not show these characteristics. The strong affinity of [Co(NH₃)₆]³⁺ to the membrane was observed.

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Purification of β2-microglobulin from human colostrum was performed by removal of fat by centrifugation and of casein by acid precipitation, followed by gel filtration and ion exchange chromatography of the whey. The β2-microglobulin was isolated in two peaks by Sephadex G-75 column chromatography. The high molecular weight fraction had the molecular weight of 48, 000 which is the same as that of lactollin, a tetrameric form of β2-microglobulin. The other fraction of β2-microglobulin showed three bands on disc gel electrophoresis at pH 8.5 and all of them were able to react with the antiserum of β2-microglobulin. They were not separated each other by gel filtration and ion exchange chromatography and apparently had the same molecular weight of 12, 000. However, they migrated as a single band in disc gel electrophoresis at pH 4.3 and it was concluded that these three bands at pH 8.5 were charge isomers.

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Detailed structural features of the Junsai (Brasenia schreberi J. F. Gmel) polysaccharide was elucidated by characterization of its degradation products from Smith degradation and partial acid hydrolysis.
Four successive Smith degradations yielded a degraded polysaccharide (7.1%), which might represent the core moiety of the polysaccharide. Methylation analysis showed that it contained a backbone chain of β-(1→3)-linked D-mannose residues, many of which were at-tached at the C-2 positions with side chains consisting of β-(1→3)-linked D-galactose and L-fucose residues. In addition, there were (1→3)-linked rhamnose, -fucose, -galactose, and (1→4)-linked D-glucuronic acid residues which appeared to occur as peripheral side chains. Partial acid hydrolysis of Junsai polysaccharide with an acid afforded a new trisaccharide, O-α-L-rhanmopyranosyl-(1→3)-O-α-D-galactopyranosyl-(1→3)-L-fucopyranose, in addition to 3-O-β-D-galactopyranosyl-D-galactose, and 2-O-β-D-glucopyranosyluronic acid)-D-mannose.
These results suggest that Junsai polysaccharide must have an extremely complicated struc-ture, to which its unique physical properties are ascribed.

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The activities of intestinal alkaline phosphatase [E. C. 3.1. 3.1.], sucrase (β-D-fructofrano-side fructohydrolase) [E. C. 3.2. 1.26.] and leucine p-nitroanilidase [E. C. 3.4. 11.2.] were equally enhanced within 4hr after single feeding of egg albumin to 4-day starved rats. They were maintained at the induced levels up to 12hr, followed by a gradual decrease. Single feeding of sucrose did not affect sucrase activity within 8hr, but gradually raised it later than 12hr.
The injection of cycloheximide diminished these dietary induction of enzyme activities to the levels in starvation. In starved controls, only sucrase activity was inhibited by actino-mycin D whose dose inhibited the incorporation of ³H-uridine into the mucosal homogenate. However, in feeding rats, the same dose of actinomycin D decreased all the induced enzyme activities below the levels in starvation without inhibiting the incorporation of ³H-uridine.
The effect of dietary protein on brush border enzymes seemed to be little in germ-free rats as compared with conventional animals.

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Effect of juvenile hormone activity of juvenile hormone I and several juvenile hormone mimics (JHM) were investigated on cell growth and macromolecular synthesis of an established insect cell line derived from mosquito (Culex molestus) ovary. The cell growth in medium with 10^-4 M methoprene (ZR-515) or 10^-4 M farnesol was perfectly inhibited. The cell shape changed into slender or fibloblast like shape. These compounds inhibited also DNA, RNA and protein syntheses. RNA synthesis was most effectively inhibited than others. Metho-prene is a stronger inhibitor of RNA and protein syntheses than puromycin or actinomycin D. The cells incubated in medium with methoprene for one day recovered the cell growth 2 weeks after the methoprene treatment and then the cell shape also recovered. RNA synthesis inhibited by methoprene was also recovered 90min after the methoprene treatment. DNA, RNA and protein syntheses in the cells treated with juvenile hormone were immediately inhibited but the inhibition was reversible.

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The effect of tripropyltin chloride (TPT) on some functional reactions in E. coli was investi-gated. The inhibition on respiration and protein, DNA and RNA syntheses were examined in vivo. Oxygen consumption by E. coli cells was scarcely inhibited even at the concentration of 30μg/ml TPT. The incorporations of ¹⁴C-labeled amino acids into protein fraction were inhibited. Especially, in the case of L-leucine, it was inhibited 60% even at 10μg/ml TPT. Both incorporations of ¹⁴C-adenine into DNA and RNA fraction were inhibited 50-60% at 20μg/ml TPT. RNA polymerase was prepared from E. coli cells and the effect of organotin compounds on the enzyme activity was examined. Organotin compounds inhibited the enzyme activity only at high concentrations (5_??_10m_M), and dialkyltin chlorides which possess no antimicrobial action showed the inhibition more intensely than trialkyltin chlorides. The effect on membrane-bound ATPase was also examined in vitro. We found that TPT has high inhibitory action on membrane-bound ATPase. However, it slightly inhibited the activity of ATPase separated from membrane.

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_k-Casein components having various carbohydrate contents were prepared by diethyl-aminoethyl-cellulose chromatography and the interactions of each _k-casein component both with α_s1, -casein and with β-casein were examined by Sepharose 4B gel chromatography, ultra-centrifugal experiments and viscosity measurements. Each _k-casein component could form complex with α_s1- and β-casein in the absence and presence of CaCl₂. Molecular weight of complexes of unfractionated _k-casein both with α_s1, -casein and with β-casein were about 70×10⁴ at 37°C in the absence of CaCl₂, while those of complexes of each _k-casein component with α_s1- and β-casein were about 50×10⁴. Stokes radii of complexes increased with increasing calcium ion. While sedimentation coefficient at 37°C of complex with β-casein had almost the same value, those of complexes with α_s1-casein decreased with increase of carbohydrate content of _k-casein components. Intrinsic viscosity of complex of _k-casein component having much carbohydrate was almost the same among tested temperatures. It is suggested that heterogeneity of _k-casein is necessary to form large complex and that the carbohydrate moiety of _k-casein contributes the stability of casein complex.

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Purified soybean glycinin (11S globulin) was acetylated at three degrees; low (21_??_51%), middle (60_??_81%) and high (90_??_92%) acetylation in lysine residues. With increasing the acetyl content, the β-structure gradually decreased and the random structure increased resulting in the exposure of tyrosine residue. These were determined from the results of optical rotatory dispersion, intrinsic viscosity, ultraviolet and fluorescence measurement. Gel filtration, ultracentrifugation and gel electrophoresis studies showed drastic conformational changes of highly acetylated 11S (over 90%), in which most of the modified protein (75%) polymerized, and the other dissociated into 3S protein. The close relation between the conformation of acetylated 11S and its emulsifying properties was discussed.

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Additions of methionine sulfoxide, psicofuranine and decoyinine resistances against an inosine-producing strain of Bacillus subtilis, 1411, were carried out successively. Finally, strain MG-1 that produced guanosine exclusively in a high yield was obtained. The correla-tions among these resistances, guanosine productivity, and regulatory mechanism of enzymes such as inosine 5'-monophosphate (IMP) dehydrogenase, guanosine 5'-monophosphate (GMP) synthetase, 5'-nucleotidase, phosphoribosylpyrophosphate (PRPP) amidotransferase and succino-adenosine 5'-monophosphate (succino-AMP) lyase were investigated.
Methionine sulfoxide resistance mainly caused decrease of specific activity of 5'-nucleoti-dase and partial losses of inhibition and repression of IMP dehydrogenase by GMP. Further-more, repression and inhibition of PRPP amidotransferase by GMP, and repression of succino-AMP lyase by GMP were also lost.
Psicofuranine resistance mainly caused variation of GMP synthetase, and partial loss of inhibition of succino-AMP lyase by AMP was observed.
Decoyinine resistance brought about complete losses of repression and inhibition of GMP synthetase by GMP, and loss of inhibition and partial loss of repression of succino-AMP lyase by AMP were also found.
From these results, it is concluded that the keys to increase guanosine production depend on losses of repression and feedback inhibition of IMP dehydrogenase, GMP synthetase, suc-cino-AMP lyase and PRPP amidotransferase, and further on degree of 5'-nucleotidase activity.

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Culture conditions for guanosine production were studied with Bacillus subtilis MG-1 that exclusively accumulated guanosine. Of components investigated, KH₂PO₄, KCl, Fe⁺⁺, Mn⁺⁺, NH₄NO₃ and sodium glutamate have played important roles for guanosine production. The optimal concentrations in the culture medium were 2.0g, 0.9g, 7.5mg, 7.5mg, 20.4g and 6.0g per liter, respectively.
In particular, the extremely minor concentration of Mn⁺⁺ 0.01ppm completely repressed guanosine production although the cells grew sufficiently. Amino acids mixture was neces-sary for cell growth, but not essential for guanosine production.
Under these conditions, MG-1 accumulated 15g of guanosine per liter in a weight yield of 18.8% of consumed sugar. However, a large amount of acetoin was also found as a bypro-duct.

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To clarify the biosynthetic pathway of maridomycin, the biosynthetic relation among 16-membered macrolide antibiotics belonging to leucomycin-maridomycin group was examined with intact cells or growing cultures of Streptomyces hygroscopicus No. B-5050-HA and its mutants.
Carbomycin A, B and leucomycin As were converted to maridomycin II in the pH range of 5.5_??_7.5 with an optimum between 6.0_??_6.5. But, maridomycin II was not converted to leucomycin A₃ or carbomycin B. These findings were confirmed by culture experiment. Leucomycin A₃, carbomycin A and B added to the culture at 48hr were almost completely converted to maridomycin II.
On the basis of these facts, we propose a biosynthetic relation among leucomycin A₃, carbomycin A, carbomycin B and maridomycin II.

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