Levan fructotransferase of Arthrobacter ureafaciens was shown to degrade Bacillus mesentericus levan up to about 40 %, leaving the levan fragment at the limit of the enzymatic degradation, while it degraded Phleum pratense phlein more than 90 %, with the respective formation of 5 and 6% reducing sugars as D-fructose. Each enzymatic digest of levan and phlein at the limit of degradation gave, in addition to the main spot of di-D-fructose 2, 6': 2', 6 dianhydride (difructose anhydride IV), two or three minor fructooligosaccharide spots on a thin-layer chromatogram. These minor oligosaccharides semiquantitatively fractionated at the limit of degradation were found to be essentially different between levan and phlein, though small amounts of fructose and levanbiose were produced in both the digests.

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We found a novel protopectinase that did not catalyze the degradation of polygalacturonic acid in the culture filtrate of Bacillus subtilis IFO 12113. The enzyme was purified by CM-Sephadex C-50 column chromatography, heat treatment at 60°C, and column chromatography on DEAE-Toyopearl 650M and Toyopearl HW-55S. The enzyme was isolated as a homogeneous protein, and was finally isolated as crystals. The molecular weight of the enzyme was estimated to be 30, 000 by gel permeation chromatography and by SDS-polyacrylamide gel electrophoresis, and 29, 800 by sedimentation analysis. Its isoelectric point was around pH 9.4. The enzyme was thermostable; its activity was not lessened by being heated at 60°C for 10 min. The enzyme catalyzed the degradation of arabinogalactan from soybean seeds. Some strains belonging to the genus Bacillus, such as B. amyloliquefaciens, B. cereus, B. circulans, B. coagulans, B. firmus, B. licheniformis, B. macerans, and _{B. pumilus}, produced the same kind of protopectinase.

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Cells of Enterobacter aerogenes IFO 12010 could grow on a medium containing alkylphosphonic acids as sole sources of phosphorus. The formation of the enzyme responsible for the liberation of inorganic phosphate (Pi) from alkylphosphonic acids was induced when Pi-grown cells were transferred to a medium containing no added Pi. The extracts prepared from cells after incubation in the absence of Pi showed high carbon-phosphorus (C-P) bond cleavage activity and catalyzed the liberation of Pi from alkylphosphonic acids most efficiently in the alkaline pH region. We named the enzyme C-P hydrolase, tentatively.

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The allergenicity of proteins interacted with oxidized lipids was examined by enzyme-linked immunosorbent assay (ELISA) using sera from soybean-sensitive individuals. Though oxidized soybean oil did not show any allergenicity, the IgE titer of sera from soybean-sensitive patients was greatly increased when oxidized soybean oil was incubated with soybean 2S-globulin. The IgE titer of patient sera became higher when greater amounts of oxidized soybean oil were used. Little difference was noted in the ELISA value of protein interacted with oxidized soybean oil when soybean 2Sglobulin was replaced by other food proteins. A similar tendency was noted when soybean oil was replaced by other vegetable oils or fatty acids. These results clearly show that proteins interacted with oxidized lipid are allergenic to soybean-sensitive patients.

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To examine the spontaneous gel-forming property of the polysaccharide of seeds of Ficus awkeotsang Makino, seeds were extracted sequentially with cold and hot water, and then aqueous ammonium oxalate. The major acidic polysaccharide ([α]²⁵_D+239°; DE = 63.6%; M.W. 3.4 × 10⁵), which was responsible for gel-formation, was obtained from the surface of heat-treated seeds and found to be composed mainly of galacturonic acid (96%). On fractionation of the major acidic polysaccharide by anion-exchange column chromatography, three kinds of methyl esterified components were obtained (P-1, DE=69.3%; P-2, 49.2% and P-3, 22.8%). Methylation analysis of the carboxylreduced polysaccharide indicated that the acidic polysaccharide is an essentially linear polysaccharide containing α-(1→4)-linked D-galacturonic acid residues and devoid of L-rhamnose residues, which confirmed it to be a polygalacturonide.
Comparison of the IR spectra and the degrees of methyl esterification of the polygalacturonide extracted from heat-treated and heat-untreated seeds suggested that the methyl ester groups in the polygalacturonide must have been released during the seed extraction at room temperature. As a model test, furthermore, when the acid polysaccharide prepared from heat-treated seeds was incubated with pectinesterase from lemon peel in the presence of 1mM CaCl₂-2H₂O, the gel-forming behavior observed was closely similar to the spontaneous gel-formation of the awkeotsang extract. These results may support the possibility that the high amount of calcium ions (4.0-5.6 %) in an aqueous extract of the seeds contributes to the "egg-box" formation, by being placed between the de-esterified structurally regular polygalacturonide chains.

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A pectinesterase (EC 3.1.1.11), responsible for the spontaneous gel-formation of the awkeotsang polygalacturonide, was isolated from the red tepals attached to the pedicels of seeds of Ficus awkeotsang Makino. The methyl esterase, partially purified by ion-exchange column chromatography on Q-Sepharose, showed an optimum pH of 5.4 for its enzyme activity in the absence of potassium or calcium ions, but the pH in the presence of these ions being sifted to 7.0-8.0.
As judged on comparison of the spontaneous gel-formation of the water extract of seeds and the enzyme-induced gel-formation of the polygalacturonide solution by treatment with the awkeotsang pectinesterase, the spontaneous gel-formation of the awkeotsang seed extract must be induced through the action of an intrinsic pectinesterase on the methoxylated linear polygalacturonide and then the formation of the so-called "egg-box" structure, calcium ions being placed between the polygalacturonide chains of the de-esterified α-(1→4)-linked D-galacturonic acid residues.

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The crude chitin-glucan complex isolated from yeast bud scars by the method of Bacon et al. was alternately treated with dilute alkali and acid to separate chitin and glucans. The respective content of chitin and glucan in the residual chitin preparation from the exhaustive chemical treatments was 83 % and 17 % by IR spectroscopy. The portion of glucans in the bud scars was constructed with (1-3)-β-D-/(1-6)-β-D-glucans. A possible construction model for the yeast scar ring is discussed.

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A 6PFK-lacking mutant of B. flavum grew on fructose but its FBPase-lacking mutant did not. The product of the PTS reaction from fructose as a substrate was not F6P but F1P. These indicate that fructose is metabolized through F1P. 1PFK and 6PFK which form F1, 6P₂, an intermediate common to both glucose and fructose metabolism, were highly specific for their respective substrates, F1P and F6P. Both the reactions followed Michaelis-Menten kinetics. Kms of 1PFK for F1P and ATP were 0.6 and 0.18 mM, and those of 6PFK for F6P and ATP were 2.4 and 0.15 mM, respectively. Reaction mechanisms of 1PFK and 6PFK were of Ordered Bi Bi and of Ping Pong, respectively. 1PFK was inhibited by F6P and the reaction products, F1, 6P₂ and ADP, with Kis of 0.9, 2.1, and 1.5 mM, respectively. 6PFK was inhibited only by ADP with a Ki of 0.45 mM, but was neither inhibited by ATP, citrate, and PEP nor activated by AMP and F2, 6P₂. The inhibition of 1PFK by F6P and F1, 6P₂ was competitive to F1P and of mixed type to ATP and that by ADP was of mixed type to both the substrates. The inhibition of 6PFK by ADP was uncompetitive to F6P and competitive to ATP, suggesting that ADP does not act as the reaction product but as an analog of the substrate, ATP.

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A variety of 6-O-(3-acyloxytetradecanoyl)-MDP methyl esters were synthesized using opticallyactive (3R)-3-acyloxytetradecanoic acid. Their immunoadjuvant activity was examined.

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Bacillus sp. KSM-635, which was isolated from soil, constitutively produced an alkaline carboxymethyl cellulase (CMCase) in quantity. Maximum cell growth was observed at an initial pH between 9 and 10, and no growth occurred below pH 8, thus illustrating the alkalophilic nature of this organism. Higher production of the CMCase required the presence of sugars (e.g., fructose, sucrose, maltose and mannitol) and complex nitrogen, with an initial pH of 8-9.
The CMCase, partially purified by precipitation with ethanol, showed an optimum pH for activity of 9.5 and an optimum temperature of 40°C, in glycine-NaOH buffer. It showed strong activity toward CMC, but very little activity toward p-nitrophenyl-β-D-glucoside or cellulosic substrates showing high crystallinity. Activity toward p-nitrophenyl-β-D-cellobioside was detected at pH 7, but it amounted to less than 2% of the maximum CMCase activity. Characteristically, the alkaline CMCase activity was not affected by various laundry components, such as surfactants, chelating agents and alkaline proteinases.

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A host-specific toxin-related compound, HV-toxin M, was isolated from a culture medium of the phytopathogenic fungus, Helminthosporium victoriae, a causal agent of Victoria blight disease of oat. The structures of six amino acid components of HV-toxin M have already been determined in Part I in this series. In this report, Part II, the sequence of these six amino acid components is clarified by analyzing the results of FAB-MS/MS (fast atom bombardment-mass spectrometry/mass spectrometry: tandem mass spectrometry), a recent MS technique, on MS fragments of Toxin M or its derivatives (Fig. 1).

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An acidic extract of egg white stimulated the DNA synthesis and proliferation of BALB/c 3T3 cells. The maximal degree of DNA synthesis in 3T3 cells in the presence of the egg white extract was half that in the presence of 10% calf serum. However, such activity was not detected in an egg yolk extract. The activity of the egg white extract was divided between two fractions of about 7 kD and 2 kD, both of them were stable in 6 M urea, from pH 2 to 12 and at high temperature. The former was inactivated by treating with 2-mercaptoethanol. The activity of both fractions is attributed to polypeptide compounds, because the activity of each was lost by pronase digestion.

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The structure of α_s1-casein at an emulsified oil/water interface was studied by immunochemical method using BALB/c mouse anti-α_s1-casein antibody. By incubating the antiserum with an α_s1-casein-stabilized emulsion, all of the specific antibodies in the serum were absorbed by the emulsion, suggesting that α_s1-casein was adsorbed to the oil surface, all of its antigenic determinants being exposed to the aqueous phase. Since the antigenic determinants of α_s1-casein for BALB/c mice are known to be present in the regions of residues 1 - 8, 33 - 54, 105 -119, 133 - 151, and 174 -199, these portions must be, at least partly, exposed to the aqueous phase and accessible to the antibodies.

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A culture liquid of Alternaria porri afforded novel bianthraquinones named alterporriol D and E, whose structures were determined by spectroscopic methods as well as by degradation to the known compound. Alterporriol D and E were found to be atropisomers of each other.

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The acid protease B from Scytalidium lignicolum, a pepstatin-insensitive acid protease, was modified with 1-diazo-3-phenyl-2-propanone (DPP) in the presence of cupric ions. About 1 mol of DPP per mol of enzyme was incorporated with a concomitant loss of the activity. The modified enzyme was digested with thermolysin and DPP-labeled peptides were separated. One peptide, called SI, was purified and its amino acid sequence was found to be Ala-Thr-Ser-Asp-Thr-Ser-Gly-Ser, which corresponds to the sequence of residue Nos. 95-102 of the enzyme. Alkali treatment of the modified peptide resulted in complete removal of the modifier, suggesting that DPP is esterlinked to Asp-98 of the enzyme. The amino acid sequence around Asp-98 of the enzyme was significantly homologous to those around the active site aspartic acid residues of penicillopepsin and porcine pepsin. These results suggest that Asp 98 is one of the active site amino acids of the enzyme.

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Cytosine deaminase (EC 3.5.4.1) was extracted from commercial compressed bakers' yeast and purified to an almost homogeneous state. The enzyme activity was more than 200 U/mg of protein, which was several times higher than reported before. The molecular weight was 41, 000 by gel permeation. The pi was at pH 4.7. 5-Fluorocytosine, 5-methylcytosine, and creatinine were other substrates for the enzyme. An experiment with inhibitors suggested that the enzyme was an SH-enzyme. The enzyme was unstable to heat, with a half-life of about 0.5 hr at 37°C. Characteristics of the enzyme, especially its substrate specificity, were compared with those reported earlier for other cytosine deaminases from bacteria and a mold.

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The chemical composition of the cell walls of Arthrobacter sp. AT-25 was investigated. The purified cell walls were composed of D-galactose, D-glucose, sulfate, phosphorus, muramic acid, glucosamine, alanine, glutamic acid, glycine, and LL-diaminopimelic acid in a molar ratio of 4.1:0.88:5.7:0.44:0.65:0.85:2.0:1.0:1.1:1.1. On mild treatment with acid, the cell walls readily released a special structure, the non-peptidoglycan portion, and gave an insoluble peptidoglycan, soluble sulfated polysaccharides, and a phosphorus-rich fraction in respective yields of 41, 40 and 6%. The peptidoglycan contained two phosphate esters, muramic acid 6-phosphate and glucosamine 6-phosphate, while the special structure (sulfated polysaccharides plus a phosphorus-rich fraction) contained three phosphate esters, glucose 6-phosphate, and glycerol 1- and 2-phosphate. These results indicate that the cell walls of the bacteria contain highly sulfated polysaccharides with low degrees of phosphorylation. This paper reports the presence of a novel special structure in the cell walls of the grampositive bacteria.

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The measurement of nicotinamide N-oxide by high-performance liquid chromatography is described. The method used a Chemcosorb 5-ODS-H (150 × 4.5 mm i.d., particle size 5 μm) column eluted with 10 mM potassium dihydrogenphosphate (pH was adjusted to 3.0 by adding concentrated phosphoric acid) containing 0.1 g/l of sodium 1-octanesulfonate-methanol (100:4, v/v) at a flow-rate of 1.0ml/min. The UV detector was set at 260nm. The detection limit for nicotinamide N-oxide was 2pmol (276 pg) at a signal-to-noise ratio of 5:1. Under these conditions, nicotinamide N-oxide was eluted at about 3.2 min. This technique was used to analyze rat urine following effective clean-up steps with chloroform, the total analysis time being about 13 min.

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Effects of gradually increasing levels of nicotinamide (Nam) in a niacin-free and tryptophan-limited diet on the blood NAD levels and the urinary excretion of Nam metabolites in rats were investigated. When the concentrations of Nam in diets were raised to 1 mg per 100 g of diet, the blood NAD levels increased in direct proportion to the intake of niacin-equivalent, but the urinary excretory metabolites of Nam such as N¹-methylnicotinamide (MNA), N¹-methyl-2-pyridone-5-carboxamide (2-py), and N¹-methyl-4-pyridone-3-carboxamide (4-py) were almost constant regardless of the Nam levels. On the contrary, when the concentrations of Nam in diets were raised to 3 mg and 5 mg per 100 g of diet, the blood NAD levels were almost constant regardless of the Nam levels and it reached nearly normal levels, but the urinary excretion of MNA, 2-py, and 4-py increased in direct proportion to the intake of niacin-equivalent. These results mean that Nam is first used for the synthesis of blood NAD and then, when it has reached nearly normal levels, Nam is used for the increase in the urinary excretion of Nam metabolites. The ratio of 2-py plus 4-py to MNA excretion remained constant regardless of the Nam levels under the conditions of the experiment.

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Chloromonilinic acids A and B were newly isolated from a culture filtrate of M. fructicola accumulating chloromonilicin in the mycelium, and their structures were elucidated on the basis of spectroscopic evidence. The unusual catabolic route of chloromonilicin is discussed.

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Male Sprague-Dawley rats aged 1 month (young) and 9 months (adult) were fed diets containing sucrose, glucose or corn starch as a carbohydrate source for 4 weeks. Dietary sucrose increased (a) the liver size in rats of both ages, (b) the serum cholesterol level in adult rats, (c) the serum triglyceride level, especially in young rats, and (d) the liver cholesterol and triglyceride levels in adult rats. Consequently, there were age-related increases in serum and liver cholesterol in rats fed sucrose. No significant difference was found in the activity of liver 3-hydroxy-3-methylgIutaryI coenzyme A reductase due to the diets in age-matched rats, whereas an age-related decrease was observed in rats fed simple sugars. Rats fed sucrose, especially adult rats, excreted more fecal steroids than ones fed glucose or corn starch. Sucrose tended to enhance the metabolism of linoleate to arachidonate, as judged from the fatty acid composition of liver microsomal phosphatidylcholine. Thus, sucrose exerts a peculiar effect on cholesterol metabolism in relation to age.

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The effects of modification of the arginine/Iysine ratio of dietary protein on the cholesterol kinetics were studied in male rats. Single amino acids (lysine to soybean protein and arginine to casein) were added to approximate the arginine/Iysine ratio in different proteins. After acclimation to these diets for 30 days, rats were administered intravenous [¹⁴C]cholesterol and oral [³H]cholesterol. Analysis of the die-away curve of [¹⁴C]cholesterol showed an apparent independence of cholesterol kinetics to the dietary manipulations, but there was a moderate reduction of the size of the slowly exchangeable pool and of the biliary concentration of cholesterol when lysine was added to soybean protein. Addition of amino acids neither influenced cholesterol absorption nor the fecal excretion of the radioactivities from labeled cholesterol. The results indicate that manipulating the arginine/Iysine ratio of dietary protein by adding single amino acids is not necessarily effective in ameliorating cholesterol metabolism in rats, although the arginine addition caused a significant reduction of serum cholesterol and triglyceride.

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Potato and yeast phosphorylase were modified with a substrate analog, the 2', 3'-dialdehyde derivative of γ- and α-cyclodextrin (dial-γ- or α-CD), respectively. Both native and dial-CD showed competitive inhibition for the two phosphorylases, though the dial-CD gave much smaller Ki values of less than 1 mM. Among the three CD, γ- and α-forms of the native and dial-CD gave the smallest Ki values for the yeast and potato enzymes, respectively. The dial-CD modification was accelerated by higher pH and higher concentrations of dial-CD. Both potato and yeast enzymes were stoichiometrically modified with the dial-CD and lost the affinity for the substrate α-glucans. Moreover, the modification was retarded by the glucan substrates, which suggested that the dial-CD might bind at the glucan binding site of phosphorylases. In contrast with the case of glucose 1-phosphate, which showed no significant change in Km values of native and modified enzymes, Km values of modified enzymes for the glucan substrates clearly increased. These results indicated that the dial-CD binding might occur at the so-called glycogen storage site of yeast enzyme or cyclodextrin binding site for the potato enzyme rather than at the active site.

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In order to find out some specific blockers of the membrane transport of amino acids and small peptides in animal small intestine and kidney, the following dihydrochalcone (DHC)-amino acid and -peptide conjugates were synthesized. [N-[(6'-hydroxy-4, 4'-dimethoxy-DHC)-2'-yloxy]acetylglycyl]glycine, N^ε-[(6'-hydroxy-4, 4'-dimethoxy-DHC)-2'-yloxy]acetylIysine, O-[(6'-hydroxy-4, 4'-dimethoxy-DHC)-2'-yl]homoserine, [O-[(6'-hydroxy-4, 4'-dimethoxy-DHC)-2'-yl]homoseryl]glycine, N-glycyl-O-[(6'-hydroxy-4, 4'-dimethoxy-DHC)-2'-yl]homoserine, and [O-[(4, 4', 6'-trihydroxy-DHC)-2'-yl]homoseryl]glycine.

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The leaves of thyme (Thymus vulgans L.) were found to contain new compounds, 4'-hydroxy5, 5'-diisopropyl-2, 2'-dimethylbiphenyl-3, 4-dione (1), 5, 5'-diisopropyl-2, 2'-dimethylbiphenyl-3, 4, 3', 4'tetraone (2), and 4, 4'-dihydroxy-5, 5'-disopropyl-2, 2'-dimethylbiphenyl-3, 6-dione (3a). These structures were determined by chemical and spectroscopic methods. The deodorant activity of compound 1 against methyl mercaptan was more effective than that of rosmanol, carnosol and sodium copper chlorophylline.

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A new acylated kaempferol glucoside, kaempferol 3-O-(2", 6"-di-O-(E)-p-coumaroyl-β-D-glucopyranoside), was isolated from the leaves of Quercus dentata Thunberg as a repellent against a fouling organism, the blue mussel Mytilus edulis L.

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Phenoloxidase was purified from pupae of the housefly, Musca domestica L. The purification procedures included ammonium sulfate precipitation, affinity chromatography and Sephadex G-200 gel filtration. The final preparations appear to be homogeneous based on results obtained from polyacrylamide gel electrophoresis. The molecular weight of phenoloxidase was estimated to be 330, 000, as determined by gel filtration. The kinetic properties of phenoloxidase were studied using six catecholamines as substrates. The preferred order of substrates for phenoloxidase was found to be N-β-alanyldopamine>dopamine>N-acetyldopamine>norepinephrine>epinephrine>DOPA.

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The signals of fatty acids in the form of triglycerides were observed in the ¹³C NMR spectrum of an intact soybean seed. The major fatty acid component composition of triglycerides in a soybean seed, which includes linoleic acid, oleic acid and palmitic acid, was estimated by subtracting the spectra of authentic fatty acids from the spectrum of the intact soybean seeds. The fatty acid compositions of seeds of 11 Japanese soybean cultivars and 5 lines bred at the Asian Vegetable Research and Development Center (AVRDC) were estimated by this rapid (within Ihr for one seed) and nondestructive analytical method.

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Steady-state inhibitory kinetic studies on almond β-glucosidase-catalyzed reactions were done to elucidate the binding subsite of several monosaccharides on this enzyme.
Glucono-l, 5-lactone (a transition-state analog), glucose, 2-deoxy glucose, fucose, and methyl α-glucoside showed mixed-type inhibition, but galactose, galactosamine, mannose, N-acetyl glucosamine, and glucosamine showed pure competitive inhibition on the hydrolysis of p-nitrophenyl β-glucoside.
These results are reasonably accounted for by assuming that the former monosaccharides (the mixed type inhibitors) bind to subsite 1 (the nonreducing-end side subsite to which the nonreducing-end glucose residue of a substrate binds in a productive binding mode), and that the latter (the competitive inhibitors) bind to subsite 2, the adjacent subsite to subsite 1.
The binding affinity (-ΔG°) of glucono-l, 5-lactone (-ΔG°=6.7kcalmol^-1 at pH 5.0, 25°C) was significantly greater than those of the others (0.3-1.6 kcalmol^-1).

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Dependency of superoxide anion (O₂^-) generation on protein kinase C (PKC) or calmodulin (CaM) in macrophages (MPs) was investigated using a O₂^--specific chemiluminescence probe with the Cypridina luciferin analog 2-methyl-6-(p-niethoxyphenyl)-3, 7-dihydroimidazo[1, 2-a]-pyrazin-3-one (MCLA). In opsonized zymosan (OZ)-stimulated MPs, 40% of the MCLA-dependent chemiluminescence disappeared when the PKC inhibitor 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7) or the CaM antagonist N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7) was added. In MPs stimulated by 12-o-tetradecanoylphorbol-13-acetate (TPA), addition of H-7 strikingly lowered MCLA-dependent Chemiluminescence, but W-7 had only a little effect. These results indicate that both PKC and CaM are involved in OZ-stimulated O₂^- generation in MPs and that TPA-stimulated O₂^- generation depends not on CaM but on PKC.

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