The endo-β-(1→3)-D-glucanase from extracts of Rhizoctonia solani has been purified to homogeneity by chromatographies on CM Bio-Gel A and Bio-Gel P-60 and finally by affinity chromatography on short-chain pachyman-AH-Sepharose. In the affinity chromatography, the adsorption of the enzyme seemed to be brought into close with the large spacer arm by prior biospecific adsorption on an attached ligand. The isoelectric point was pH 9.8 and the molecular weight was 29, 000. The optimum pH for short-chain pachyman was 5.5. The enzyme was fairly stable at pH 5 to 8.5 and at temperatures below 50°C. The enzyme favors streches of β-D-(1→3)-linkages, and also depolymerize β-(1→3)-β-(1→4) mixed-linked glucan. It has transglycosylase activity.

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A dextransucrase with high affinity for Sephadex G-100 gel was purified from Leuconostoc mesenteroides NRRL B-512F. The purified enzyme was very similar in its subunit molecular weight and properties to those of the main component of dextransucrase from B-512F. The stability of the enzyme was significantly increased by glycerol (33%) and bovine serum albumin (0.1%). Addition of exogenous dextran stimulated the sucrase activity (reducing sugar production) 1.3-fold, and the transferase activity (dextran synthesis) was increased 9.6-fold. Thus, the exogenous dextran clearly improved the efficiency of polymer synthesis. A micro-determination of enzyme activity by a fluorescent reagent BEC (borate ethanolamine complex) allowed for kinetic examination at lower substrate concentrations, which gave biphasic double reciprocal plots for the substrate sucrose.

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Vitellogenin, a female-specific serum protein, as a carrier of carotenoid was studied using chum salmon, Oncorhynchus keta.
Levels of carotenoid in muscle markedly decreased during spawning migration, while serum carotenoid increased markedly. Vitellogenin appeared in the serum at the start of spawning migration and disappeared during upstream migration. Appreciable amounts of a carotenoid, probably astaxanthin, was bound to the Vitellogenin. The ovaries of chum salmon matured during spawning migration. During upstream migration, the ovaries had much carotenoid.
These results suggested that Vitellogenin might be closely related to the transport of carotenoid from muscle into the ovaries.

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Arctic acid (1), arctinone-a (2), arctinone-b (3) and methyl arctate-b (8), minor components of Arctium lappa L., were synthesized via 5-(1-propynyl)-2, 2'-bithienyl (18) starting from 2, 2'-bithienyl (11).

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A nondialyzable polyphenolic substance having antiplasmin activity was isolated from the bark of Pterocarpus indicus, Willd as a brownish powder. The results of ultracentrifuging, HPLC and TLC of its acetate indicated a narrow distribution of its molecular weight, i.e. its relative homogeneity. This observation, and the pattern seen in its ¹³C-NMR spectrum that indicated a difference of its constituent units from the known coniferous polyflavanols, suggested some uniqueness of this substance (called PI in this paper) in the polyflavanol group. Its large apparent molecular weight, which was calculated from the sedimentation data, also seems to support this suggestion.
The substance inhibited plasmin esterolytic activity at ED₅₀ = 2.5 μg/ml, and also showed a carcinostatic effect on mice bearing ascites Ehrlich carcinoma at a dose of 2 mg/kg.

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Three proteases designated P-I, P-II, and P-III were separated from larval gut juice of the silkworm, Bombyx mori, and the effects of these proteases on the two morphologically different inclusions produced by Bacillus thuringiensis kurstaki HD-1 strain were investigated. Bipyramidal inclusions were solubilized by each protease at pH 10.2, and proteins with a molecular weight of about 60, 000 were produced, while cuboidal inclusions were resistant to the proteases. Protease P-III appeared to be most important in the dissolution of the bipyramidal inclusions. Based on the affinity to various synthesized substrates, P-III was considered to be a chymotrypsin-like protease. After dissolution of inclusions by P-III, dissolved and undissolved fractions were separated, and the insecticidal activity of each fraction was tested. Results indicate that bipyramidal inclusions are preferentially toxic to the silkworm and the cuboidal ones carry the mosquitocidal activity.

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In a study of the relationship between the dynamic changes in cellular organelles and biosynthesis of soybean storage proteins, we found that as the protein components accumulated, the a and a'-subunits of 7S globulin appeared first, followed successively by the acidic and basic subunits of US globulin and finally by the β-subunit of 7S globulin. Labeling experiments using tissue slices showed that the radioactivities of (³H)leucine and (¹⁴C)N-acetyl-D-glycosamine were incorporated into the trichloroacetic acid insoluble fraction and that (¹⁴C)N-acetyl-D-glycosamine was specifically incorporated into 7S.

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A 3.0-kb Hind III fragment of Thermus flavus AT-62 DNA cloned in Escherichia coli was shown to direct the synthesis of thermophilic malate dehydrogenase. The enzyme produced by the recombinant clone was purified by a relatively simple procedure of heat treatment and affinity chromatography nearly to homogeneity. The enzyme preparation had practically the same heat stability and kinetic and immunological properties as those of the enzyme prepared from T. flavus cells.

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The frequent occurrence of lethal specimens of Zosimus aeneus and Atergatis floridus on southern Negros Island, Philippines, was confirmed by mouse lethality tests. Among eight specimens of Z. aeneus analyzed by fluorometric liquid chromatography, tetrodotoxin and its derivatives were dominant in five indicating the possible involvement of tetrodotoxin in human intoxication. The three other specimens of Z. aeneus and one specimen of A. floridus contained paralytic shellfish toxins as the major toxins.

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Bacillus subtilis AJ 3250 is capable of accumulating a remarkable amount of α-amylase in the culture liquid on pH-stat submerged fermentation. This microorganism requires sufficient oxygen as in other aerobic fermentations, and amylase production can only reach the maximum when the agitation and aeration conditions maintain the dissolved oxygen concentration above the critical level in pH controlled cultures. However, in pH uncontrolled cultures, amylase production as well as cell mass activity increase under low aeration conditions such as 1/20 vvm. Studies were carried out to analyse this phenomenon, and some new ways to express the relationship between the behaviour of pH and that of carbon dioxide partial pressure in a culture system were introduced. In this study, it was found that the differentiation in the carbon dioxide evolution rate reflects the dynamic characteristics of the pH change in this type of fermentation. An appropriate method of neutralization for managing pH-stat cultures on submerged fermentation can be obtained using this theory.

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To study the preliminary localization of the antigenic sites of bovine α_s1-casein, we separated fragments 1-54, 55-60, 61-123, 124-135, 136-196 and 197-199 from α_s1-casein cleaved with cyanogen bromide, and examined the antigenic reactivities of the fragments with antiserum to intact α_s1-casein.
On quantitative precipitation between a_sl-casein and its antiserum, a mixture of fragments 1-54, 55-60, 61-123, 124-135, 136-196 and 197-199 inhibited the formation of immune precipitates by 84.2%. Four of these six fragments, i.e. fragments 1-54, 61-123, 124-135 and 136-196, still showed high antibody-binding activities in the quantitative immunoprecipitin inhibition test and an enzyme-linked immunosorbent assay. Furthermore, on immunodiffusion analysis, only fragment 61-123 formed a single and sharp precipitin arc.
These results indicate that at least one antigenic site of α_s1-casein is associated with each of regions 1-54, 124-135 and 136-196, and more than two with region 61-123.

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The title compounds were synthesized by employing 2-aminohexadecanoic acid and serine as the chiral sources, and served for an assay of fruiting-inducing activity on Schizophyllum commune. The structure-bioactivity relationship among closely related synthetic ceramides is discussed.

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Structure-activity relationships of antimutagens were studied by assaying analogues and derivatives of 2(5H)furanone designed on the basis of the structures of natual antimutagens by the method of observing UV-induced reversion of E. coli WP2 B/r uvr A^- trp E^-. It is shown that an α, β-unsaturated carbonyl system is required for antimutagenic activity. Further modification of 3(2H)furanones resulted in the development of a highly potent antimutagen. The possibility that the antimutagenic effects may be due to alteration of proteins involved in the DNA repair system by trapping thiol groups is also discussed.

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When Aspergillus ory, zae IAM 2640 was grown in liquid medium, a high molecular weight acid CPase was produced exclusively in the medium. The enzyme was purified and characterized. The purified enzyme was homogeneous by PAGE at pH 9.4, by isoelectric focusing, and by FPLC, and its molecular weight was 155K by gel filtration. When the 155K enzyme was electrophoresed in SDS-polyacrylamide gel, a single band with a molecular weight of 73K was obtained. The enzyme was inhibited by TPCK and PMSF but not IAA or PCMB. The Km and k_cat values of the enzyme for Z-Glu-Tyr, angiotensin, and bradykinin at pH 3.7 and 30°C were 0.48mM, 86 sec^-1, 0.1 mM, 0.34sec^-1, and 0.06mM, 14.7 sec^-1, respectively. The enzyme hydrolyzed proangiotensin and bradykinin sequentially at pH 3.7 from their C-terminus. The enzyme shared an antigen with low molecular weight A. oryzae acid CPase O-1.

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Eight amylase inhibitors were isolated from a transglucosylation mixture of 1-deoxynojirimycin, soluble starch and bacterial saccharifying amylase. The inhibitors were found to be a series of compounds and were designated as (glucose)_n • 1-deoxynojirimycin, from the results of constituent and reducing end analyses (n-1 to 8).
The linkage between glucoses was determined to be α-1, 4 by the permenthylation or β-amylase digestion method. The glucose-1-deoxynojirimycin linkage was determined to be α-1, 4 by ¹H and ¹³C-NMR spectroscopy. The inhibitors inhibited various amylases depending on the number of glucose residues. The size of the active site (subsite) of an amylase might influence its susceptibility to the inhibitors.

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The present study was undertaken to define more precisely the mechanisms involved in the toxicity of ingested Concanavalin A (Con A) in rats, by observation of its behavior in the gastrointestinal tract. Con A was observed to have a high affinity for water-insoluble components of feces. The addition of a massive amount of the water-insoluble components to a high sucrose diet (HSD) containing Con A did not ameliorate the adverse effect of ingested Con A on the adaptive response of the intestinal, sucrase activity to dietary sucrose. The addition of Con A bound feces to HSD also prevented the adaptive response of the intestinal sucrase activity to dietary sucrose. The luminal pH of the stomach 5 hr after HSD administration was 3 to 4, which extremely reduced the affinity of Con A for carbohydrate moieties of the cell membrane, whereas those of the jejunum and cecum ranged from 6 to 7. These results substantiate the proposed mechanism, that is, that the toxicity of ingested Con A involves its binding to the luminal surface of the small intestine, and it then in turn disturbs the functional formation of the brush border membrane in response to dietary alterations.

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S-PI(Pepstatin Ac)-insensitive acid proteinase was found in a culture filtrate of Scytalidium Hgnicolum ATCC 24568 and designated as acid proteinase C. Acid proteinase C was purified and about 320 mg of the purified enzyme was obtained from 41.5 liters of culture filtrate, a recovery of 0.9%. Acid proteinase C was monodispersive by physicochemical criteria such as ultracentrifugal analysis and disc electrophoresis.
Acid proteinase C was most active at pH 2.0 toward casein, and stable in the pH range of 3 to 5 under treatment at 37°C for 20 hr. The molecular weight was 4.06×10⁵ by sedimentation equilibrium analysis, and 3.6 × 10⁵ by Andrew's gel nitration method. A subunit structure was suggested by SDS-PAGE.
Acid proteinase C was characterized by the following points: (1) the proteolytic activity was not inactivated by any of such acid proteinase inhibitors as S-PI, DAN and EPNP; (2) the molecular weight was extraordinarily high in comparison with those of other proteinases; (3) acid proteinase C was inactivated weakly by alpha-MAPI, a potent inhibitor of serine- and thiolproteinases; (4) acid proteinase C hydrolysed at a high rate MAPI-analogous peptide substrates (Suc-Phe-Arg-Ala-Phe-NPhNO2: k_cat/Km, 5.8 × 10⁴M^-1 • sec^-1).

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The kinetics of S-PI (Pepstatin Ac)-insensitive acid proteinases A-2 and C from S. lignicolum were studied to elucidate the essential groups for catalysis. The hydrolysis of Z-Phe-Leu-Ala-Ala by acid proteinase A-2 was studied from pH 1 to 5 at 30°C, and the Michaelis constant, Km, and the maximum velocity, V, were found for each pH. The pH-k_cat/Km profile was bell-shaped. The pKe₁ and pKe₂ for the free enzyme were 1.53 and 3.77, respectively. Acid proteinase A-2 does not contain histidihe residues in the molecule. Accordingly, both of the essential groups are considered to be carboxyl residues. Similar experiments were done with acid proteinase C using Suc-Phe-Arg-Ala-Phe-NPhNO₂ as substrate. The pKe₁ and pKe₂ at 30°C were 2.89 and 5.43, respectively. Based on the data at 30°C and 40°C, the heat of dissociation, ΔHe, was calculated by the equation of Van't Hoff. Both of the groups were regarded as carboxyl residues, since the ΔHe were -0.8 and 1.2kcal/mol, respectively.
These results suggest that Scytalidium enzymes have active carboxyl residues participating in the actions, as do the Pepstatin-sensitive acid proteinases.

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The lipids of rice in storage, mainly triacylglycerols which accumulate in spherosomes, began to decrease after 6 months of storage, while the level of free fatty acids gradually increased from the early stage of storage. Furthermore, phosphatidylcholine, which is one of the major constituents of the spherosome membrane, altered to phosphatidic acid after 6 months of storage, indicating that the spherosome membrane underwent deterioration. It is suggested from these results that the spherosome membrane becomes leaky due to the alteration of its phospholipid composition, and consequently triacylglycerols that ooze out of the spherosomes, are degraded to free fatty acids and glycerol through mono- or di-acylglycerols. After 6 months of storage, degradation of the rice bran lipids proceeded rapidly by the accompanying deterioration of the other constituents.

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(±-2-(4-Substituted phenyl)-3-methylbutanoic acids (2a-c) were optically resolved by preferential crystallization in the form of their diethylamine salts (3a-c).
The molar ratio of the acid to the amine in these salts was found to be 2: 1 by chemical analyses, and was confirmed by analyzing the crystal structure of one of these salts (3b) with X-ray single crystallography.

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A soil isolate, strain S93B1, identified as Pseudomonas cruciviae could grow on more than ten biphenyl-related compounds including p-chlorobiphenyl.
Biphenyl was converted to benzoic acid and γ-benzoylbutylic acid. Mono-substituted biphenyl compounds such as o-bromobiphenyl, p-chlorobiphenyl, o-nitrobiphenyl and m-nitrobiphenyl were converted to o-bromobenzoic acid, p-chlorobenzoic acid, o-nitrobenzoic acid and m-nitrobenzoic acid, respectively. Phenol and 2-phenoxymuconic acid were produced on the degradation of biphenyl ether. The formation of 2-phenoxymuconic acid demonstrated that biphenyl ether was degraded through an ortho cleavage pathway and the formation of γ-benzoylbutylic acid suggested the degradation of biphenyl through a meta cleavage pathway.

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We selected Aspergillus niger GRM3 because it secreted a novel enzyme that hydrolyzed glycyrrhizinic acid. The enzyme was purified to the electrophorctically homogeneous state and an activity more than 890-fold that of culture broth. The molecular weight of the enzyme was estimated to be 150, 000 by gel filtration and it was most active over the range of pH 4.1 to 4.5. The enzyme hydrolyzed glycyrrhizinic acid to produce glycyrrhetinic acid and D-glucuronyl-β-1, 2-Dglucuronic acid. The hydrolytic action of this enzyme was different from those of β-glucuronidases reported so far.

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Asymmetric ring opening of the cyclic acid anhydrides cis-2, 3-6 with the axially dissymmetric binaphthyldiamines (S)-1a-d and subsequent esterification gave diastereoisomeric mixtures of the amide-esters 7a-h. Successive reduction of the ester group and ring closure by hydrolysis afforded (-)-cis-2, 4-dimethyl-δ-valerolactone (8, 92% e.e.), (-)-mevalonolactone (9, 58% e.e.), (+)-3-isopropyl-(5-valerolactone (10, 42% e.e.), and (+)-2, 3-methylene-γ-butyrolactone (11, 46% e.e.). Through kinetic resolution of the racemic anhydride trans-2, (-)-trans-2, 4-dimethyl-dvalerolactone (12) was yielded in a 74% e.e., whose absolute configuration was established to be 2R, 4R.

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This study aimed to ascertain whether the dietary fat-dependent modification of the distribution of intestinal HMG-CoA reductase activity in rats is related to differences in the site of absorption of dietary fat. Either [³H]triolein or [³H]tripalmitin and [¹⁴C]β-sitosterol were used as an absorbable and a non-absorbable marker and the absorption site was estimated by comparing the [³H]/[¹⁴C] ratio in the lumen of the gastrointestinal tract with that of the diet. The small intestine was divided into four segments of equal length. When triolein was used, the marker ratio ([³H]/[¹⁴C]) was essentially comparable up to the jejunum, and reduced significantly in the upper ileum where mucosal radioactivity was also fairly high. When [³H]tripalmitin was used, the luminal marker ratio was slightly increased as the digest moved down to the ileum. A distinct decrease of the ratio was also observed in the upper ileum but to a lesser extent than in the case of triolein. Incorporation of radioactivity into the mucosa was most substantial in the upper ileum. Although the digestibility differed somewhat, both triolein and tripalmitin were considered to be absorbed mainly in the ileum. Hence the distribution of HMG-CoA reductase activity along the small intestine was not always consistent with the localization of the site of fat absorption. This study did not indicate the stimulatory effect of dietary fat on intestinal cholesterogenesis and the production of chylomicrons to process the absorbed fat.

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The total lipids of Rails Janet apples' mitochondria after six-month storage at 0°C were examined by differential scanning calorimetry (DSC). The phase change temperature was -85°C, differing from that of Starking Delicious apples. The total lipids were extracted from the pulp of Rails Janet apples after eleven months of storage and fractionated into neutral lipid, glycolipid, and phospholipid fractions by column chromatography on silicic acid. These fractions were measured by DSC. The glycolipid fraction contributed to the phase change temperature at -85°C. Furthermore, the spot having an Rf value of 0.43 on thin-layer chromatography contributed to the phase change temperature. The glycolipid fraction may contribute to cold tolerance.

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A 418 base pair DNA duplex consisting of a gene for human lysozyme with a synthetic ribosome binding site of E.coli was assembled by stepwise ligation of 56 chemically synthesized oligodeoxyribonucleotides. The synthesized gene was expressed under the control of the lambda pRpL promoter and the gene product accumulated as several percent of the total cellular proteins in E. coli. The expressed product existed as insoluble and biologically inactive aggregates in the cells, but the biological activity was regenerated by solubilization and renaturation of the aggregated protein. The results of the N-terminal amino acid sequence analysis of the product showed the human lysozyme synthesized in E. coli has an additional methionine residue at the N-terminal of the mature human lysozyme due to the translational initiation codon of E. coli.

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The enzyme system of Corynebacterium equi IFO 3730 catalyzed the diastereo-selective oxidation of β-methyl-sec-alcohols. For example, only the erythro isomer of 5-methyl-6-tetradecanol was oxidized to the corresponding ketone, the threo-alcohol remaining. Moreover, the resulting ketone was metabolized enantioselectively by the same microbe. Thus, it was possible to obtain optically active α-methylketones. This biochemical kinetic resolution method was applied for some 2-phenyl-3-alkanones, the expected chiral ketones of moderate to high optical purities being obtained. The degradation pathway was deduced to involve Baeyer-Villiger type oxidation, on the basis of the isolation of small amouts of α-phenylethyl alcohol and acetophenone.

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β-(1→4)-D-Glucanase(II) was isolated and purified to homogeneity on SDS-electrophoresis from brewing barley malt. This enzyme had an endo-hydrolase action pattern on β-glucan prepared from the same malt, while the enzyme formed precipitates in the reaction mixture. These precipitates (P-I) as a whole were composed of β -(1→3) and (1→4) linkages in a molar ratio of 27.6: 72.4. The P-I was separated into two parts and analyzed; they had mainly linear β-(1→4) linkages. When cellotetraose or 3-O-β-cellotriosyl-D-glucose was used as a substrate for β-(1→4)-D-glucanase(II), insoluble materials were similarly formed in reaction mixture. These water-insoluble materials were cellooctaose and 3-O-β-cellooctaosyl-D-glucose, respectively. These results suggest that β-(1→4)-D-glucanase(II) catalyzed the transfer of β-D-glucosyl residues from each oligosaccharide to C₄-OH of the non-reducing residue in each of them, and the resulting products from the transglucosylation precipitated in the reaction mixture.

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Both enantiomers of 1-hydroxyethylphosphinic acid were obtained by resolution of the racemic acid using (+)- and (-)-1-(1-naphthyl)ethylamine as the resolving agents, and the (+)-isomer was assigned to be (S)-configuration. The four diastereomers of butyl 1-hydroxyethylphosphinates were prepared in an optically pure state by reacting the resolved acid with 1-butanol in benzene and then separating the diastereomeric products by chromatographic means.

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An extracellular polysaccharide was isolated from the culture medium of a virulent strain (U-7) of Pseudomonas solanacearum. The polysaccharide caused tomato cuttings to wilt. It seemed to be composed mainly of N-acetylgalactosamine together with small amounts of rhamnose, glucose, and protein. This is, to our knowledge, the first example of homo-N-acetylgalactosaminoglycan in nature.

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