Two kinds of cell-bound Upases were purified from S. lipolytica with a total recovery of 8%. One was adsorbed and the other not to CM-Sepharose CL-6B at pH 5.0. The method of purification involved chromatography on CM-Sepharose CL-6B, DEAE-Sepharose CL-6B and Sephadex G-100 columns. Sodium dodecylsulfate-polyacrylamide gel electrophoresis of the purified lipases gave a single band. The purified lipases required an activator, such as oleic acid for hydrolysis of triglycerides. The lipases hydrolyzed tricaprylin at the largest initial rate and the optimum pH for hydrolysis of olive oil was about 8.0 under the experimental conditions. They were stable for 20 min below 37°C and in a pH range from 4.5 to 8.0 for 22hr at 5°C. The molecular weight of Lipase I and II was estimated to be 39, 000 and 44, 000, respectively, and both lipases were a monomeric enzyme.

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The mitochondria were prepared from apples cv. Starking Delicious which were sensitive to chilling injury and apples cv. Rails Janet which were not sensitive to chilling injury. The content of unsaturated fatty acids in the Rails Janet mitochondria was higher than that in the Starking Delicious mitochondria. Phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylinositol, phosphatidic acid, phosphatidylserine, diphosphatidylglycerol (cardiolipin) and mono- and digalactosyldiglyceride were identified as the conjugated lipid components. The PC/PE ratios were 0.74 and 0.14, respectively, in the mitochondrial phospholipid fraction of Rails Janet and Starking Delicious apples. The higher ratio of PC/PE and unsaturated fatty acid contents in Rails Janet than those in Starking Delicious may cause the resistance to chilling injury in Rails Janet by playing a role in lowering of the phase transition temperature. These facts were thought to contribute largely to the flexibility, fluidity and the function of transport of the membranes in the cold state.

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We isolated a new aerobic gram negative Collagenolytic bacterium, strain CRZV₁. This strain is yellow pigmented, non motile; it poorly degrades the sugars and its proteolytic activity corresponds to one enzyme, a collagenase which is produced in media with collagen, or collagen like substrates. The strain CRZV₁, presents many similarities with Flavobacteria. However, the GC content of the DNA, which is 65%, excludes our Collagenolytic strain from the genus Flavobacterium, where all the species have GC content between 30 and 42%. All the strains, already described as Flavobacteria, the GC contents of which are situated between 55 and 70%, are actually unclassified. They could be included in the genus Empedobacter. This genus is not in the approved list of bacterial names, so we only consider this new Collagenolytic bacteria as a gram negative, aerobic, yellow pigmented bacteria.

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Fumarate reductase was highly purified from the cytosol fraction of the cells of baker's yeast, which were anaerobically cultured, with a yield of 6% by 7 steps. The purified enzyme is homogeneous in disc-gel electrophoresis, and has a molecular weight of 58, 800. Its isoelectric point is 5.3. The enzyme contains 1 molecule of non-covalently bound FAD per molecule of protein. Non-heme iron was not detected. The amino acid composition was determined. The spectra of the enzyme showed absorption peaks at 273, 380 and 456 nm with shoulders at 370, 440 and 478 nm. The fluorescence spectra showed excitation peaks at 452 and 368 nm and an emission peak at 500 nm. Double-reciprocal plots of the reaction rate against the FADH₂ and FMNH₂ concentrations curved upwards. The hill coefficient values and S_0.5 for FADH₂ and FMNH₂ were estimated to be 1.6 and 0.21 μM, and 1.5 and 0.38μM, respectively. The Km for fumarate was 0.2mM.

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Properties of a glutathione transport system in T. ferrooxidans strain AP-44 were investigated using a reduced form of ³⁵S-glutathione (³⁵S-GSH). About 71.2% of the total radioactivity taken up into the cells was distributed in the cytosol fraction. The amount of GSH taken up into the cells was in proportion to the amount of ferrous iron oxidized. However, a high concentration of silver ions (50mM), which completely inhibited an iron-oxidizing activity, did not inhibit the GSH transport. The results suggest that GSH was transported by using a proton electrochemical gradient formed across the cytoplasmic membrane. Since growth inhibition by silver nitrate was decreased by the addition of GSH to both silver ion sensitive-cells and resistant-cells, the GSH transport system may play some role in the silver ion resistance mechanism of the bacterium.

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A β-cyano-L-alanine (β-CNAla)-assimilating bacterium was isolated from soil samples, and was identified as belonging to the genus Pseudomonas. Under the growing conditions used, this strain consumed β-CNAla without accumulating any detectable metabolite. However, both intact cells and cell-free extracts of the strain formed aspartic acid from β-CNAla. By affinity chromatography using AHA (N-ω-aminohexyl-L-aspartic acid)-Sepharose 6B gel a β-CNAla-degrading enzyme was purified from the cell-free extract, and it was found that the enzyme was specific for the degradation of β-CNAla.
The strain also utilized the aliphatic nitriles, aceto-, propio-, n-butyro- and succinonitrile, as the sole nitrogen source. Cell-free extracts from cells grown on various aliphatic nitriles possessed both nitrilase and amidase activity but not β-CNAla-degrading activity, and in this strain β-CNAla was found to be degraded via a pathway different from that for common nitriles.

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Five kinds of thermo-labile antigens (TLA a, TLA b, TLA c, TLA d and TLA e) of yeast cell surface were isolated. Each TLA was homogeneous on polyacrylamide gel electrophoresis. The molecular weights of TLA a, TLA b, TLA c, TLA d and TLA e were 68, 000, 86, 000, 68, 000, 170, 000 and 82, 000, respectively, as estimated by gel filtration on Sephadex G-200. When gel electrophoresis was performed in 0.1% sodium dodecyl sulfate (SDS), TLA a, TLA b, TLA c and TLA e gave a single band of molecular weight at about 62, 000, 38, 000, 33, 000 or 79, 000 in each test, whereas TLA d gave two bands of molecular weights at about 81, 000 and 40, 000. Among the five kinds of antigens, only TLA e contained 9.7% sugar, the other antigens containing no sugar moiety. TLA a, TLA c, TLA d and TLA e did not give a precipitin line against anti-TLA antisera when heated at 70°C for 10 min, whereas TLA b lost antigenicity by heating at 90°C. the isoelectric points for the five kinds of antigens were at pH 6.4 (TLA a), pH 6.1 (TLA b), pH 5.2 (TLA c), pH 4.7 (TLA d) and pH 4.0 (TLA e). All antisera specific to each TLA agglutinated fresh yeast cells, suggesting that these five kinds of antigens are located on the cell surface.

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The preceding paper described that when succinylated α_s1-casein, ca. 25, 000 daltons, was modified with papain in the presence of L-leucine n-dodecyl ester (Leu-OC₁₂), an approximately 20, 000-dalton macropeptide was formed as the main product. In the present work we have investigated its chemical structure and surface function. A treatment for purification at the petroleum ether/water interface gave an electrophoretically homogeneous 20, 000-dalton macropeptide which functioned as a surfactant to emulsify corn oil as well as "-octane. Pulsed NMR and ESR studies demonstrated that the macropeptide, when used to emulsify n-octane in water, acted to restrict the mobility of those molecules involved in the emulsion. Various data from chemical analyses coupled with knowledge about the primary structure of α_s1-casein showed that the 20, 000-dalton macropeptide was structured as succinyl-Arg¹-....-Phe¹⁴⁵-Leu-OC₁₂. A discussion is included to explain the surface function of this peptide in relation to its amphiphilic structure.

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The difference spectra before and after oxidation of L-ascorbic acid (AsA) by ascorbate oxidase (EC 1.10.3.3) were measured using a recording spectrophotometer. A linear relationship was found between the peak height of difference spectra and the concentration of AsA. The AsA content in plant and animal foods was evaluated by this difference spectral method and was compared with that obtained by the 2, 4-dinitrophenylhydrazine method and the indophenol method. The values of AsA estimated by the present method and these official methods agreed well in all samples. The effect of pH and the interference of various compounds on the AsA assay were also examined.

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Four temperate phages, SPA 10, SPA 18, SPA38 and SPA48 were isolated from soil with Streptomyces parvulus 2297 as a host. A cleavage survey of DNAs from these phages was undertaken using 18 restriction endonucleases. By agarose gel electrophoretic analyses, the genome sizes of the SPA10, SPA18, SPA38 and SPA48 phages were estimated to be 31, 31, 25 and 29 magadaltons, respectively. A cleavage map of SPA10 phage DNA was constructed with restriction endonucleases PvuII, BglII and BclI.

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Five aminotransferases, TA-A, -B, -T, -P_I, -P_II, which might participate in the biosyntheses of aspartate, branched-chain amino acids, tyrosine, and phenylalanine, respectively, were separated from one another in Brevibacterium flavum, and examined for their keto-acid substrate specificities and the apparent Michaelis constants (Km') for their main substrates. TA-A specifically reacted with oxalacetate, among the keto-acids tested, and accounted for 90% of the total activity in the cellular extracts. TA-B was found to be the only aminotransferase that reacted with branchedchain keto-acids, and also showed some activity with prephenate and phenylpyruvate but having a much larger Km' than the other enzymes. TA-T specifically reacted with prephenate and oxalacetate, among the keto-acids tested, and accounted for 80 and 2% of the total activities of the cellular extracts, respectively, but showed a much larger Km' for oxalacetate than did TA-A. TA-P_I and -P_II showed similar substrate-specificities and Km's to each other. They were active with phenylpyruvate, p-hydroxylphenylpyruvate, prephenate, and oxalacetate. Their activity with phenylpyruvate accounted for only 36% of the total activity, but showed a markedly smaller Km' than did TA-B having 64% of the total activity. An aspartate auxotroph, No. 102-7, was found to lack TA-A and TA-P_I, and to have a reduced amount of TA-P_II. The relative activity of the mutant TA-P_II with oxalacetate decreased to about 1/10 that of the wild-type TA-P_II. Two groups (I and II) of prototrophic revertants were derived from No. 102-7: Group I restored TA-A but neither TA-P_I nor TA-P_II, while group II restored none of the three enzymes but had a new type of aminotransferase which was active with aspartate, but showed different chromatographic properties from TA-A.

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A novel metallo-proteinase inhibitor (FMPI) was purified about 10, 000 times from the culture filtrate of Streptomyces rishiriensis NK-122 through column chromatographies on Dowex 1 × 2 (OH^-) form, aluminum oxide, Dowex 1 × 2 (OH^-) form, XAD-2, Cellulofine GC-15 and Sephadex LH-20, successively. FMPI was homogeneous on thin-layer and high performance liquid chromatographies. FMPI is composed of L-phenylalanyl-L-arginine and phosphoric acid, its color reaction is positive for Rydon-Smith, Sakaguchi and ammonium molybdate-perchloric acid reagents and negative for ninhydrin, Pauly, Ehrlich, phenol-sulfuric acid, Folin-Ciocalteau and Tollens reagents. FMPI is stable in a strongly alkaline solution (pH > 11) but very labile below pH 11 even at room temperature.

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The nondialyzable melanoidins prepared from glucose-butylamine (I) and xylose-butylamine (II) reaction systems, freeze-dried powder obtained from the dialyzable fraction of the glucose-butylamine reaction system (III) and N-butyl-glucosylamine (IV) were pyrolyzed at 350°C for 0.5-2hr and the volatile pyrolysate was investigated. To trap the volatile compounds, Tenax GC trapping and cold trapping methods were used. Identification of these volatiles was made by gas chromatography-mass spectrometry, using a glass capillary column. The volatile components in the pyrolysates of I, II, III and IV were qualitatively similar to each other. The major volatile components in the pyrolysates of I, II, III and IV were identified as two furans, 1 -butanol, two 1-butylpyrroles, 1-butylpyrrolidine, 1-butylaziridine and two N-butylamides. The results are discussed in relation to those obtained from previously investigated sugar-amino acid melanoidins.

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The antioxidative and prooxidative activities of sugars and sugar analogs were investigated on the autoxidation of methyl linoleate and safflower oil. Autoxidation of methyl linoleate was conducted in either the dry state or aqueous emulsion state. Although all sugars and sugar analogs inhibited the autoxidation of methyl linoleate in the dry system, the reducing sugars accelerated oxidation in the aqueous emulsion system. When glyceraldehyde, dihydroxyacetone and glycerol were added in the oxidation system, glyceraldehyde and dihydroxyacetone with water accelerated the formation and decomposition of methyl linoleate monohydroperoxide. On the other hand, glycerol inhibited the formation and decomposition of hydroperoxide. These results indicate that the carbonyl group of sugars accelerates lipid peroxidation and the hydroxy group of sugars and sugar analogs inhibits the oxidation. The effect of sugars in the safflower oil-sugar-cellulose oxidation system was also examined. Sugars at low humidity inhibited the autoxidation of safflower oil, but reducing sugars at high humidity accelerated the oxidation.

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3-Chloro-D-alanine chloride-lyase, which occurs in the cells of Pseudomonas putida CR 1-1, catalyzes not only the α, β-elimination reaction of 3-chloro-D-alanine to form pyruvate, but also its β-replacement reaction in the presence of a high concentration of sodium hydrosulfide to form D-cysteine. Using the β-replacement reaction, the enzymatic synthesis of D-cysteine by resting cells was investigated. The culture conditions for cell production of the bacterium with high D-cysteine-producing activity and the reaction conditions for D-cysteine production were optimized. Under these optimal reaction conditions, 100% of the added 3-chloro-D-alanine could be converted to D-cysteine and, as the highest yield, 20.6mg of D-cysteine per 1.0ml of reaction mixture could be synthesized.

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The distribution of heavy metals (Fe, Mn, Zn, Cu, Pb, Ni and Cd) were investigated in various organs and tissues of striped dolphin, Stenella coeruleoalba. The animals were caught alive at Taiji, on the coast of Kii Peninsula, during the open season in December 1978. Determination of the metals was made by atomic absorption spectrophotometry and significant differences of metal concentration in the positions of the muscle, blubber and skin, respectively, were observed. The front ventral muscle of matured dolphins showed the highest concentrations of Zn and Cd and lowest Fe when compared to other parts of the muscle. Most of the metals recorded relatively low concentrations in melon rather than in the other lipid layers of blubber. In skin tissue, the concentrations of Fe, Mn and Zn were significantly higher in black-colored skin than in white skin. Moreover, a difference in the concentrations of metals according to bone position was observed. In general, high concentrations of most of the metals were found in liver, kidney and bone, with low concentrations in brain and the lipid layer of blubber. Furthermore, relatively high concentrations of Cu, Mn and Zn were found in skin, and for Mn, Zn, Ni and Cd it was likewise in pancreas and the reproductive organs. Based upon these results, the nature of the organ(s) of a dolphin that has to be selected for ecological and hygienic comparison was discussed.

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Myofibrillar protein degradation was measured by the rate of N^τ-methylhistidine (MeHis) release from the-perfused hindquarters in normal and streptozotocin-induced diabetic rats. In diabetic rats, the rate of MeHis release to the perfusate was elevated 2-fold compared with normal rats. The daily excretion of MeHis into urine was also increased 2-fold in the diabetic rats.
Insulin in the perfusate did not suppress the release of MeHis from the perfused muscle in normal rats. On the other hand, in diabetic rats, MeHis release was suppressed by insulin. The high concentration of free MeHis in the diabetic muscle was decreased to the normal level with insulin added to the perfusate. These results give further evidence to show that myofibrillar protein degradation is controlled by insulin.

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Two glycerol dehydrogenases, GD-I and GD-II, were purified from Geotrichum candidum by precipitation of ammonium sulfate, sequential column chromatography on Blue-Sepharose CL-4B and DEAE-Sepharose CL-6B and gel filtration on Cellulofine GC-700. The purified enzyme preparations were homogeneous upon disc gel electrophoresis. The molecular weights were estimated to be 135, 000 for GD-I and 130, 000 for GD-II, and their isoelectric points were 5.9 and 6.2, respectively. The specific activity of GD-I was twice that of GD-II. However, their other properties were very similar: Their optimum pHs for the oxidation of glycerol were 10.5, and those for the reduction of dihydroxyacetone were 5.5. The enzyme activities were highly activated by Cl^-, but inhibited by PO₄^3-, BO₃^3- and 2-mercaptoethanol. The enzymes showed highest activity for glycerol, but also acted on several analogs of glycerol.

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The absolute configurations of fenvalerate and other related Cyanohydrin esters were studied by circular dichroism (CD) measurements and by high performance liquid Chromatography (HPLC). Fenvalerate has UV absorption peaks around 278nm associated with the ¹L_b phenyl transitions and corresponding positive CD peaks were observed around 281nm for the enantiomers of (S)-configuration at the Cyanohydrin chiral center. Most of the other Cyanohydrin esters also gave positive CD peaks for the enantiomers of (S)-connguration. CD spectra in the 180 to 250nm range were also studied.
By HPLC, the elution order of the diastereoisomers of Cyanohydrin esters were closely correlated with their absolute configuration and the (RS, SR)-pair consistently eluted earlier than the (RR, SS)-pair for α-substituted phenylacetic acid esters.

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Some kinetic properties of a dipeptidase purified from a cell-free extract of Streptococcus cremoris H 61 were investigated. The Km values of this enzyme for various dipeptides were divided into 3 groups. Group 1 comprised mainly of neutral dipeptides, such as Leu-Gly, Leu-Leu and Leu-Ala, which had relatively low Km values (in the range 4.0-6.6 mM). Group 2 consisted of dipeptides with aromatic large amino acids either at the N- or C-terminal positions, like Leu-Phe, Phe-Ala and Leu-Tyr, which had very low Km values (in the range 1.0-2.4mM). Group 3 was made up by dipeptides with acidic or basic amino acids at the N-terminals; His-Ala and Glu-Val were typical of this group. These had very high Km values (in the range 10-20mM). Substantial substrate competition was found to exist in the presence of His-Ala. Bestatin inhibited the enzyme competitively with Leu-Gly and was found to have an apparent Ki value of 3.0 × 10^-8M for the enzyme. Further, the enzyme was completely inhibited by EDTA at a concentration of 2.0 × 10^-5M. On the other hand, once the activity was inhibited by EDTA, it could be restored by Co²⁺ and Zn²⁺ in the acidic pH side, and by Ca²⁺ and Mn²⁺ in the alkaline pH side.

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The cis-abienol-transformable bacterium JTS-162 was identified as Nocardia restricta. This bacterium has three plasmids (pCA1, pCA2 and pCA3). Two types of cured strains were obtained by mitomicin C treatment or growth at the maximum temperature; one type had two plasmids (pCA2 and pCA3) and the other type had no plasmid. These two types of cured strains lost the ability to oxidize C-18 methyl and to split the A-ring of cis-abienol. These two types of cured strains were also devoid of the. ability to oxidize the C-18 methyl of sclareol. From these results, it was considered that C-18 methyl oxidation and A-ring splitting of these labdanes were determined by plasmid pCA1 in N. restricta JTS-162.

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A system to improve the yield of neoviridogrisein II, in which the cis-4-hydroxy-D-proline is replaced with D-proline, was devised by selective inhibition of the hydroxylation of L-proline with zinc ion in a chemically defined medium. Zinc ion opposed the ferrous ion in the hydroxylation system of Streptomyces griseoviridus P8648.

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To elucidate the role of the methyl substituent on the side chain of abscisic acid (ABA), we synthesized (2Z, 4E)-3-demethyl-α-ionylideneacetic acid (4) and its related analogs, methyl (2Z)-3-demethyl-β-ionylideneacetate 1', 2'-epoxide (9) and methyl (2Z) and (2E)-3-demethyl-abscisate (12) and (13). The biological assay of these compounds suggested that the 3-methyl group on the side chain of ABA was indispensable to biological activity.

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