Biochemical Characterization of Secreted Proteases During Growth in Tetrahymena pyriformis WH-14: Comparison of Extracellular with Intracellular Proteases

Tetrahymena pyriformis strain WH-14 secreted large quantities of intracellular proteases into its culture medium during growth. Extracellular enzymes were purified to homogeneity from cell-free medium by ammonium sulfate precipitation, CM-Sephadex column chromatography, gel filtration, and DEAE-cellulose column chromatography. The DEAE-cellulose eluates were separated into four peaks (P-I, P-II, P-III, and P-IV), each of which exhibited a different specific activity toward azocasein and α-N-benzoyl-DL-arginine-ρ-nitroanilide (Bz-Arg-Nan). These four forms of the protease showed similarity in amino acid composition, molecular weight (21,000–24,000), and antigenic reactivity. They had pH optima at neutral range. P-I showed the highest specificity to azocasein whereas P-IV was most effective toward the synthetic substrates. The Km values for hydrolysis of Bz-Arg-Nan were 2.4, 1.6, 1.3, and 1.4 mM for P-I, P-II. P-III, and P-IV, respectively, and the corresponding Kcat/Km values were 5.0, 9.4, 28.5, and 114.3 S^-1.M^-1. These properties of secreted proteases were compared with those of intracellular proteases purified by the same procedure except for the initial Triton X-100 extraction. There were similarities in specific activity toward two substrates, molecular weight, Km, pH optima, and antigenic reactivity between the proteases from two sources, providing evidence that the intracellular proteases may be secreted into the extracellular medium without modification.     

Correlation Between the Molecular Structure and the Biological Activity of Co-ARIS, a Cofactor for Acrosome Reaction-Inducing Substance

Two components in the egg jelly are required for inducing the acrosome reaction in starfish; a sulfated glycoprotein called acrosome reaction-inducing substance (ARIS) and its cofactor called Co-ARIS. Three distinct molecules were isolated as the major Co-ARIS' and designated as Co-ARIS' I, II and III. Structural analysis of Co-ARIS' revealed that they are steroidal saponins comprising a sulfated steroid and a pentasaccharide chain. Co-ARIS' I and II differ only in the steroidal side chain. In the presence of ARIS, each Co-ARIS induced the acrosome reaction with a maximal effect at 100–200 μM (Co-ARIS I) or 25–50 μM (Co-ARIS' II and III). Mixtures of Co-ARIS' I, II and III were more effective than the individuals. The activity of Co-ARIS was considerably reduced by solvolytic desulfation but was not affected at all by periodate oxidation. Reduction by NaBH₄ decreased the activity of Co-ARIS I and enhanced that of Co-ARIS II. Treatment of Co-ARIS III with NaBH₄ did not affect the activity as anticipated from its structure. These results suggest that the sulfate moiety and the side chain of steroid are important for the activity of Co-ARIS. The saccharide chain, however, seems not necessarily to be strictly specified for the activity.     

Optical emission spectrographic studies on the distribution and accumulation of nanogram amounts of 15N in rice seedlings

An optical emission spectrographic method was used to studythe distribution and accumulation of nanogram quantities of¹⁵N in leaves and roots of individual rice plants. Gaseous nitrogenthat was prepared from plant samples, not weighing more than100 µg, was trapped in an electrodeless tube. The excess¹⁵N was determined by exciting the gas with a high frequencygenerator, then measuring the light intensities of the molecularbandheads at 2977, 2983 and 2989 ?. For leaves that contained4 atom % ¹⁵N the precision obtained was ?1.33%. A comparisonof emission and mass spectrographic ¹⁵N values were obtainedwith a number of second leaves of rice plants that were grownunder similar environmental conditions. The ¹⁵N contents obtainedat a 2 atom % level by the 2 methods were in reasonable agreement. ¹Permanent address: University of Tokyo, Dept. of Agr. Chem.,Japan. ²Permanent address: Argonne National Lab., Argonne, Illinois,U. S. A. (Received April 2, 1969; )     

Studies on the change of the respiratory system in roots in relation to the growth of plants II. Activity of iron-containing oxidases in roots of cereal crops with the aging of plants

Distribution of iron-containing oxidases in aging nodal rootsof rice and wheat was studied. Activities of cytochrome c oxidase(1.9.3.1 [EC] , cytochrome c : O₂ oxidoreductase), catalase (1.11.1.6 [EC] ,H₂O₂: H₂O₂ oxidoreductase) and peroxidase (1.11.1.7 [EC] , donor:H₂O₂ oxidoreductase) in wheat roots were comparatively higherthan were those in rice roots at corresponding stages. Cytochromec oxidase in roots remained active throughout the lives of therice and wheat crops. In rice roots, catalase seemed to playa distinct role around the panicle formation stage. Decay ofcatalase activity took place earlier than did that of peroxidaseand cytochrome c oxidase activities. In wheat roots similarenzyme activity changes were not observed. Data may suggestthat the high activity of iron containing oxidases at the panicleformation stage (I) may be chiefly due to catalase activityin rice roots. ¹Paper presented at the 14th Annual Meeting of the Society ofthe Science of Soil and Manure, Japan (1968). (Received November 21, 1968; )     

Purification and Characterization of Phospholipase C Preferentially Hydrolysing Phosphatidylcholine in Tetrahymena Membranes

A phospholipase C (PLC) activity that preferentially hydrolyses phosphatidylcholine to diacylglycerol and phosphorylcholine was found to be present in Tetrahymena pyriformis, strain W and most of its activity was recovered in the membrane fraction. This enzyme was extracted with 1% Triton X-100 from the membrane fraction and purified to apparent homogeneity by sequential chromatographies on Fast Q-Sepharose, hydroxyapatite HCA-100S, Mono Q and Superose 12 gel filtration columns. The purified enzyme had specific activity of 2083 nmol of diacylglycerol released/mg of protein/min for dipalmitoylphosphatidylcholine hydrolysis. Its apparent molecular mass was 128 kDa as determined by SDS-polyacrylamide gel electrophoresis and was 127 kDa by gel filtration chromatography, indicating that the enzyme is present in a monomeric form. The enzyme exhibited an optimum pH 7.0 and the apparent K_m value was determined to be 166 μM for dipalmitoylphosphatidylcholine. A marked increase was observed in phosphatidylcholine hydrolytic activity in the presence of 0.05% (1.2 mM) deoxycholate. Ca²⁺ but not Mg²⁺ enhanced the activity at a concentration of 2 mM. This purified phospholipase C exhibited a preferential hydrolytic activity for phosphatidylcholine but much less activity was observed for phosphatidylinositol (～ 9%) and phosphatidylethanolamine (～ 2%).     

Ecological distribution and phenology of an invasive species, Cardamine hirsuta L., and its native counterpart, Cardamine flexuosa With., in central Japan

Cardamine hirsuta is a European species that was recently introduced into Japan and its wide distribution has been confirmed in the Kanto district. To understand mechanisms of the recent spread of C. hirsuta in Japan, a comparative study of the alien species and its native congeneric species, C. flexuosa, was conducted. Habitat preferences, phenology and seed germination were examined. Cardamine hirsuta and C. flexuosa showed distinctive habitat-preferences; the former was most common in open habitats created by recent man-made constructions, and the latter was common in rice paddy fields and surrounding areas. The results indicate that C. flexuosa is a year-long annual, with a mixed phenology of summer and winter germination and growth. Seed dormancy during summer was relatively weak for C. flexuosa, and some plants that germinated early in summer reproduced during the same summer–autumn period. Plants that germinated in late summer and autumn behaved as winter annuals. In rice paddy fields, C. flexuosa is a winter annual because germination is prevented by submergence during summer. Plants flower during the following spring and complete their life cycle before the fields are flooded for rice cultivation. Cardamine hirsuta showed strong seed dormancy during summer and behaved as a typical winter annual. Seeds of C. hirsuta were intolerant to submergence in water, a condition that breaks seed dormancy of C. flexuosa. The results explain the absence of C. hirsuta from rice paddy fields. It was concluded that the spread of C. hirsuta is attributable to the recent expansion of urban habitats created by human activity and has occurred without direct competition with C. flexuosa. Considering recent urbanization in many areas, it is suggested that C. hirsuta has been spreading rapidly in Japan.     

Determination and Regulation Capacities of the Wing Disc of the Fleshfly, Sarcophaga peregrina

Unilateral extirpation of the wing discs was performed on mid- and late-third instar larvae of Sarcophaga peregrina to investigate the regulation capacities of the thoracic discs. Out of 636 operated larvae, 175 became adult and all of them exhibited hemithoracic structures, lacking any adult structures in the operated side, which were normally produced from a wing disc. These findings suggest that the wing disc does not possess the capacity of bicentric regulation to produce the thoracic structures of the opposite side.     

Methane-flux-dependent lateral faunal changes in a Late Cretaceous chemosymbiotic assemblage from the Nakagawa area of Hokkaido, Japan

A Late Cretaceous carbonate body (2 m in maximum diameter) surrounded by clastic rocks, recently discovered in the Nakagawa area (Hokkaido, Japan), is interpreted as a methane‐seep deposit, on the basis of negative carbon isotopic composition (as low as ?43.5‰), variable sulphide sulphur isotopic composition, high carbonate content, and in situ fractures. It most likely formed owing to methane‐bearing pore‐water diffusion. We estimate that the concentration of methane decreased toward the margin of the carbonate body, and that only small carbonate concretions were precipitated at a certain distance from the methane‐seep centre. These spatial characteristics coincide well with the observed pattern of faunal distribution. The gastropod‐dominated association (indeterminate abyssochrysids and ataphrids and the acmaeid limpet Serradonta sp. are most common) co‐occurs with lucinid and thyasirid bivalves (Thyasira sp., Myrtea sp., and Miltha sp.), and was found within and just above the methane‐derived carbonate body. Acharax and Nucinella (solemyoid bivalves) are more typical of the peripheral part of the methane‐influenced sediments. We suggest that this pattern of faunal distribution reflects the decreasing concentration of methane and apparently also hydrogen sulphide when moving from the centre of discharge toward the periphery of the methane seep.     

Studies on the change of the respiratory system in roots in relation to the growth of plants V. Effect of root pruning terminal oxidase activity in roots of cereal crops

This experiment studies the comparative activities of terminaloxidases (Fe-containing oxidases, Cu-containing oxidases andCN-resistant oxidases) in roots of rice and wheat seedlingsand the effect of root pruning on these terminal oxidases inthe roots of those crops. Data indicate that (i) excepting achange in the activity of Cu-containing oxidases no appreciabledifference in respiration or in Fe-containing and CN-resistantoxidases was noticed in the two types of seedling roots and(ii) root pruning did not produce any noticeable change in terminaloxidase activities in roots of wheat seedlings. A drastic changein terminal oxidase activities in roots of rice seedlings wasobserved, suggesting that a close relationship exists betweenCN-resistant oxidase and transplanting time. (Received November 21, 1968; )