Purification and properties of a nitrite reductase from Porphyra yezoensis Ueda

Nitrite reductase was extracted from the red alga Porphyra yezoensisUeda and purified through precipitation with ammonium sulfate,column chromatographies, and polyacrylamide gel disk electrophoresis.The enzyme preparation thus obtained showed a single band ondisk electrophoresis. The absorption spectrum had three maxima at 385 nm (Soret band),580 nm (-band), and 278 nm; the ratio of absorbance of the Soretband to the -band was 4.3. The molecular weight and the numberof amino acid residues were estimated to be 63,000 and 601,respectively. The enzyme activity was optimal at around pH 7.5, and its activitywas heat labile as indicated by reduction of activity by about70% when heated at 37°C for 10 min. The enzyme used ferredoxin and methyl viologen, but not NADP⁺or NAD⁺, as the electron carriers. Moreover, reduced forms ofthe latter two showed no effect on its activity. Km values ofthis enzyme for NO₂^–, Fd, and MV were 8.1 x 10^–4M, 4.3 x 10^–8 M, and 3.7 x 10^–4 M, respectively.Almost half of its activity was lost when potassium cyanidewas added at a concentration as low as 10^–5 M, and theKi value was 1.8 x 10^–5 M. Thus, the nitrite reductaseof Porphyra must be systematically grouped in EC 1.7.7.1 [EC] . Itresembled closely that of Chlorella, except for the amountsof some amino acids. ¹ Present address: Department of Biological Sciences, Universityof Tsukuba, Sakura-Mura, Ibaraki, 300-31 Japan. ² Present address: Department of Fisheries, College of Agricultureand Veterinary Medicine, Nihon University, Shimouma, Setagaya-ku,Tokyo, 154 Japan. (Received June 10, 1975; )     

Nitrite-reducing activity of modified cytochrome c-553 from the red alga Porphyra yezoensis Ueda

Crystalline cytochrome c-553 was obtained from Porphyra yezoensisUeda. The cytochrome in areduced form was modified to show anitrite-reducing activity after appropriate treatment with heat,hydrogen peroxide, or photooxidation using methylene blue asthe electron acceptor, but the reducing activity was far lowerthan that of the nitrite reductase isolated from this alga.The modified cytochrome c-553 was autooxidizable and showedan absorption spectrum resembling that of cytochrome c-553 inthe oxidized form except for slight shifts of the absorptionmaximumin the -band region toward shorter wavelengths. ¹ Present address: Department of Biological Sciences, Universityof Tsukuba, Sakura-Mura, Ibaraki, 300-31 Japan. ² Present address: Department of Fisheries, College of Agricultureand Veterinary Medicine, Nihon University, Shimouma, Setagaya-ku,Tokyo, 154 Japan. (Received June 10, 1975; )     

The enzyme system for the entrance of 14CO2 in the dark CO2-fixation of brown algae

High activity of phosphoenolpyruvate (PEP)-carboxykinase, orADP: oxalacetate (OAA) carboxy-lyase activity (a kind of EC4. 1. 1. 32) was discovered in enzyme extracts or partiallypurified preparations obtained from the brown algae, Eiseniabicyclis, Dictyota dichotoma, Spatoglossum pacificum; and Hizikiafusiformis. Enzyme activities were determined by measuring theradioactivity incorporated in the products of dark ¹⁴CO₂-fixationand by spectrophotometric determinations. Except for the lowactivity of "malic enzyme" (EC 1. 1. 1.40), no activities ofother carboxylases, i.e. PEP-carboxylase, PEP-carboxytransphosphorylase,and pyruvate carboxylase could be detected in algal extractsprepared under various conditions. Malate dehydrogenase (EC1. 1. 1. 37), fumarase (EC 4. 2. 1. 2), and glutamic: oxalacetictransaminase (EC 2. 6. 1. 1) were also detected. The algal PEP-carboxykinase required ADP and Mn²⁺ for maximumactivity in the carboxylation reaction; and ATP and Mn²⁺, butnot GTP, for maximum activity in both the decarboxylation andOAA-¹⁴CO₂-exchange reactions. The optimum pH of purified PEP-carboxykinase was in the regionof 7.0 to 7.3 in both the carboxylation and decarboxylationreactions, and its Km values for HCO₃^–, PEP, and ADP were10 mM, 0.3 mM, and 0.07 mM, respectively, in the carboxylationreaction, and values for OAA and ATP were 0.05 mM and 0.4 mM,respectively, in the decarboxylation reaction. Furthermore,the decarboxylation reaction was markedly inhibited by 20 mMHCO₃^–. The physiological role of PEP-carboxykinase as the enzyme responsiblefor the entrance reaction of the dark CO₂-fixation is discussed. ¹ Contributions from the Shimoda Marine Biological Station ofTokyo Kyoiku University, No. 236. This work was supported inpart by a Grant-in-Aid for Co-operative Research from the Ministryof Education, Japan and Matsunaga Science Foundation (to T.Ikawa). ² Present address: Department of Antibiotics, the National Instituteof Health, Shinagawa, Tokyo, Japan. (Received February 22, 1972; )     

Incorporation of14C-radioactivity into various lipid classes at different growth-stages of culturedLaminaria japonica

A study is reported on the incorporation of¹⁴C-acetate into lipid classes from three different growth stages ofLaminaria japonica, a species long used for food in Japan. This was done because of the possible utilization of its lipids.Radioactivity incorporated into whole lipids in the three growth stages under the same experimental conditions (10 °C, 500 lux) increased with maturity of the thalli. The radioactivity was found mainly in PC, TG and 1,2-DG and subsequently distributed into other lipid classes (PG,PI,PE,MGDG,SQDG and DGDG) to a lesser extent. The incorporation patterns of the former group were similar at all stages, but those of the latter group differed slightly according to growth stage.In juvenile thalli,¹⁴C was incorporated to a much higher extent into PG, MGDG, PI and fucosterol than PE, SQDG, DGDG and MG, while the¹⁴C-incorporation into MG, SQDG, DGDG and PS in the mature growth stage was higher than into the other lipid classes. The absolute level of incorporation was higher for all these compounds in mature thalli than the thalli of other growth stages.     

Changes in M:G ratios of extracted and residual alginate fractions on boiling with water the dried brown alga Kjellmaniella crassifolia (Laminariales,Phaeophyta)

Kjellmaniella crassifolia, the edible macro-brown alga in Japan contained nearly 27% of alginates of which nearly 7% was extractable from the fronds with boiling water for 6 h and the residual alginates in the frond were almost exhaustively extracted with a dilute alkali at 60 °C for 6 h. The alginates dissolved in all these extracts with both boiling water and dilute alkali were purified by fractionation with MgCl₂ and alcohol.The content of MM blocks in the boiling water-soluble alginate sample increased remarkably during heating for 6 h while that of GG blocks from the same sample decreased. In contrast, MM blocks in the alkali-soluble alginate sample decreased during 6 h heating while GG blocks continued to increase. Since the amounts of MG blocks showed slight fluctation, the M:G ratio of alginates extracted with boiling water increased towards the end of extraction whereas the reverse is true for the alkali-soluble alginates.     

Enzymes involved in the last steps of the biosynthesis of mannitol in brown algae

Mannitol-1-phosphate dehydrogenase (EC 1.1.1.17 [EC] ) and mannitol-1-phosphatase(EC number yet unassigned) were detected in the brown algae,Spatoglossum pacificum and Dictyota dichotoma. The enzymes wereextracted from algal fronds and their properties were investigatedusing partially purified preparations. Mannitol-1-phosphatase shows maximum activity at pH 7. The enzymehad a narrow substrate specificity. The Km value for mannitol-1-phosphateis 8.3x10^–4 M (30°C, pH 7.0). The enzyme is activatedby Mg⁺⁺ and Mn⁺⁺and is strongly inhibited by PCMB, Hg⁺⁺and NaF. Mannitol-1-phosphate dehydrogenase showed maximum activitiesat pH values 6.5 and 10.2 in reductive and oxidative reactions,respectively. The dehydrogenase also showed narrow substratespecificity; mannitol-1-phosphate and NAD or fructose-6-phosphateand NADH₂ are utilized, respectively, in oxidative and reductivereactions by the enzyme. Km values for these substrates andthe coenzymes are 2.5x10^–4 M and 7.1x10^–5 M forthe first pair and 2.8x10^–4 M and 1.3x10^–5 M forthe latter pair. This enzyme was strongly inhibited by PCMBand Hg⁺⁺, but was only slightly affected by adenosine phosphates. Possible roles of these enzymes in the biosynthesis of mannitolin brown algae are discussed. ¹ Contributions from the Shimoda Marine Biological Station ofTokyo Kyoiku University, No. 233. This work was supported inpart by a Grant-in-Aid for Co-operative Research from the Ministryof Education, Japan and in part by a grant to one of us (T.Ikawa) from the Matsunaga Science Foundation. ² Present address: Chemical and Physical Laboratory, HoechstJapan Research Laboratory, Minamidai, Kawagoe, Japan. (Received February 22, 1972; )     

Content of arachidonic and eicosapentaenoic acids in polar lipids from Gracilaria (Gracilariales,Rhodophyta)

Fatty acid composition, especially the distribution of eicosapolyenoic acids in several species of Gracilaria, was analyzed in relation to their taxonomy. The species have been grouped into two types based on distribution of these polyenoic acids: Type 1, which contains palmitic, oleic and arachidonic acids as the major components, and Type II, which contains eicosapentaenoic acid in addition to Type I fatty acids. Octadecapolyenoic acids were detected only in trace amounts in each Type. A similar remarkable difference also was observed in the fatty acid composition of lipid classes. The major component of eicosapolyenoic acids in Type I was arachidonic acid in all lipid classes. In Type II, eicosapentaenoic acid was the major component in monogalactosyl diacylglycerol, digalactosyl diacylglycerol, sulfoquinovosyl diacylglycerol and phosphatidylglycerol. Arachidonic and eicosapentaenoic acids were contained in large amounts in Type II phosphatidylcholine. Grouping of Gracilaria species into Type I and Type II is not entirely consistent with morphological and taxonomic features, but the difference in fatty acid composition is likely due to genetic rather than to environmental factors.     

Cellulases of a marine mollusc, Dolabella sp

1. A crude cellulase extract was prepared from the hepatopancreas of a marine mollusc, Dolabella sp., and partially purified by ammonium sulphate fractionation. 2. The optimum pH values of the partially purified preparation were 6.5 and 8.0 for Walseth cellulose and CM-cellulose respectively. It was most stable at pH6.0 and showed moderate thermostability. 3. The partially purified preparation was subjected to starch-zone electrophoresis, and incompletely resolved into several fractions that contained one or more cellulase components of different substrate specificity. 4. Some of these cellulase fractions showed practically no aryl beta-glucosidase activity and hydrolysed aryl beta-cellobioside with difficulty. From substrates such as higher cello-oligosaccharides, cellodextrin, CM-cellulose, Walseth cellulose and cotton fibre, they produced cellobiose as the major and cellotriose as the minor end products, both of which were resistant to further attack by cellulase. 5. From the slope of the curves of viscosity-reducing power for CM-cellulose, the cellulase components from Dolabella were presumed to be of a ;more-random' or a ;less-random' type in the mode of action. 6. In the hepatopancreas of this mollusc, beta-glucosidases were also present, which hydrolysed cellobiose as well as aryl beta-glucosides. The optimum pH values of these enzymes were about 5.5.