Studies on chlorophyllase of Chlorella protothecoides IV. Some properties of the purified enzyme

Some properties of chlorophyllase (chlorophyll-chlorophyllido-hydrolase,EC 3.1.1. 14) from Chlorella protothecoides are described. Themolecular weight estimated by sodium lauryl sulfate (SDS)-polyacrylamidegel electrophoresis was 38,000, and the isoelectric point determinedby the isoelectric focusing technique was 4.5. The enzyme hada constant high activity over a wide pH range from 6.0 to 8.5,and was stable from pH 4.0 to 9.2. (Received May 12, 1979; )     

Studies on chlorophyllase of Chlorella protothecoides II. Formation of atypical chlorophyllide a

When chlorophyll a was incubated with a preparation of chlorophyllaseextracted with Triton X-100 from methanol-acetone powder ofChlorella protothecoides, the substrate was changed to chlorophyllidea, and subsequently to an atypical form of chlorophyllide a.The formation of an atypical form of chlorophyllide was notdetected in the reaction with chlorophyll b as substrate, norin the reaction with another preparation of chlorophyllase extractedfrom the algal cells. (Received August 18, 1969; )     

Studies on chlorophyll formation in Chlorella protothecoides III. Effects of chloramphenicol, cycloheximide, and ethionine on chlorophyll formation

Effects of chloramphenicol, cycloheximide, puromycin and ethionineon the light-independent and subsequent light-dependent processesof chlorophyll formation in "glucose-bleached" cells of Chlorellaprotothecoides were studied. These substances, except puromycin,strongly suppressed different phases of chlorophyll formation.Ethionine most strongly suppressed the light-independent phaseand chloramphenicol an early, relatively short process in thelight-dependent phase of chlorophyll formation. Cycloheximideseverely suppressed all phases of chlorophyll formation. Possibleimplications of these results for the biosynthesis of chlorophyllin algal cells are discussed. ¹ Present address: National Food Research Institute, Ministryof Agriculture and Forestry, Koto-ku, Tokyo 135, Japan. ² Laboratory of Entomology, Faculty of Agriculture, TamagawaUniversity, Machida-shi, Tokyo, Japan (Received October 5, 1972; )     

Studies on nucleic acids in chloroplasts isolated from Chlorella protothecoides

1. 1. Subcellular fractions of Chlorella protothecoides were separatedby fractional centrifugation of the algal cell homogenate inmixtures of cyclohexane and CCl₄. The base composition, meltingprofiles and IRC-50 column chromatographic patterns of DNA preparationsfrom the chloroplast and non-chloroplast fractions were examined.It was shown that the algal chloroplast contains at least oneDNA species which is different from the nuclear DNA.
2. 2. RNApreparations from the subcellular fractions were subjectedtoMAK column chromatography, sucrose density gradient centrifugationand analysis for base composition. It was demonstrated thatthe chloroplast contains ribosomal RNA and soluble RNA. Twocomponents of the chloroplast ribosomal RNA were found to havethe same patterns as those of the E. coli ribosomal RNA in MAKcolumn chromatography and zone centrifugation. The major componentof the chloroplast ribosomal RNA was distinctly different fromthat of the non-chloroplastic (cytoplasmic) ribosomal RNA inall properties examined.

¹This work was partly reported at the Symposium on Mitochondriaand Chloroplasts as Self-duplicating Units sponsored by theBotanical Society of Japan in August, 1966, and at the Symposiumon Biogenesis of Subcellular Particles, the 7th Internatl. Congressof Biochemistry, Tokyo, 1967.     

小球藻RubisCO的纯化及其在异养向自养转变过程中的含量变化

经硫酸铵分部沉淀、ＳｅｐｈａｃｒｙｌＳ－３００和ＤＥＡＥ－纤维素柱层析纯化了小球藻ＲｕｂｉｓＣＯ,得率为１５％,比活力达１．２３２μｍｏｌＣＯ２ｍｓ－１ｍｉｎ－１,分子量是５００ｋＤ,它和菠菜叶片ＲｕｂｉｓＣＯ在分子量、亚基组成和免疫特性等方面相似,反映ＲｕｂｉｓＣＯ在高等和低等植物中有较高的同源性。自养小球藻ＲｕｂｉｓＣＯ占细胞可溶性蛋白质的２４％。而异养转变后的小球藻细胞内不含ＲｕｂｉｓＣＯ。异养小球藻向自养生长转变过程中,２０ｈ后细胞内叶绿素含量逐渐增加,２４ｈ时细胞内出现ＲｕｂｉｓＣＯ,２４ｈ后大量增加,至４１ｈ时含量达最高峰;标志着小球藻细胞光合作用能力的恢复和加强。     

Purification and properties of chlorophyllase from greened rye seedlings

1. Chlorophyllase [EC 3.1.1.14] was extracted from the acetone-dried powder of the chloroplasts of greened rye seedlings with 1% cholate, and purified 870-fold with a yield of about 30%. The purification procedure was composed of fractionations with acetone and ammonium sulfate, and hydrophobic chromatography on a phenyl-Sepharose CL-4B column. 2. The purified enzyme was pure as analyzed by molecular-sieve chromatography and isoelectric electrophoresis. It had an isoelectric point of 4.5 and a molecular weight of 39,000. 3. The purified enzyme was stable at pH 6-9 and 4 degrees C. At pH 7.5, it was stable in the presence and absence of 30% acetone. However, at 30 degrees C, it was not stable above a 10% concentration of acetone. 4. The purified enzyme hydrolyzed chlorophylls a and b from spinach into chlorophyllides a and b and phytols, respectively; and bacteriochlorophyll a from Rhodospirillum rubrum into bacteriochlorophyllide a and a derivative of phytol, possibly all-trans-geranylgeraniol. The hydrolysis rates were stimulated to their maxima in the presence of 30% acetone; maximum stimulation was about 50% with bacteriochlorophyll a and about 400% with chlorophyll a. 5. At pH 7.5 and 30 degrees C in the presence of 30% acetone, the Km values and specific activities were 12 microM and 480 nmol . min-1 . mg-1 for chlorophylls a, and 4 microM and 170 nmol . min-1 . mg-1 for R. rubrum bacteriochlorophyll a, respectively.     

Studies on chlorophyllase in tea leaves III. Properties of soluble and insoluble chlorophyllases

In contrast to previous knowledge of chlorophyllase activityin higher plants, significant enzyme activity was isolated fromtea leaves in a soluble state. Soluble chlorophyllase was partially purified by proceduresincluding ammonium sulfate fractionation (Preparation I). Theinsoluble fraction was extracted, by solubilizing it with SDC,from the methanol-acetone powder of sediments of the leaf homogenate,from which the water-soluble enzyme had been completely removedby repeated extraction. This initially insoluble enzyme wasalso partially purified (Preparation II). Specific activities(mg chlorophyll a hydrolyzed per hr per mg protein, 7.2 forPreparation I, and 12.4 for Preparation II), were much higherthan those reported for other plant material. The soluble enzyme was more resistant to PCMB, lipase and heattreatment. The two enzymes differed in optimum temperature andoptimum acetone concentration needed for the reaction, but showedthe same optimum pH, and same Km value. The Km value was thesame (7 µM) for reactions with 30% and 50% acetone. These results suggest that, in spite of differences in locationand extractability, activities of the soluble and insoluble(solubilized) chlorophyllase in tea leaves are attributableto the same enzyme. (Received March 8, 1972; )     

Heterotrophic growth and lipid production of Chlorella protothecoides on glycerol

During the production of biodiesel, a significant amount of glycerol is generated which currently has little commercial value. A study on the growth and lipid production of Chlorella protothecoides using glycerol as the carbon source was performed to demonstrate the utility of recycling crude glycerol created during biodiesel production. Glycerol was examined as both the sole carbon source and in combination with glucose. Algae cultures grown on only glycerol in shake flasks showed a specific growth rate and final lipid yield of 0.1/h and 0.31 g lipid/g substrate, respectively. The values were similar to those observed on pure glucose, 0.096/h and 0.24 g lipid/g substrate. When the media contained a mixture of glycerol and glucose, simultaneous uptake of the two substrates was observed. Due to the difference in rates of lipid storage, lipid production was 0.077 g lipid/(l h) during growth on glycerol, while growth on glucose had a productivity of 0.096 g lipid/(l h). During growth on the 9:1 mixture of both glucose and glycerol, lipid productivity was 0.098 g lipid/(l h). In order to simulate the use of waste glycerol from biodiesel production the experiments were repeated and similar growth rates, yields, and lipid productivities were achieved. Therefore, we have demonstrated the promise for simultaneous high growth rates and lipid yields of C. protothecoides heterotrophically grown on mixtures of glycerol.     

Studies on phospholipases from Streptomyces. III. Purification and properties of Streptomyces hachijoensis phospholipase C.

1. Phospholipase C [EC 3.1.4.3] found in the growth medium of Streptomyces hachijoensis was purified about sixty-fold by dialysis and column chromatography on Sephadex G-50. 2. The active fraction was separated by isoelectric focusing into two fractions, phospholipase C-I (pI 6.0) and phospholipase C-II (pI 5.6). 3. Both purified phospholipases C were homogeneous by immunodiffusion and were not differentiated as regards antigencity. 4. Phospholipase C-I had maximal activity at pH 8.0 and the optimal temperature was 50degree. Phospholipase C-I was stable at 50degrees for 30 min and was stable at neutral pH. 5. The activity of phospholipase C-I was inhibited by high concentrations of various detergents such as Triton X-100, sodium, cholate, SDS and was also inhibited by Ca2+, Ba2+, Al3+, and EDTA, but was stimulated by Mg2+, and ethyl ether. 6. The Km value of phospholipase C-I was 0.9 mM, using phosphatidylcholine as a substrate. 7. By the gel filtration procedure, the molecular weights of phospholipase C-I and -II were both determined to be 18,000. 8. Phosphatidylcholine, phosphatidylinositol, cardiolipin, sphingomyelin, and lysophosphatidylcholine were hydrolyzed by phospholipase C-I, but phosphatidylethanolamine and phosphatidylserine were hydrolyzed with difficulty under the same conditions, Phospholipase C-I also hydrolyzed phosphatidic acid.     

小麦叶绿素酶生化动力学特性研究

采用分光光度法对小麦Chlase的生化性质及其动力学特征进行了部分研究.结果表明:Chlase的Km值为11.6μmol/L,Vmax为1.14μmol·min-1·g-1FW;最适pH为7.5,pH在7～8的范围内,Chlase催化Chl脱植基反应的活性较高;最适温度为45℃,在60℃、70℃、80℃保温时,该酶活力丧失50%所需的时间分别为27min、22min、18min;研究发现不同的金属离子及络合剂对该酶活性有影响,低浓度的Ca2+、Zn2+和较高浓度的Mg2+对Chlase有激活作用,而Fe3+、EDTA则较明显地抑制了Chlase活性;Chlase可催化Chla、b的脱植基反应,对Chlb的亲和性较高.     

Compaitive Study on Liposoluble Compoands in Autotrophic and Heterotrophic Chlorella protothecoides

Both autotrophically and heterotrophically grown Chlorella protothecoides cells have been obtained in cell cultures. The content of liposoluble compounds in the cells of heterotrophic algae occupied 72% of the total cells in dry weight, which was more than 4 times as high as that in the autotrophic algal cells. There existed remarkbly different distribution patterns of the hydrocarbons in thesetwo kinds of cells. The hydrocarbons in autotrophic cells were characterised by the predominance of C17 normal alkanes, wheraes the heterotrophic cells were rich in normal alkanes of higher molecular weight or longer carbon chain with C25 as the dominant carbon. The structure of the compounds in benzene fraction is not quite clear, but the compounds in autotrophis sample may be related to the degeneration of the pigments. The compounds in heterotrophic sample probably come from lipid acids. The visible--ultraviolet absorption spectrum of the pigment compounds demonstrated the absorption peaks of the acetone extract from the autotrophic cells at 432.5, 451.5, 472.5 and 661.5 nm, reflecting the existence of chlorophyll and carotenoid, both with a rather high concentration. However, the acetone extract from the hetertrophic algal cells only showed absorption peaks at 427.4, 450.8 and 477.5 nm. The absorption peaks of the original green cells completely disappeared at 432.5 and 661.5 nm, reflecting the disappearance of chlorophyll in cells on the whole; the remaining absorption peaks only reflected the existence of carotenoid, but its concentration had already been greatly reduced. The resuls from comparative experiments were of essential significance on the study of physiological metabolism in heterotrophically grown C. protothecoides and on the exploration and application of the lipid compounds in this kind of algae.     

Insect apolipophorin III. Purification and properties

The hemolymph of adult Manduca sexta (tobacco hornworm) contains a 17,000-dalton protein that can associate reversibly with the insect lipoprotein lipophorin. The protein is abundant in the hemolymph of the adult, but is found in larval hemolymph in only small amounts, and does not associate with larval lipophorin. On the basis of its association with adult lipophorin, we have designated the protein apolipophorin III. Apolipophorin III was dissociated from adult lipophorin by guanidinium chloride treatment and isolated by gel permeation and ion exchange chromatography. The unassociated apolipophorin III was also purified from lipophorin-free hemolymph by gel permeation, ion exchange, and lectin chromatography. Both preparations have identical isoelectric points and amino acid composition as well as the following properties. Apolipophorin III is a non-glycosylated polypeptide lacking cysteine and tryptophan. The 17,000-dalton polypeptide dimerizes in solution to a protein of Mr = 34,000.     

不同营养条件下原始小球藻对蒽的富集和降解研究

研究了自养与异养条件下原始小球藻对蒽的降解和富集能力 .结果表明 ,自养条件下 ,浓度为1 0mg·L-1的蒽有 48.18%被降解 ,其中 2 8.81%属于自然光降解 ,仅有 19.37%被原始小球藻降解 .而异养条件下的原始小球藻对浓度为 2 .5mg·L-1的蒽降解率达到 33.5 3%,说明异养原始小球藻不仅能耐受高浓度蒽 ,而且表现出比自养原始小球藻更强的蒽降解能力 .两种条件下 ,80 %以上残留的蒽都被富集到藻细胞中 .虽然自养条件下原始小球藻对蒽的生物富集系数达 90 6 4,远大于异养条件下的生物富集系数(1899) ,但异养条件下藻对蒽的绝对富集量 (2 0 2 .2 9μg)远远高于自养条件下的 6 9.6 87μg .     

Mucolipidosis III beta-N-acetyl-D-hexosaminidase A. Purification and properties

Mucolipidosis III acid hydrolases possess an altered carbohydrate recognition marker needed for their lysosomal localization. As a result of this alteration, a portion of these enzymes is secreted from the cell to the extracellular spaces. The structural changes that may have occurred to one of these secreted enzymes, beta-N-acetyl-d-hexosaminidase A (EC 3.2.1.52) were investigated. Normal and mucolipidosis III urinary beta-N-acetyl-d-hexosaminidase A were purified to apparent homogeneity by using affinity [Sepharose-2-acetamido-N-(epsilon-aminocaproyl)-2-deoxy-beta- d-glucopyranosylamine] and ion-exchange (DEAE- and CM-cellulose) chromatography. Sodium dodecyl sulphate/polyacrylamide-slab-gel electrophoresis showed that both enzymes had similar subunit patterns consisting of apparent mol.wts. of 68000, 60000-58000, 55000 and 29000. Differences, however, were noted in the relative proportions of the protein bands where the normal urinary beta-N-acetyl-d-hexosaminidase A contained predominantly the smaller subunits, whereas the mucolipidosis III enzyme had a predominance of the larger subunits. The binding of mucolipidosis III beta-N-acetyl-d-hexosaminidase A to Ricinus communis lectin and concanavalin A with and without endo-beta-N-acetyl-d-glucosaminidase H treatment indicated that the mutation leads to a modification of a portion of the normally occurring high-mannose-type oligosaccharide units to the complex-type. This was further supported by carbohydrate compositional analysis, which revealed a mannose/galactose ratio of 2.1 for the mucolipidosis III beta-N-acetyl-d-hexosaminidase A compared with a ratio of 3.5 for the normal enzyme. Our results indicate that as a result of their inability to be properly localized to the lysosome the majority of the mucolipidosis III lysosomal hydrolase high-mannose oligosaccharide units are further processed to the complex-type before secretion of predominantly higher-molecular-weight subunits from the cell.     

The Respiratory Chain of Chlorella protothecoides: II. Isolation and Characterization of Mitochondria

Mitochondria were isolated from glucose-bleached Chlorella protothecoides Krüger, Indiana strain 25. These mitochondria oxidized succinate, NADH, and l-malate at high rates. Oxygen uptake with these substrates was partially inhibited by 1 mmm-chlorobenzhydroxamic acid (mCLAM). Respiratory control was seen with succinate as substrate in the presence of mCLAM. The apparent Km for succinate was determined to be 0.83 mm. Chlorella mitochondria catalyzed the oxidation of reduced horse heart cytochrome c.     

Studies on red pigments excreted by cells of Chlorella protothecoides during the process of bleaching induced by glucose or acetate I. Chemical properties of the red pigments

During the process of bleaching of Chlorella protothecoidescells induced by the addition of glucose or acetate in a nitrogen-deficientmineral nutrient medium, at least four red pigments were foundto be excreted into the incubation medium. These pigments wereseparated by thin-layer chromatography on silica gel, and theirabsorption spectra in visible and infrared regions as well astheir NMR spectra were measured. Elementary composition andthe Gmelin colour reaction of the pigments were also examined.The results obtained suggest that the four pigments are certainacidic substances—containing a structure similar to thatof a bile pigment—which are most probably derived fromchlorophyll through oxidative rupture of the tetrapyrrole ringof the latter. (Received July 23, 1968; )