A STATISTICAL CHARACTERIZATION OF GROWTH AMONG CLONES OF SYNURA PETERSENII (SYNUROPHYCEAE)1

Each of four clones from the Synura petersenii complex was grown at different pHs (5.5, 6.5, 7.5, 8.5) in batch culture experiments. Growth response curves and exponential growth rates were compared among clones and pH treatments in order to examine growth trend variation among the clonal groups. The clones were isolated from geographically distant North American localities. The clonal groups represented distinct mating types, an isolate and its subisolate, and S. petersenii- and S. glabra-like scale morphologies. No consistent relationship existed between growth response curve, and culture medium pH. Additionally, the trends across time differed according to clone and pH combination. Pairwise comparisons of linear trends from transformed growth response curves indicated two distinct clonal associations. Although the clonal associations corresponded with the final cell density of the cultures, growth response curves did not correspond with mating type, the parent-isolate and subisolate, or scale morphology. Clones with glabra-like scales had greater growth rates than the clone with petersenii-like scales. The conflicting results generated from growth response curve and growth rate analyses support the concept that S. petersenii and S. glabra form a highly variable, homogeneous grouping.     

HETEROTHALLIC SEXUALITY AND DENSITY DEPENDENT ENCYSTMENT IN THE CHRYSOPHYCEAN ALGA SYNURA PETERSENII KORSH.1

Sexual reproduction of the common planktonic chrysophyte Synura petersenii is described from observations made on clonal isolates grown in defined culture. Sexual fusion was isogamous and heterothallic, with cells of normal appearance from compatible clones serving as hologametes. No special culture conditions were required to induce sexual behavior; actively growing cell populations appeared to be continually receptive to mating when mixed with a sufficient number of cells from a compatible clone. A single, bipolar mating group was documented containing five of the seven clones tested. Zygotic statospores were found to be binucleate and to contain 4 chloroplasts at maturity. Production rates of zygospores were low for even the most highly compatible clones, with batch culture yields ranging from 1-20% of final cell density under the culture conditions utilized. Six of the clones tested were also capable of very low frequency (0.001-0.01%) homothallic statospore production but the reproductive significance of these cysts remains enigmatic. The dynamics of sexual encystment suggest that the process proceeds during periods of active population growth and is density dependent. Based on the characteristics of cyst induction and encystment dynamics, it is concluded that chrysophycean flagellates may have a perennation strategy quite different from that of the majority of planktonic diatoms, dinoflagellates, and green algae for which resting cyst production requires an exogenous trigger usually associated with physiological stress and periods of negative growth.     

THE REQUIREMENT FOR SILICON IN SYNURA PETERSENII (CHRYSOPHYCEAE)1,2

A silicon requirement for growth is demonstrated for an alga other than a diatom. Concentrations of less than 1 μM silicate greatly decreased the growth rate of Synura petersenii Korshikov and caused morphological changes. Half of maximum growth rate (μ_max= 1.12 divisions/day) appeared at a concentration of only 0.23 μM silicate. Germanium dioxide inhibited, growth considerably; the degree of inhibition varying from none to almost 100% depending upon both Si and Ge concentrations. The amount of silicon deposited in the external scales of S. petersenii is comparable per unit area to that of diatoms. S. petersenii is able to deplete the medium of silicate to very low levels. The feasibility of batch culture techniques for this kind of work is discussed briefly.     

COLINEARITY OF CHLOROPLAST GENOMES IN DIVERGENT ECOTYYES OF THE MARINE DIATOM SKELETONEMA COSTATUM (BACILLARIOPHYTA)1

The chloroplast genomes of three isolates of the marine diatom Skeletonema costatum (Grev.) Cleve were mapped and found to be 131 ± 2 kb with inverted repeats (IRs) of approximately 20 kb. In contrast to higher plants, the psbA gene mapped to the IR, and rbcS mapped to the same fragment as the rbcL gene in the large single-copy region. The maps of the three isolates were colinear and revealed as many as 20 site mutations out of a total of 47 sites. The number of site mutations among isolates was consistent with previous data on their genetic diversity and physiology. Comparisons of gene order among our maps and those of three other diatom species showed that closely related genera retained similar gene orders but that more distantly related taxa exhibited extensive rearrangements. We conclude that simple restriction fragment analysis of chloroplast DNA is useful in comparative studies of diatom populations and species but that other analytical methods are more appropriate for phylogenetic studies at higher levels.     

SEXUALITY AND UNIPARENTAL INHERITANCE OF CHLOROPLAST DNA IN THE ISOGAMOUS GREEN ALGA ULVA COMPRESSA (ULVOPHYCEAE)1

Ulva compressa L. is a heterothallic macroalga considered to be in the early evolutionary stage between isogamy and anisogamy. Two genetic lines of this species, each consisting of gametophytes with opposite mating types, were collected on the coasts of Ehime and Iwate prefectures: MGEC‐1 (mt⁺) and MGEC‐2 (mt^?) from Ehime, and MGEC‐5 (mt⁺) and MGEC‐6 (mt^?) from Iwate. Their relative gamete sizes (i.e., cell volumes) do not correspond to their mating types: MGEC‐6 (19.8 μm³) > MGEC‐1 (18.6 μm³) > MGEC‐5 (17.0 μm³) > MGEC‐2 (10.1 μm³). The pattern of organelle inheritance is an important sexual characteristic in many eukaryotes. We therefore investigated the relationship between gamete size and the inheritance of chloroplast DNA (cpDNA). Polymorphisms between the cpDNA of the two lines were used as markers. We found a 24 bp insertion between psbF and psbL, and the substitution of a StyI site (from C CAAGG to T CAAGG) in the intergenic region between petD and accD. Two interline crosses (MGEC‐1 × MGEC‐6 and MGEC‐2 × MGEC‐5) produced 42 and 38 zygotes, respectively. PCR and PCR–RFLP analyses showed that the cpDNA of the mt⁺ gametes was consistently inherited in both crosses. The cpDNA is inherited from one parent only, and it depends not on gamete size but on being mt⁺. The cpDNA was observed during crossing and in the zygotes 6 h after mating. In 6% of the zygotes, the cpDNA derived from the mt^? gametes disappeared 3–4 h after mating. Preferential digestion of the cpDNA in the zygote’s mt^? gamete may form the basis for uniparental inheritance of cpDNA.     

ORGANIZATION OF THE CHLOROPLAST GENOME OF THE FRESHWATER CENTRIC DIATOM CYCLOTELLA MENEGHINIANA1

We constructed a complete physical map and a partial gene map of the chloroplast genome of Cyclotella meneghiniana Kützing clone 1020-1a (Bacillariophyceae). The 128-kb circular molecule contains a 17-kb inverted repeat, which divides the genome into single copy regions of65 kb and 29 kb. This is the largest genome and inverted repeat found in any diatom examined to date. In addition to the 16S and 23S ribosomal RNA genes, the inverted repeat contains both the ndhD gene (as yet unexamined in other diatoms) and the psbA gene (located similarly in one of two other examined diatoms). The Cyclotella chloroplast genome exists as two equimolar populations of inversion isomers that differ in the relative orientation of their single copy sequences. This inversion heterogeneity presumably results from intramolecular recombination within the inverted repeat. For the first time, we map the ndhD, psaC, rpofi, rpoCl, and rpoC2 genes to the chloroplast genome of a chlorophyll c-containing alga. While the Cyclotella chloroplast genome retains some prokaryotic and land plant gene clusters and operons, it contains a highly rearranged gene order in the large and small single copy regions compared to all other examined diatom, algal, and land plant chloroplast genomes.     

SILICEOUS SCALE PRODUCTION IN CHRYSOPHYTE AND SYNUROPHYTE ALGAE. I. EFFECTS OF SILICA-LIMITED GROWTH ON CELL SILICA CONTENT,SCALE MORPHOLOGY,AND THE CONSTRUCTION OF THE SCALE LAYER OF SYNURA PETERSENII1

The cells of synurophyte flagellates (algal class Synurophyceae, formerly included in the Chrysophyceae) are enclosed within a regularly imbricate layer of ornamented siliceous scales. Scale morphology is of critical taxonomic importance within this group of algae, and the scales are valuable indicator microfossils in paleolimnological studies. The data presented here demonstrate that scale morphology and the integrity of the scale layer can exhibit extreme variability in culture as a function of the cellular quota of silica under silica-limited growth. Silica-limited, steady-state populations of the colonial flagellate Synura petersenii Korsh. were maintained over a range of specific growth rates (μ= 0.11–0.69 days^?1) and silica cell quotas (Q_si= 0.13–2.40 pmoles Si · cell¹). Scale morphology and the organization of the scale layer became increasingly aberrant as silica stress increased. Under severe stress, scale deposition was completely suppressed so that cells appeared scale-free. This depression of scale deposition was reversible; populations of silica-starved, scale-free cells rapidly regenerated new scale layers when placed in batch culture and spiked with dissolved silica. During recovery from silica stress, cell division was repressed for 24 h while mean cell silica quota increased 25-fold. The first new scales appeared within 2 h after the silica addition, and development of the new scale layer proceeded in an approximately synchronous manner, residting in normal scale layers on virtually all cells after 48 h of recovery in Sirich medium. Silica content of silica-replete Synura cells is comparable to freshwater diatoms of siynilar size, but Synura has much greater potential quota variability than diatoms and no apparent threshold silica requirement. Silica-limited growth kinetics and competition between diatoms and Synura for silica are discussed. The results suggest that morphological variability of siliceous scales in natural populations of synurophyte flagellates may result from silica stress and that the experimental approach developed here has great potential value as a means for circumscribing ecotypic variation in scale morphology. Results also demonstrate that scale production can be uncoupled from cell division, suggesting that cell cycle regulation of silica biomineralization in the Synurophyceae may be fundamentally different from that of diatoms (algal class Bacillariophyceae). This experimental system has application in the future study of the intracellular membrane systems and the regulatory processes involved in silica biomineralization.     

MOLECULAR PHYLOGENY OF THE GENUS CAULERPA (CAULERPALES,CHLOROPHYTA) INFERRED FROM CHLOROPLAST tufA GENE1

The genus Caulerpa consists of about 75 species of tropical to subtropical siphonous green algae. To better understand the evolutionary history of the genus, a molecular phylogeny was inferred from chloroplast tufA sequences of 23 taxa. A sequence of Caulerpella ambigua was included as a potential outgroup. Results reveal that the latter taxon is, indeed, sister to all ingroup sequences. Caulerpa itself consists of a series of relatively ancient and species‐poor lineages and a relatively modern and rapidly diversifying clade, containing most of the diversity. The molecular phylogeny conflicts with the intrageneric sectional classification based on morphological characters and an evolutionary scheme based on chloroplast ultrastructure. High bootstrap values support monophyly of C. mexicana, C. sertularioides, C. taxifolia, C. webbiana, and C. prolifera, whereas most other Caulerpa species show para‐ or polyphyly.     

ROLE OF MULTIPLE FTSZ RINGS IN CHLOROPLAST DIVISION UNDER OLIGOTROPHIC AND EUTROPHIC CONDITIONS IN THE UNICELLULAR GREEN ALGA NANNOCHLORIS BACILLARIS (CHLOROPHYTA,TREBOUXIOPHYCEAE)1

Chloroplasts of the unicellular green alga Nannochloris bacillaris Naumann cultured under nutrient‐enriched conditions have multiple rings of FtsZ, a prokaryote‐derived chloroplast division protein. We previously reported that synthesis of excess chloroplast DNA and formation of multiple FtsZ rings occur simultaneously. To clarify the role of multiple FtsZ rings in chloroplast division, we investigated chloroplast DNA synthesis and ring formation in cells cultured under various culture conditions. Cells transferred from a nutrient‐enriched medium to an inorganic medium in the light showed a drop in cell division rate, a reduction in chloroplast DNA content, and changes in the shape of chloroplast nucleoids as cells divided. We then examined DNA synthesis by immunodetecting BrdU incorporated into DNA strands using the anti‐BrdU antibody. BrdU‐labeled nuclei were clearly observed in cells 48 h after transfer into the inorganic medium, while only weak punctate signals were visible in the chloroplasts. In parallel, the number of FtsZ rings decreased from 6 to only 1. When the cells were transferred from an inorganic medium to a nutrient‐enriched medium, the number of cells increased only slightly in the first 12 h after transfer; after this time, however, they started to divide more quickly and increased exponentially. Chloroplast nucleoids changed from punctate to rod‐like structures, and active chloroplast DNA synthesis and FtsZ ring formation were observed. On the basis of our results, we conclude that multiple FtsZ ring assembly and chloroplast DNA duplication under nutrient‐rich conditions facilitate chloroplast division after transfer to oligotrophic conditions without further duplication of chloroplast DNA and formation of new FtsZ rings.     

植物叶绿体4.5S核糖体rRNA作为分子杂交探针的研究

我们提取纯化了芹菜,菠菜和蕃茄叶绿体核糖体4.5SRNA(4.5SrRNA)并在其5’端标记~(32)P,作为探针与菠菜,蕃茄和芹菜叶绿体DNA(ctDNA)进行分子杂交。结果不仅证明4.5SrRNA可作为公用分子杂交探针,而且也说明不同植物4.5SrRNA序列有相当大的同源性。     

MOLECULAR DIVERGENCE AND CHARACTERIZATION OF TWO CHLOROPLAST DIVISION GENES,FTSZ1 AND FTSZ2, IN THE UNICELLULAR GREEN ALGA NANNOCHLORIS BACILLARIS (CHLOROPHYTA)1

Two FtsZ paralogues (NbFtsZ1 and NbFtsZ2) were isolated from the unicellular green alga Nannochloris bacillaris Naumann. These sequences encoded proteins of 435 and 439 amino acids with tubulin signature motifs (GGGTG[T/S]G), which are important for GTP binding activity. NbFtsZ1 and NbFtsZ2 had four and three introns, respectively, and two different putative core promoters; a TATA box (TATAAAA) and an initiator element (CCCAGG) were located 40 bp and 80 bp upstream of the coding regions of NbFtsZ1 and NbFtsZ2, respectively. Southern blot hybridization and contour‐clamped homogeneous electric field electrophoresis showed that N. bacillaris contained at least one copy of each gene and that NbFtsZ1 was located on chromosome 5 and NbFtsZ2 on chromosome 3 or 4. Phylogenetically, NbFtsZ1 and NbFtsZ2 belong to the vascular plant protein families FtsZ1 and FtsZ2, respectively. The FtsZ1 proteins do not contain carboxy‐terminal consensus sequences, whereas all FtsZ2 proteins possess the consensus sequence (I/V)PxFL(R/K)(K/R)(K/R). Our study has shown that NbFtsZ2 possesses a similar consensus sequence (VPDFLRRK), whereas NbFtsZ1 does not, further supporting their classification as FtsZ2 and FtsZ1. Escherichia coli ftsZ mutants transformed with cloned NbFtsZ1, and NbFtsZ2 cDNAs were restored for the capacity to divide by binary fission, suggesting that the proteins retained the ability to function in the bacterium. An anti‐NbFtsZ2 antibody specifically recognized a single protein band of approximately 51 kDa on an immunoblot of N. bacillaris cellular proteins. Immunostaining of the algal cells with this antibody produced an intense fluorescent signal as a ring near the middle of the cell, which corresponded to the chloroplast division site.     

A PRELIMINARY COMPARISON OF RESTRICTION FRAGMENT PATTERNS IN THE GENUS CAULERPA (CHLOROPHYTA) AND THE UNIQUE STRUCTURE OF THE CHLOROPLAST GENOME OF CAULERPA SERTULARIOIDES1

Comparisons of chloroplast DNA restriction fragments in four species of Caulerpa revealed that patterns between the species were different, with few and possibly no homologous bands. Two forms of Caulerpa sertularioides also revealed different patterns, and it is possible that the forms are separate species. The chloroplast genome in Caulerpa sertularioides f sertularioides (S. G. Gmelin) Howe is 131.4 kb in size and lacks large repeat units. The discovery of another green-algal chloroplast genome that lacks an inverted repeat indicates that this feature is either not ancestral to the Chlorophyceae or has been lost several times. Several gene clusters commonly found in chloroplast DNAs were found to occur in Caulerpa chloroplast DNA, for example, psbD/C, atpF/H, and psaA/B. The 16S and 23s rRNA, which are typically adjacent, contained in an inverted repeat, and cotranscribed, are over 40 kb apart. Genes rps12 and tufA, members of the str operon in eubacteria, are over 50 kb in distance from each other in Caulerpa. The gene order in Caulerpa is unlike any other chloroplast genome characterized to date.     

FRACTIONATION OF NUCLEAR,CHLOROPLAST, AND MITOCHONDRIAL DNA FROM POLYSIPHONIA BOLDII (RHODOPHYTA) USING A RAPID AND SIMPLE METHOD FOR THE SIMULTANEOUS ISOLATION OF RNA AND DNA1

Extraction of nucleic acids from red algae is complicated by the presence of phycocolloids. For this reason, methods used for nucleic acid isolation from other organisms are not always amenable to use with red algal preparations; modifications in some cases lead to protocols that are time consuming and complicated, often requiring large amounts of algal tissue for starting material. Here we describe the isolation of both RNA and DNA followed by fractionation and identification of nuclear, chloroplast, and mitochondrial DNAs from a single preparation of Polysiphonia boldii Wynne and Edwards using a simple method that yielded approximately 100 μg of total RNA and 20 μg of total DNA from 1 g of frozen powdered algae. The potent protein denaturant guanidinium thiocyanate and the detergent sarkosyl were used to gently lyse the cells and organelles and immediately inhibit nuclease activity in the extract. The nucleic acids were isolated by ultracentrifugation into a dense solution of CsCl; the RNA was recovered as a pellet and the DNA as a band within the CsCl solution. Agarose gel electrophoresis of the total RNA showed discrete ribosomal RNA bands, indicating little nonspecific degradation. The nuclear, chloroplast, and mitochondrial DNAs were fractionated by density gradient ultracentrifugation in the presence of the DNA binding dye, bisbenzimide H (Hoechst 33258), which binds preferentially to DNA with a high A + T:G + C ratio, thus altering its density to a greater degree than it does that of DNA with a lower nucleotide ratio. The three fractions were identified by Southern blot analysis using heterologous gene probes specific for the different genomes. The protocol should be applicable to different types of algae. The simple nucleic acid isolation step can be performed on multiple samples simultaneously without subsequent fractionation of DNA, allowing comparison of DNA from different individuals, populations, or species.     

MORPHOLOGICAL CHANGES IN MITOCHONDRIAL AND CHLOROPLAST NUCLEOIDS AND MITOCHONDRIA DURING THE CHLAMYDOMONAS REINHARDTII (CHLOROPHYCEAE) CELL CYCLE1

Morphological changes in the organellar nucleoids and mitochondria of living Chlamydomonas reinhardtii Dang were examined during the cell cycle under conditions of 12:12 light:dark. The nucleoids were stained with SYBR‐Green I, and the mitochondria were stained with 3,3‐dihexyloxacarbocyanine iodide. An mocG33 mutant, which contains one large chloroplast nucleoid throughout the cell cycle, was used to distinguish between the mitochondrial and chloroplast nucleoids. Changes in the total levels of organellar DNA levels were assessed by real‐time PCR. Each of the G₁, S, M, and Smt,cp phases was estimated. At the start of the light period, the new daughter cells were in G₁ and contained about 30 mitochondrial and 10 chloroplast nucleoids, which were dispersed and had diameters of 0.1 and 0.2 μm, respectively. During the G₁ phase of the light period, and at the start of the S phase, both nucleoids formed short thread‐like or bead‐like structures, probably divided, and increased continuously in number, concomitantly with DNA synthesis. The nucleoids probably became smaller due to the decrease in DNA of each particle and were indistinguishable. The cells in the S and M phases contained extremely high numbers of scattered nucleoids. However, in the G₁ phase of the dark period, the nucleoids again formed short thread‐like or bead‐like structures, probably fused, and decreased in number. The mitochondria appeared as tangled sinuous structures that extended throughout the cytoplasm and resembled a single large mitochondrion. During the cell cycle, the numbers of mitochondrial nucleoids and sinuous structures varied relative to one another.     

THE LACK OF CHLOROPLAST DNA IN ACETABULARIA MEDITERRANEA (ACETABULUM) (CHLOROPHYCEAE): A REINVESTIGATION1

Three features of chloroplast DNA (cpDNA) in plastids isolated from Acetabularia mediterranea (acetabulum) were analyzed after staining the organelles with the fluorochrome 4′6-diamidino-2-phenyl indole (DAPI): (1) number of chloroplasts exhibiting DNA fluorescence, (2) number of nucleoids per plastid, and (3) nucleoid morphology. In vegetative Acetabularia cells only half of the total chloroplast population comprising several millions displayed the whitish-blue fluorescence of the DNA/DAPI complex. This percentage remained stable independent of whether cells were grown in supplemented natural sea water or enriched synthetic sea water. A single nucleoid, widely differing in size and morphology among the organelles, was characteristic of 76–81% of chloroplasts with DNA. Less than 20% contained two nucleoids, and in rare cases three or four nucleoids were present. The pattern of nucleoid numbers followed a Poisson distribution in one experiment, if calculated with the intrinsic mean of the observed data. In two other experiments, however, a significant difference existed between observed and expected values for a Poisson distribution according to the Chisquared test. After secondary enlargement of portions of the negatives, the nucleoids’substructure was disclosed and found to consist of brightly fluorescent spots interspersed by unstained regions The lack of cpDNA in Acetabularia cells appears to be brought about by (1) the polarized pattern of growth and translation confined to the apical region of the single cell and (2) the cpDNA arrangement in a single nucleoid acentrically located in the organelle. A scheme for the evolution of a chloroplast population having plastids without DNA is proposed. In theory the lack of cpDNA could arise in each plant, since chloroplasts never evolved a mitotic-like spindle to ensure the equal distribution of genetic material. The different nucleoid arrangement in most other plants, however, efficiently counteracts this ‘carelessness of nature’     

STRUCTURAL CHARACTERIZATION OF THE TERMINAL DOMAINS OF LINEAR PLASMID‐LIKE DNA FROM THE GREEN ALGA ERNODESMIS (CHLOROPHYTA)1

Terminal regions of linear plasmid‐like DNA molecules from chloroplasts of the green alga Ernodesmis verticillata (Kützing) Børgesen were cloned and structurally characterized. Phosphorylation experiments with polynucleotide kinase indicated the presence of a 5′‐phosphate, but the data did not reveal any protective protein(s) at the 5′ end. To characterize the 3′ end of these molecules, homopolymer‐ (poly(G)‐) tailed molecules were annealed to and cloned into a linearized vector that was poly(C) tailed with terminal deoxynucleotidyl transferase. Sequencing analyses verified the heterogeneous nature of the molecules. Two distinct clones displayed extensive terminal inverted repeats (TIRs) at the 3′ end (94 and 433 nt). Shorter TIRs (3–6 nt) were identified at the 3′ end of most clones, which may serve to protect the ends. In fact, exonuclease III and λ exonuclease digested the plasmid‐like DNAs only after heat denaturation, signifying that conformational changes due to such treatment potentially make the 3′ and 5′ ends (respectively) susceptible to degradation. Multiple tandem and direct repeats were evident near the 3′ ends. A consensus sequence of 18+ nt was discovered in nearly every clone opposite the poly(G) tail, suggesting that this sequence has structural and/or functional significance. Pair‐wise sequence comparisons and the presence of repeats indicated that these novel molecules may be highly recombinant.     

迷蛱蝶属及相关物种细胞色素b基因序列及其分子系统学研究

迷蛱蝶属Mimathyma隶属于蛱蝶科Nymphalidae闪蛱蝶亚科Apaturinae,该属所包含的种类复杂,其分类学地位存在争议.本文对迷蛱蝶属、闪蛱蝶属Apatura和带蛱蝶属Athyma7个种共19个个体的线粒体DNA细胞色素b基因部分序列进行测定分析,并以花斑螯蛱蝶Charaxes kahruba (Moore)作为外群用PAUP软件构建MP和NJ分子系统树.结果显示迷蛱蝶Mimathyma chevana(Moore)、夜迷蛱蝶Mimathyma nycteis(Ménétriès)、白斑迷蛱蝶Mimathyma schrenckii (Ménétriès)和环带迷蛱蝶Mimathyma ambica Kollar形成1个聚类簇,支持Moore将这4个种由闪蛱蝶属移出并建立迷蛱蝶属的观点.同时,尽管迷蛱蝶在形态上与该属其余3种相似,但研究发现聚类簇Ⅰ中夜迷蛱蝶、白斑迷蛱蝶和环带迷蛱蝶首先相聚,然后再与迷蛱蝶聚在一起,表明迷蛱蝶与这3种亲缘关系较远.此外,本文的研究结果还显示迷蛱蝶属与闪蛱蝶属关系密切,而与带蛱蝶属的关系较远.     

PURIFICATION AND CHARACTERIZATION OF GLYCOLATE OXIDASE FROM THE BROWN ALGA SPATOGLOSSUM PACIFICISM (PHAEOPHYTA)1

Glycolate oxidase was purified to apparent homogeneity from the brown alga Spatoglossum pacificum Yendo. The 1326-fold purified glycolate oxidase enzyme exhibited a specific activity of 22. 4 micromoles glyoxylate formed ·min^?1·mg protein^?1. The molecular weight of the native enzyme was estimated to be 230,000 by gel filtration. The subunit molecular weight of the enzyme was determined to be 49,000 by sodium dodecyl sulfate–polyacrylamide gel electrophoresis, suggesting that the native enzyme is a tetramer. There were two absorption peaks at 345 and 445 nm, indicating that glycolate oxidase is a flavoprotein. This enzyme had a high isoelectric point (pI 9.6) and a high pH optimum (pH 8.3). The K_m values for glycolate and l -lactate were 0.49 and 5.5 mM, respectively. This enzyme also had a broad specificity for other straight-chain α-hydroxy acids but not for β-hydroxyacids. Cyanide, azide, N-ethylmaleimide, and p-chloromercuribenzoic acid did not affect the enzyme, whereas 2-pyridylhydroxymethanesulfonic acid strongly inhibited it. These properties of glycolate oxidase from the brown alga S. pacificum are similar to the properties of the glycolate oxidasesfrom higher plants. Polyclonal antibodies raised against the polypeptide fragment of Spatoglossum glycolate oxidase could recognize glycolate oxidase from Spinacia oleracea L., although the cross-reactivity was weak. The N-terminal sequence of two internal polypeptide fragments of the enzyme from S. pacificum showed a high degree of similarity to that of glycolate oxidase from higher plants. These results suggest that glycolate oxidase from higher plants and brown algae share the same ancestral protein.