The Isolation and Characterization of a Cytochrome b6f Complex from the Cyanobacterium Spirulina sp.

A cytochrome b₆f complex was isolated and purified from Spirulinasp. The complex was solubilized with n-heptyl ß-D-thioglucosideand chromatographed on a DEAE-Toyopearl 650M column. The purifiedcomplex contained a small amount of chlorophyll and carotenoid.At least four polypeptides were present in the complex: cytochromef (29 kDa), cytochrome b₆(23 kDa), iron-sulfur protein (ISP,23 kDa), and a 17 kDa polypeptide. Each polypeptide was separatedfrom the complex treated with 2-mercaptoethanol or urea. Theabsorption spectra of cytochrome b₆ and cytochrome f were similarto those of Anabaena and spinach as expected. The complex wasactive in supporting ubiquinol-cytochrome c oxidoreductase activity.Fifty percent inhibition of the activity was accomplished by1 µM dibromothymoquinone (DBMIB). The K_m values for ubiquinol-2and cytochrome c (horse heart) were 5.7 µM and 7.4 µM,respectively. (Received August 15, 1988; Accepted November 14, 1988)     

Succinoxidase and Cytochrome Oxidase in Mitochondria from the Spadix of Arum

1. Mitochondria prepared from young Arum spadix have enoughcytochrome c oxidase to account for the rate at which succinateis oxidized, but succinoxidase activity increases markedly asthe plants mature so that in old material cytochrome oxidaseactivity is only 10 per cent, of succinoxidase. 2. Disintegration of the mitochondria by vibration with ballotini,treatment with digitonin or incubation in the warm reveals anintra-mitochondrial cytochrome c oxidase probably active enoughto account for the fastest rates of succinate oxidation. 3. Succinic dehydrogenase activity is demonstrated and experimentswith p-chloromercuribenzoate indicate that it plays a part inthe oxidation of succinate. 4. Cyanide completely inhibits both external and internal cytochromeoxidase but even at the earliest stages it only reduces succinoxidaseby about 50 per cent. Antimycin A also inhibits succinoxidaseby about 50 per cent.     

Partial Purification and Characterization of the Quinol Oxidase Activity of Arum maculatum Mitochondria

The menadiol oxidase activity of Arum maculatum mitochondria has been solubilized and fractionated. A preparation has been obtained which has an increased specific activity and a greatly decreased polypeptide composition when compared to the mitochondria. This preparation retains normal inhibitor sensitivities in that the oxidation of menadiol remains insensitive to cyanide and is inhibited by aromatic hydroxamates. Metal analyses of the preparation showed that only iron was closely correlated with the oxidase activity. No unusual lipid components were detected in the preparation. The results are discussed in relation to chemical quinol oxidation mechanisms and to several recent hypotheses concerning the nature of the higher plant alternative oxidase.     

RNA Editing of Cytochrome Oxidase Subunit III in Sunflower Mitochondria

Direct sequencing of cytochrome oxidase subunit III (coxIII) mRNA with a specific primer confirms RNA editing in sunflower (Helianthus annus) mitochondria. Six instances of mRNA editing could be verified, one of these specific to this species. All the editing events involve C to U transitions in the coxIII mRNA causing codon changes that lead to amino acids better conserved in evolution than those encoded in the genomic DNA. This observation confirms RNA editing to be widespread in higher plant mitochondria.     

Isolation and Characterization of Rhodobacter capsulatus Mutants Affected in Cytochrome cbb3 Oxidase Activity

The facultative phototrophic bacterium Rhodobacter capsulatus contains only one form of cytochrome (cyt) c oxidase, which has recently been identified as a cbb₃-type cyt c oxidase. This is unlike other related species, such as Rhodobacter sphaeroides and Paracoccus denitrificans, which contain an additional mitochondrial-like aa₃-type cyt c oxidase. An extensive search for mutants affected in cyt c oxidase activity in R. capsulatus led to the isolation of at least five classes of mutants. Plasmids complementing them to a wild-type phenotype were obtained for all but one of these classes from a chromosomal DNA library. The first class of mutants contained mutations within the structural genes (ccoNOQP) of the cyt cbb₃ oxidase. Sequence analysis of these mutants and of the plasmids complementing them revealed that ccoNOQP in R. capsulatus is not flanked by the oxygen response regulator fnr, which is located upstream of these genes in other species. Genetic and biochemical characterizations of mutants belonging to this group indicated that the subunits CcoN, CcoO, and CcoP are required for the presence of an active cyt cbb₃ oxidase, and unlike in Bradyrhizobium japonicum, no active CcoN-CcoO subcomplex was found in R. capsulatus. In addition, mutagenesis experiments indicated that the highly conserved open reading frame 277 located adjacent to ccoNOQP is required neither for cyt cbb₃ oxidase activity or assembly nor for respiratory or photosynthetic energy transduction in R. capsulatus. The remaining cyt c oxidase-minus mutants mapped outside of ccoNOQP and formed four additional groups. In one of these groups, a fully assembled but inactive cyt cbb₃ oxidase was found, while another group had only extremely small amounts of it. The next group was characterized by a pleiotropic effect on all membrane-bound c-type cytochromes, and the remaining mutants not complemented by the plasmids complementing the first four groups formed at least one additional group affecting the biogenesis of the cyt cbb₃ oxidase of R. capsulatus.The gram-negative facultative photosynthetic bacterium Rhodobacter capsulatus has a highly branched electron transport chain, resulting in its ability to grow under a wide variety of conditions (52). Its light-driven photosynthetic electron transfer pathway is a cyclic process between the photochemical reaction center and the ubihydroquinone cytochrome (cyt) c oxidoreductase (cyt bc₁ complex) (30). On the other hand, the respiratory electron transfer pathways of R. capsulatus are branched after the quinone pool and contain two different terminal oxidases, previously called cyt b₄₁₀ (cyt c oxidase) and cyt b₂₆₀ (quinol oxidase) (3, 27, 29, 53). The branch involving cyt c oxidase is similar to the mitochondrial electron transfer chain in that it depends on the cyt bc₁ complex and a c-type cyt acting as an electron carrier. The quinol oxidase branch circumvents the cyt bc₁ complex and the cyt c oxidase by taking electrons directly from the quinone pool to reduce O₂ to H₂O. The pronounced metabolic versatility, including the ability to grow under dark, anaerobic conditions (50, 52), makes these purple non-sulfur bacteria excellent model organisms for studying microbial energy transduction.Marrs and Gest (29) have reported the first R. capsulatus mutants which were defective in the respiratory electron transport chain. Of these mutants, M5 was incapable of catalyzing the α-naphthol plus N′,N′-dimethyl-p-phenylenediamine (DMPD) plus O₂→indophenol blue plus H₂O reaction (NADI reaction) and unable to grow by respiration (Res⁻), and hence was deficient in both terminal oxidases. Another mutant, M4, was also NADI⁻ but Res⁺ due to the presence of an active quinol oxidase. Marrs and Gest have also described two different spontaneous revertants of M5, called M6 and M7, which regained the ability to grow by respiration (29). M6 regained cyt c oxidase activity and became concurrently NADI⁺ and sensitive to low concentrations of cyanide and the cyt bc₁ inhibitor myxothiazol, but remained quinol oxidase⁻. On the other hand, M7 regained the quinol oxidase activity but remained cyt c oxidase⁻ (thus, NADI⁻ and resistant to myxothiazol, a phenotype identical to that of M4). All of these mutants remained proficient for phototrophic (Ps) growth.The cyt c oxidase of R. capsulatus has been purified previously and characterized as being a novel cbb₃-type cyt c oxidase without a Cu_A center (15). It is composed of at least a membrane-integral b-type cyt (subunit I [CcoN]) with a low-spin heme b and a high-spin heme b₃-Cu_B binuclear center, and two membrane-anchored c-type cyts (CcoO and CcoP). It has a unique active site that possibly confers a very high affinity for its substrate oxygen (49). The structural genes of this enzyme (ccoNOQP) have been sequenced recently from R. capsulatus 37b4 (45) and aligned to the partial amino acid sequence of the purified enzyme from R. capsulatus MT1131 (15). Although a ccoN mutant of strain 37b4 was reported to lack cyt c oxidase activity (45), the observed discrepancies between the amino acid sequence and the nucleotide sequence do not entirely exclude the possible presence of two similar cb-type cyt c oxidases in this species. The presence of a similar cyt c oxidase has also been demonstrated in several other bacteria, including P. denitrificans (9), R. sphaeroides (13), and Rhizobium spp. In the latter species, the homologs of ccoNOQP have been named fixNOQP (23, 34) and are required to support respiration under oxygen-limited growth during symbiotic nitrogen fixation (36).The biogenesis of a multisubunit protein complex containing several prosthetic groups, such as cyt cbb₃ oxidase, is likely to require many accessory proteins involved in various posttranslational events, including protein translocation, assembly, cofactor insertion, and maturation (46). Thus, insights into this important biological process, about which currently little is known, may be gained by searching for mutants defective in cyt c oxidase activity. In this work, we describe the isolation of such mutants and their molecular genetic characterization, including those already available, such as M4, M5, and M7G. These studies indicate that in R. capsulatus, gene products of at least five different loci are involved in the formation of an active cyt cbb₃ oxidase.     

Characterization of the cydAB-Encoded Cytochrome bd Oxidase from Mycobacterium smegmatis

The cydAB genes from Mycobacterium smegmatis have been cloned and characterized. The cydA and cydB genes encode the two subunits of a cytochrome bd oxidase belonging to the widely distributed family of quinol oxidases found in prokaryotes. The cydD and cydC genes located immediately downstream of cydB encode a putative ATP-binding cassette-type transporter. At room temperature, reduced minus oxidized difference spectra of membranes purified from wild-type M. smegmatis displayed spectral features that are characteristic of the gamma-proteobacterial type cytochrome bd oxidase. Inactivation of cydA or cydB by insertion of a kanamycin resistance marker resulted in loss of d-heme absorbance at 631 nm. The d-heme could be restored by transformation of the M. smegmatis cyd mutants with a replicating plasmid carrying the highly homologous cydABDC gene cluster from Mycobacterium tuberculosis. Inactivation of cydA had no effect on the ability of M. smegmatis to exit from stationary phase at 37 or 42 degrees C. The growth rate of the cydA mutant was tested under oxystatic conditions. Although no discernible growth defect was observed under moderately aerobic conditions (9.2 to 37.5 x 10(2) Pa of pO(2) or 5 to 21% air saturation), the mutant displayed a significant growth disadvantage when cocultured with the wild type under extreme microaerophilia (0.8 to 1.7 x 10(2) Pa of pO(2) or 0.5 to 1% air saturation). These observations were in accordance with the two- to threefold increase in cydAB gene expression observed upon reduction of the pO(2) of the growth medium from 21 to 0.5% air saturation and with the concomitant increase in d-heme absorbance in spectra of membranes isolated from wild-type M. smegmatis cultured at 1% air saturation. Finally, the cydA mutant displayed a competitive growth disadvantage in the presence of the terminal oxidase inhibitor, cyanide, when cocultured with wild type at 21% air saturation in an oxystat. In conjunction with these findings, our results suggest that cytochrome bd is an important terminal oxidase in M. smegmatis.     

Isolation and Partial Characterization of the NADH Dehydrogenase Complex from Barley Chloroplast Thylakoids

Using chloroplasts from barley leaves we attempt to purify andpartially characterize the NADH dehydrogenase complex. The enzymaticactivity was assayed as NADH-ferricyanide and NADH-nitro bluetetrazolium oxidoreductase. Analyzed by SDS-polyacrylamide gelelectrophoresis and subsequent enzymatic renaturation, the chloroplastsoluble fraction contains principally a 66 kDa enzyme. The membranousfraction solubilized with deoxycholate and analyzed by nativeelectrophoresis and NADH-nitro blue tetrazolium staining revealedthree enzymes: one with similar electrophoretic mobility tothat described for the soluble enzyme, another one which isa complex separated in 3% polyacrylamide gel and a third one,another complex separated in the top of the 5–22% linearpolyacrylamide gel gradient. The complex polypeptidic patternswere similar but different to those found for any thylakoidalproteinic complex known. Nine major polypeptides were detectedin the complex polypeptidic patterns, four of them constituentsof the small size thylakoid enzyme. The molecular masses ofsix polypeptides agreed with those indicated as encoded by 6chloroplast ndh genes. All the enzymes, including the 66 kDasoluble enzyme, contained a 53 kDa polypeptide, which is probablythe NADH-binding complex subunit. Isoelectric focusing of thethylakoidal enzyme points to a basic isoelectric point. Ion-exchangeor hydroxylapatite column chromatography followed by nativeelectrophoresis of the active fractions only separated the smallsize enzyme, which showed complex inactivation. (Received March 16, 1995; Accepted September 18, 1996)     

Host-Pathogen Interactions: XIV. Isolation and Partial Characterization of an Elicitor from Yeast Extract

An elicitor of glyceollin accumulation in soybeans (Glycine max L.) has been isolated from a commercially available extract of brewers' yeast. Yeast is not a known pathogen of plants. The elicitor was isolated by precipitation in 80% (v/v) ethanol followed by column chromatography on DEAE-cellulose, sulfopropyl-Sephadex, and concanavalin A-Sepharose. Compositional and structural analysis showed the elicitor to be a glucan containing terminal, 3-, 6-, and 3,6-linked glucosyl residues. The yeast elicitor stimulates the accumulation of glyceollin in the cotyledons and hypocotyls of soybeans when as little as 15 nanograms or 100 nanograms of the elicitor is applied to the respective tissues. The yeast elicitor is very similar in both structure and absolute elicitor activity to an elicitor isolated from the mycelial walls of Phytophthora megasperma var. sojae, a pathogen of soybeans. These and other results of this laboratory suggest that plants are able to respond to the presence of a wide range of fungi by recognizing, as foreign to the plant, structural polysaccharides of the mycelial walls of the fungi.     

hCOA3 Stabilizes Cytochrome c Oxidase 1 (COX1) and Promotes Cytochrome c Oxidase Assembly in Human Mitochondria

Cytochrome c oxidase (COX) or complex IV of the mitochondrial respiratory chain plays a fundamental role in energy production of aerobic cells. In humans, COX deficiency is the most frequent cause of mitochondrial encephalomyopathies. Human COX is composed of 13 subunits of dual genetic origin, whose assembly requires an increasing number of nuclear-encoded accessory proteins known as assembly factors. Here, we have identified and characterized human CCDC56, an 11.7-kDa mitochondrial transmembrane protein, as a new factor essential for COX biogenesis. CCDC56 shares sequence similarity with the yeast COX assembly factor Coa3 and was termed hCOA3. hCOA3-silenced cells display a severe COX functional alteration owing to a decreased stability of newly synthesized COX1 and an impairment in the holoenzyme assembly process. We show that hCOA3 physically interacts with both the mitochondrial translation machinery and COX structural subunits. We conclude that hCOA3 stabilizes COX1 co-translationally and promotes its assembly with COX partner subunits. Finally, our results identify hCOA3 as a new candidate when screening for genes responsible for mitochondrial diseases associated with COX deficiency.     

Subunit Composition of Cytochrome c Oxidase in Mitochondria of Zea mays

Cytochrome c oxidase has been purified from Zea mays mitochondria by a solubilization with dodecyl maltoside followed by a simple and rapid two step fast protein liquid chromatographic method involving anion exchange on Mono Q and size exclusion chromatography on Superose 12. The preparation obtained was resolved by urea sodium dodecyl sulfate-polyacrylamide gel electrophoresis and had a subunit composition comprising polypeptides of apparent molecular masses of 48, 31, and 25 kilodaltons at least one at 16 and 11 kilodaltons and three subunits below 10 kilodaltons. Comparison with a purified yeast cytochrome c oxidase revealed that the four largest subunits showed similar electrophoretic mobilities. Subunits I and II cross-reacted with antibodies raised against the yeast homologous polypeptides. Polypeptides of the plant ubiquinone:cytochrome c reductase complex have also been identified by cross-reaction with antibodies raised against yeast cytochrome b and c₁ subunits and by inference from comigration.     

The Isolation of a Functional Cytochrome b 6 f Complex: from Lucky Encounter to Rewarding Experiences

The recognition that, in photosynthesis, the plastoquinol oxidizing cytochrome b (6f ) complex resembles the ubiquinol oxidizing cytochrome bc1 complex in respiration is one of the examples of exciting universalization in biological research. A peripheral observation towards the end of 1979 initiated an intensive investigation, which is still ongoing today: next to the ATP synthase the cytochrome b (6f ) complex could be selectively solubilized from the chloroplast membrane by the combined action of octyl glucoside and cholate. It was mere luck that the isolate was substantially active as an electrogenic, proton translocating plastoquinol-plastocyanin oxidoreductase, and that it also catalyzed oxidant-induced reduction of cytochrome b (6), a signature of the Q-cycle mechanism. The basic findings during the first characterization of the complex are summarized, and the excitement among the collaborating groups is remembered. More recent developments, including the impact of gene technology and the elucidation by the crystal structure, are additionally traced here.     

Isolation and Partial Characterization of a Glycoprotein Complex with Sperm-Receptor Activity from Ciona intestinalis Ovary1

Sperm-egg interaction in the ascidian Ciana intestinalis is mediated by a fucosyl-glycoprotein (FP) component of the egg vitelline coat. FP are responsible for sperm binding, sperm activation and the acrosome reaction. In this paper we report a detailed biochemical and functional characterization of FP purified from the ovaries by affinity chromatography. thic component with sperm receptor activity is a high molecular weight glycoprotein complex (>10⁷) with a protein-carbohydrate ratio of 2:1, which inhibits the binding of the spermatozoa to the vitelline coat and induces sperm activation and the acrosome reaction. Exhaustive proteolytic digestion of FP yields high molecular weight glycopeptides (> 4×10⁵), which contain N-acetylgalactosamine, fucose, galactose and rhamnose. These glycopeptides retain some receptor activity, thus raising the question of the involvement of the polypeptide backbone in the sperm-egg binding process. However, the glycopeptide fraction fails to induce the acrosome reaction: we suggest that the polypeptide fraction plays a role in the induction of sperm activation and the acrosome reaction.     

Respiratory Activities, Cytochrome Composition and Cytochrome c Oxidase Subunit II Levels of Mitochondria from Alloplasmic Wheats Having Aegilops Cytoplasms

Respiratory activities, cytochrome composition and cytochromec oxidase (EC 1.9.3.1 [EC] ) subunit II (COXII) levels of isolatedmitochondria in one euplasmic and four alloplasmic lines ofwheat (Triticum aestivum), having cytoplasms of Aegilops columnaris,Ae.crassa (4x),Ae. mutica and Ae. triuncialis, which show variousgrowth abnormalities, were studied. Cytoplasm-specific variationon the respiratory activities has been revealed among the cytoplasmsof five Triticum and Aegilops species. The cyanide-resistantrespiratory pathway does not seem to contribute to the variationin activity. The low level of state 3 and cytochrome oxidaseactivities were evident in lines having Ae. columnaris and Ae.triuncialis cytoplasms, which also show growth inhibition. Thelow cytochrome oxidase activity in both cytoplasms is consistentwith the low amount of cytochrome aa₃ and the severe reductionin COXII levels. The relatively low amount of cytochrome aa₃in Ae. mutica is also consistent with the reduced level of theCOXII. On the other hand, relatively low amounts of cytochromeb (b-557) are found in the Ae. crassa cytoplasmic line. Theseresults suggest that the growth abnormalities found in the alloplasmicwheat lines are related to the variation in respiratory activitiesand cytochrome composition. In particular, growth inhibitionfound in the alloplasmic wheats having Ae. columnaris and Ae.triuncialis may be caused by the severe depression of respiratoryactivity due to the impaired cytochrome oxidase activity oftheir mitochondria. ³Present address: Department of Biochemistry, Dalhousie University,Halifax, NS, Canada B3H 4H7     

7H-Pyridocarbazole, a Probe to Study the Role of Diphosphatidylglycerol in the Functioning of the Cytochrome Oxidase Complex of Mitochondria

Ditercalinium, the generic name for 7H-pyridocarbazole dimer,is a planar aromatic cation which exhibits strong inhibitoryeffects on the respiratory chain of plant mitochondria. By enzymaticassays and spectroscopic studies, the inhibition is shown tooccur at the level of the cytochrome c oxidase complex. It issuggested that the mechanism of inhibition could be due to thecomplexation of the aromatic cation with the diphosphatidylglycerolenvironment essential for cytochrome c oxidase activity ratherthan to a drug-enzyme direct interaction. 7H-pyridocar-bazolecan be used as an appropriate tool to study the role that negativelycharged acidic phospholipids, as diphosphatidylglycerol, playin the functioning of cytochrome c oxidase. (Received July 10, 1989; Accepted February 19, 1990)     

Isolation and Partial Characterization of a Mycotoxin from Penicillium roqueforti

Extracts of pure cultures of Penicillium roqueforti isolated from toxic feed samples and of P. roqueforti NRRL 849 were lethal to rats by either intraperitoneal or oral administration. Purification studies guided by this test led to the isolation of a major toxin which showed intraperitoneal and oral median lethal dose values in weanling rats of 11 and 115 mg/kg, respectively. Partial characterization of the crystalline compound, C(17)H(20)O(6), by infrared, ultra violet, PMR, and mass spectroscopy, and by several chemical transformations indicated the presence of three C-methyl substituents plus one acetoxy, one aldehyde, and one alpha,beta-unsaturated ketone group. Two oxygen atoms are present either in epoxide or ether form.     

Properties of the Cytochrome c Oxidase Activity of Cytochrome b561 from Photoanaerobically Grown Rhodopseudomonas sphaeroides

Cytochrome b₅₆₁ from Rhodopseudomonas sphaeroides had cytochromec (c2) oxidase activity and a pH optimum at 6.0 for this activity.The activity was affected by the ionic strength of the reactionmixture. The apparent K_m and maximal velocity (V_max) valuesin the absence of addea salts were 14 µM and 120 nmoloxidized per min per mg protein for horse heart cytochrome c.Reduced horse heart cytochrome c was reoxidized in first-orderkinetics by this cytochrome b₅₆₁. The specific activity was0.7 s^–1 per mg protein at 20°C at the concentrationof 30 µMM cytochrome c. Activity was inhibited by KCN and NaN₃, but not by antimycin.The addition of a low concentration of KCN to the cytochromeb₅₆₁ produced a change in the absorption spectrum, evidencethat KCN interacts with the heme moiety of cytochrome b₅₆₁.Results of this and preceeding studies show that the cytochromeoxidase (cytochrome "o") described earlier (Sasaki et al. 1970)is cytochrome b₅₆₁. (Received May 16, 1983; Accepted September 8, 1983)     

Isolation and Characterization of a Thermotolerant Methanol-Utilizing Yeast

A yeast capable of growth on methanol as its sole carbon-energy source was isoalted from soil samples and identified as a strain of Hansenula polymorpha. A continuous enrichment culture at 37 C with a simple mineral salts medium was used to select this organism. The isolate, designated DL-1, has a maximal specific growth rate of 0.22 per h, at pH 4.5 to 5.5 and temperatures of 37 to 42 C, in simple mineral salts medium with methanol (0.5%), biotin, and thiamine. Growth occurred in a chemostat at temperatures up to 50 C, with strong growth at 45 C. The maximal growth yield of the yeast on methanol was 0.36 g of dry cell weight per g of methanol, and the yield on oxygen was 0.37 g of dry cell weight per g of O(2). Protein content of the isolate is 46%, and total nucleic acid content varies from 5.0 to 7.0% with increasing growth rate from 0.08 to 0.20 per h. The amino acid profile of this yeast protein indicates that it could serve as a good source of food protein. Feeding studies with rats show the yeast to have no toxic effects.     

Import Pathway of Nuclear-Encoded Cytochrome c Oxidase Subunit 2 Using Yeast as a Model

Abstract: Subunit 2 of cytochrome c oxidase (Cox2) is a mitochondrial-encoded protein in most organisms. In soybean Glycine max a second Cox2 gene was identified in the nucleus which is functional, whereas the mitochondrial-encoded cox2 gene is silent. For import and sorting of the nuclear-encoded soybean Cox2 protein ( Gm Cox2p) into mitochondria, the protein has acquired an N-terminal extension of 136 amino acid residues that is cleaved off in three steps during import. To study the function and processing of the Gm Cox2p leader peptide, we used yeast as a model system. Using different leader peptide-GFP constructs, we were able to show that the i₁ intermediate is generated in the mitochondrial matrix and the mature protein is generated in the inner membrane space. Mitochondrial processing peptidase (MPP) is involved in processing the first part of the leader peptide, processing of the last part is catalysed by the inner membrane peptidase (IMP). Oxa1p is necessary for insertion of the protein into the inner mitochondrial membrane. Gm Cox2p therefore utilises many of the same components as its mitochondrial-encoded predecessor, for sorting and maturation, following its import into the mitochondria.     

Isolation and Characterization of a Boron-Polysaccharide Complex from Radish Roots

A boron-polysaccharide complex was purified from a Driselasedigest of cell walls of radish roots. The complex had a molecularweight of 7.5 KDa and contained boron (0.232%, w/w), uronicacid (52.3%, w/w) and neutral sugars (32.4%). ¹¹B-NMR spectroscopicanalysis suggested that the boron was present as a tetravalent1 : 2 borate-diol complex. ¹This work was supported by a Grant-in-Aid (no. 04660069) fromthe Ministry of Education, Science and Culture. ⁴Present address: Kasai Experimental Farm, Sumitomo ChemicalCo., Ltd. Kasai, Hyogo, 675-23 Japan.