Flavonoids and Some Other Phenolics as Substrates of Peroxidase: Physiological Significance of the Redox Reactions

2 O₂ without accumulating oxidation products of phenolics. Scavenging of H₂O₂ by the systems can proceed in vacuoles and the apoplast, because phenolics, AA and POX are normal components of the compartments. AA seems to control lignification because it reduces radicals of lignin monomers which are formed by POX-dependent reactions. On lignification, oxidation of sinapyl alcohol is enhanced by radicals of coniferyl alcohol and hydroxycinnamic acid esters when apoplastic POX rapidly oxidizes coniferyl alcohol and the esters but slowly oxidizes sinapyl alcohol. POX seems to participate in the browning of tobacco leaves and onion scales on aging. H₂O₂, which is required for the POX-dependent reactions, can be formed by autooxidation of the phenolics that are transformed to brown components. It is discussed that browning involves the formation of antimicrobial substances. Received 5 June 2000/ Accepted in revised form 1 July 2000     

A Possible Mechanism for the Oxidation of Sinapyl Alcohol by Peroxidase-Dependent Reactions in the Apoplast: Enhancement of the Oxidation by Hydroxycinnamic Acids and Components of the Apoplast

Apoplastic peroxidase isoenzymes from stems of Nicotiana tabacumrapidly oxidized sinapic acid and sinapyl alcohol, in additionto 4-coumaric acid, ferulic acid and coniferyl alcohol. By contrast,the peroxidase isoenzymes from stems of Vigna angularis oxidizedsinapic acid and sinapyl alcohol quite slowly but rapidly oxidizedcompounds with a 4-hydroxyphenyl or a guaiacyl group. However,the oxidation of sinapyl alcohol was greatly enhanced by 4-coumaricacid, ferulic acid and an ester of ferulic acid. Intercellularwashing fluid of V. angularis, which contained apoplastic components,also enhanced the oxidation of sinapyl alcohol. Based on theseresults, a possible mechanism for the oxidation of sinapyl alcoholis discussed on the assumption that the biosynthesis of ligninproceeds mainly via peroxidases which cannot oxidize sinapylalcohol in V. angularis. (Received October 23, 1995; Accepted April 3, 1996)     

Characterisation of Acidic and Basic Apoplastic Peroxidases from Needles of Norway Spruce (Picea abies, L., Karsten) with Respect to Lignifying Substrates

Acidic and basic peroxidases, termed as POD-A and POD-B, wereisolated from the apoplastic space of spruce (Picea abies, L.)needles and purified by acetone precipitation and anion exchangechromatography to apparent homogeneity. The molecular massesof POD-A and POD-B were 39.6 and 29.0 kDa, respectively. ThepH optimum of both isozymes ranged from 4.5 to 6. The apparentK_m values of POD-A and POD-B were 460 and 210 µM for coniferylalcohol. Both isozymes acted also as NADH oxidases with apparentK_m-values of 103 µM (POD-A) and 70 µM (POD-B). NAD⁺but not NADH was found in the apoplastic space of lignifyingneedles. Based on the lignification rate, the contents and kineticproperties of PODs, NADH oxidation by POD is not the major sourceof H₂O₂ required for lignin polymerisation. (Received December 21, 1996; Accepted March 3, 1997)     

Apoplastic Solute Concentrations of Organic Acids and Mineral Nutrients in the Leaves of Several Fagaceae

Ion chromatographic methods determined organic acids and mainnutrient minerals in the apoplastic solution from leaves ofseveral Fagaceae (Quercus ilex L., Quercus cerris L., Quercusvirgiliana (Ten.) Ten, and Fagus sylvatica L.). The anions oforganic acids found in high amounts (250 to 650 µM) werequinate, malate, and oxalate. Lactate, pyruvate, formate andacetate were detected in relatively low amounts with concentrationsbetween 20 and 200 µM. The total concentration of organicacids in the apoplastic sap ranged between 1.5 and 2 mM. Thetotal concentration of inorganic cations (K⁺, Mg²⁺, NH₄⁺, Ca²⁺,Na⁺) and anions (C1^–, NO₃^–, SO^2–₄ and PO^3–₄)in the apoplastic sap varied between 5 and 10 mM, and 0.35 and1.8 mM, respectively. We conclude that the concentration oforganic acid ions in the leaf apoplast depends mainly on theexchange with the leaf cells and is influenced by the electrochemicalgradient between the symplast and the apoplast in relation tothe water potential of the leaf. The determination of formateand acetate in the apoplastic compartment of leaves lend weightto the argument that the production of these acids by treesis a important emission source to the atmosphere. (Received June 9, 1998; Accepted April 8, 1999)     

Regulation of Peroxidase-Dependent Oxidation of Phenolics by Ascorbic Acid: Different Effects of Ascorbic Acid on the Oxidation of Coniferyl Alcohol by the Apoplastic Soluble and Cell Wall-Bound Peroxidases from Epicotyls of Vigna angularis

Intercellular washing fluid (IWF) and washed cell walls obtainedfrom epicotyls of Vigna angularis catalyzed the oxidation ofconiferyl alcohol in the presence of hydrogen peroxide, indicatingthe presence of both soluble and bound peroxidases in the cellwalls. The products of oxidation of coniferyl alcohol were identicalin both cases. Ascorbic acid inhibited the oxidation of coniferylalcohol. The inhibition was due to the rapid reduction of anoxidized intermediate of coniferyl alcohol by ascorbic acid,with resultant regeneration of coniferyl alcohol. However, theinhibitory effects of ascorbic acid were different in the caseof IWF and cell walls. Ascorbic acid completely inhibited theoxidation of coniferyl alcohol by IWF peroxidase as long asascorbic acid was available, whereas the oxidation of coniferylalcohol by cell wall-bound peroxidase was competitively inhibitedby ascorbic acid. Ascorbic acid was present in cell walls andlignin was formed in cell walls during aging of stem. Basedon these results, a possible function for ascorbic acid in theregulation of oxidation of phenolics in cell walls is discussed. (Received March 19, 1993; Accepted May 24, 1993)     

SH oxidation coordinates subunits of rat brain ryanodine receptor channels activated by calcium and ATP

We have reported that ryanodine receptor (RyR) channels display three different responses to cytoplasmic free Ca²⁺ concentration ([Ca²⁺]) depending on their redox state (Marengo JJ, Hidalgo C, and Bull R. Biophys J 74: 1263–1277, 1998), with low, moderate, and high maximal fractional open times (P_o). Activation by ATP of single RyR channels from rat brain cortex was tested in planar lipid bilayers with 10 or 0.1 µM cytoplasmic [Ca²⁺]. At 10 µM [Ca²⁺], low-P_o channels presented lower apparent affinity to activation by ATP [[ATP] for half-maximal activation (K_aATP) = 422 µM] than moderate-P_o channels (K_aATP = 82 µM). Oxidation of low-P_o channels with thimerosal or 2,2'-dithiodipyridine (DTDP) gave rise to moderate-P_o channels and decreased K_aATP from 422 to 82 µM. At 0.1 µM cytoplasmic [Ca²⁺], ATP induced an almost negligible activation of low-P_o channels. After oxidation to high-P_o behavior, activation by ATP was markedly increased. Noise analysis of single-channel fluctuations of low-P_o channels at 10 µM [Ca²⁺] plus ATP revealed the presence of subconductance states, suggesting a conduction mechanism that involves four independent subchannels. On oxidation the subchannels opened and closed in a concerted mode. subconductance states; calcium ion release channels; calcium ion regulation; thimerosal; 2,2'-dithiodipyridine     

The Influence of Apoplastic Ascorbate on the Activities of Cell Wall-Associated Peroxidase and NADH Oxidase in Needles of Norway Spruce (Picea abies L.)

Cell wall-associated peroxidases (EC 1.11.1.7 [EC] ) were extractedfrom the current year's needles of Norway spruce trees (Piceaabies L.) in two fractions, namely soluble apoplastic peroxidasesand covalently wall-bound peroxidases. Peroxidase activitieswere determined with two substrates: coniferyl alcohol, whichis important for lignification, and NADH, which is necessaryfor the production of H₂O₂. Coniferyl alcohol peroxidase activitywas detected in both the soluble apoplastic fraction and thewall-bound fraction, whereas NADH oxidase activity was foundonly in the soluble apoplastic fraction. Net oxidation of coniferylalcohol and NADH was inhibited by ascorbate, which reduced theoxidized intermediates of the peroxidase- and oxidase-catalyzedreactions. Since ascorbate itself was oxidized in these reactions,the inhibition was not persistent and it was released once theascorbate present in the assay mixture had been oxidized. Ascorbatedelayed the oxidation of NADH 10-fold more efficiently thanthe oxidation of coniferyl alcohol. Although the level and theredox state of apoplastic ascorbate were lower in lignifyingneedles than in mature needles, the concentration, which was1.17 mM in apoplastic washing fluids, was sufficiently highto inhibit peroxidase activity in vitro. These results suggestthat peroxidases can catalyze lignification only if local differencesexist in the concentration of reduced ascorbate between lignifyingand non-lignifying tissues. (Received April 21, 1994; Accepted September 26, 1994)     

Age-Dependent Changes in Levels of Ascorbic Acid and Chlorogenic Acid, and Activities of Peroxidase and Superoxide Dismutase in the Apoplast of Tobacco leaves: Mechanism of the Oxidation of Chlorogenic Acid in the Apoplast

In order to understand browning in tobacco plants during aging,age-dependent changes in the levels of ascorbic acid (AA) andchlorogenic acid (CGA) and its isomers were investigated inthe apoplast and the symplast of the leaves. Also activitiesof peroxidase (POX) and superoxide dismutase (SOD) were determined.AA decreased during aging until it was no longer detectablein the apoplast, while symplastic AA remained although the leveldecreased on aging. In contrast, levels of CGA and its isomersand activity of POX in the apoplast increased on aging, whilethose in the symplast remained nearly constant in mature andold leaves. The activity of SOD in the apoplast increased duringaging, while that in the symplast decreased. Oxidation of CGAby the apoplastic solution was observed in the absence of externallyadded H₂O₂ and the oxidation was inhibited by SOD and catalase.Brown components, which contained caffeic acid moieties, accumulatedin the apoplast on aging and the components produced O–₂and H₂O₂ by autooxidation. From these results, we conclude (i)that brown components are formed in the apoplast by the CGA/POXsystem, (ii) that the H₂O₂ required for the reaction can beprovided by the CGA/POX system itself and by autooxidation ofthe brown components, and (iii) that apoplastic SOD functionsto generate H₂O₂ from apoplastically formed O–₂. (Received February 8, 1999; Accepted May 7, 1999)     

Apoplastic pH of intact leaves of Vicia faba as influenced by light

The fluorochrome FITC-dextran was used to measure the effectof light on the apoplastic pH of intact Vicia faba leaves withthe ratio imaging technique. In darkadapted leaves the apoplasticpH varied depending on the leaf between 5.2 and 5.9. Red light(660 nm, 4–12 W m^–2) leads to multiphasic responses:in the first seconds an alkalinization ({small tilde}0.3 pHunits), and thereafter an acidification of the leaf apoplast({small tilde}0.4 pH units) were observed. Both effects couldbe inhibited by DCMU. While variation of CO₂ concentration revealedno effect on light-induced apoplastic pH changes, a decreasein O₂ concentration decreased the effect. On the basis of ourdata it is suggested that the influence of photosynthesis onplasmalemma H⁺ ATPase is responsible for the observed effects,rather than altered CO₂ uptake. Key words: Leaf apoplast, apoplastic pH, light, ratio imaging, pH-sensitive fluorescent dye, Vicia fab     

Effects of Ascorbate on the Oxidation of Derivatives of Hydroxycinnamic Acid and the Mechanism of Oxidation of Sinapic Acid by Cell Wall-Bound Peroxidases

A cell wall fraction isolated from epicotyls of Vigna angularis,which contained both ionically and covalently bound peroxidases,rapidly oxidized p-coumaric, caffeic and ferulic acids and slowlyoxidized sinapic acid. The oxidation of sinapic acid was greatlyenhanced in the presence of p-coumaric, caffeic or ferulic acid.Ascorbate (20 µM) inhibited the oxidation of ferulic acidby about 70% and completely inhibited the oxidation of p-coumaricand ferulic acids. The cell wall fraction was capable of bindingferulic and sinapic acids but not caffeic acid. p-Coumaric acidbound only slightly to cell walls. The oxidation of p-coumaricand ferulic acids by KCl-washed cell walls was inhibited byabout 60% and 10%, respectively, by 20 µM ascorbate, butthe oxidation of caffeic acid was completely inhibited by ascorbateat less than 20 µM. The oxidation of derivatives of hydroxycinnamicacid by peroxidases released from cell walls by washing with1 M KCl was completely inhibited by ascorbate. These resultssuggest that the inhibition by ascorbate depends on the substituentgroup of the phenyl ring of the derivatives of hydroxycinnamicacid when the oxidation reaction is catalyzed by cell wall-boundperoxidases and that the oxidation of sinapic acid is mediatedby phenoxyl radicals of derivatives of hydroxycinnamic acidother than sinapic acid. (Received December 2, 1993; Accepted March 3, 1994)     

Purification and properties of UDP-glucose: coniferyl alcohol glucosyltransferase from suspension culturesof Paul's scarlet rose.

UDP-glucose:coniferyl alcohol glucosyltransferase was isolated from 10-day-old, darkgrown cell suspension cultures of Paul's scarlet rose. The enzyme was purified 120-fold by (NH₄)₂SO₄ fractionation and chromatography on DEAE-cellulose, hydroxyapatite, and Sephadex G-100. The enzyme has a pH optimum of 7.5 in Tris-HCl buffer, required an -SH group for activity, and is inhibited by ?-chloromercuribenzoate and EDTA. Its molecular weight is estimated to be 52,000. The enzyme is specific for the glucosylation of coniferyl alcohol (K_m 3.3 × 10^?6 M) and sinapyl alcohol (K_m 5.6 × 10^?6 M). With coniferyl alcohol as substrate the apparent K_m value for UDP-glucose is 2 × 10^?6m. The enzyme activity can be detected in a number of callus-tissue and cell-suspension cultures. The role of this enzyme is believed to be to catalyze the transfer of glucose from UDPG to coniferyl (or sinapyl) alcohol as storage intermediates in lignin biosynthesis.     

Methylation of Xenobiotic Thiols by Euglena gracilis: Characterization of a Cytoplasmic Thiol Methyltransferase

The green alga Euglena gracilis contains a thiol methyltransferasethat catalyzes the S-adenosylmethionine-dependent methylationof pentachlorobenzenethiol. The enzyme was localized in thecytoplasm and partially purified. The pH optimum for the enzymewas 6.5. The enzyme methylated a number of foreign thiols, butnot the cellular thiols, glutathione or cysteine. Phenols andanilines were not substrates. When pentachloro-benzenethiolwas the methyl acceptor the K_m was found to be 82 µM andthe corresponding K_m for S-adenosylmethionine was 140 µM.The molecular weight of the enzyme was 21,000, as determinedby gel filtration. A role for this enzyme in detoxifying xenobioticthiols is proposed. (Received September 28, 1984; Accepted April 25, 1985)     

Metabolism of p-Hydroxybenzoate via Hydroxyquinol by Trichosporon cutaneum WY2-2: Characterization of the Pathway using Superoxide Dismutase as a Stabilizer of Hydroxyquinol

Trichosporon cutaneum WY2-2 was shown to metabolize p-hydroxybenzoatevia protocatechuate and hydroxyquinol. Using superoxide dismutaseas a stabilizer of hydroxyquinol, the conversion of protocatechuateto hydroxyquinol and the ring fission process of hydroxyquinolwere confirmed. Hydroxyquinol was chemically identified as theproduct of protocatechuate hydroxylase reaction. Partially purifiedprotocatechuate hydroxylase was highly specific for protocatechuate;its K_m values for protocatechuate and NADH were 17.6 and 12.4µM, respectively. It catalyzed equimolar CO₂ formation,NADH oxidation and O₂ consumption from protocatechuate. Hydroxyquinoldioxygenase was highly specific for hydroxyquinol, with a K_mof 2.9 µM. ¹A preliminary account of this work was presented at the 81stMeeting of the Chubu-branch of Agricultural Chemical Societyof Japan, Gifu, October, 1980. ²Present address: Biological Institute, Faculty of Science,Nagoya University, Nagoya 464, Japan. ³Present address: Shin Nihon Chemical Co. Ltd... 19-10, Showa-cho,Anjoh, Aichi 446, Japan. (Received November 15, 1985; Accepted August 27, 1986)     

A Comparison between the Uptake of Potassium by Plants from Solutions of Constant Potassium Concentration and during Depletion

A comparison was made between two methods of measuring the relationshipbetween the external [K⁺] and the flux of K⁺ into whole plantsof Lolium perenne and Raphanus sativus. The values of flux obtainedfrom solutions of 1.2 µM K⁺ held constant around the rootswere three and six times greater for Lolium and Raphanus respectivelythan the values obtained at the same concentration in a depletionexperiment in which the solutions, initially 100 µM K⁺,were depleted to below 1.2 µM K⁺ by plant uptake. In thedepletion experiment with Lolium, the flux was higher into plantsgrown at low [K⁺] than into plants grown at 100 µM eventhough [K⁺] within the plant was about the same for all groupsof plants. It is suggested that Lolium grown at low [K⁺] hasan efficient mechanism for K⁺ uptake which continues to operatefor some time after the plants have been transferred to a higherconcentration. With both species, K_m was 15–20 µMin the depletion experiment and below 1 µM when concentrationswere held constant.     

Measurement of the Apoplastic Activity of K+ and Cl- in the Leaf Epidermis of Commelina communis in Relation to Stomatal Activity

Bowling, D. J. F. 1987. Measurement of the apoplastic activityof K⁺ and Cl^– in the leaf epidermis of Commelina communisin relation to stomatal activity.–J. exp. Bot. 38: 1351–1355. Ionic activity of K⁺ and Cl^– in the apoplast of the lowerepidermis of the leaf of Commelina communis was measured usingion selective micro-electrodes. Large gradients across the stomatalcomplex were observed which were related to stomatal aperture.On stomatal closure the activity of K⁺ and Cl^– in theapoplast of the guard cell rose from 3·0 mol m^–3to 100 mol m^–3 and 33 mol m^–3 respectively. It wasconcluded that the apoplast is an important pathway for iontransport between the cells. Key words: Stomata, ionic activity, leaves, apoplast     

Variations in the Contents and Kinetic Properties of Ribulose-1,5-bisphosphate Carboxylases among Rice Species

The K_m(CO₂) ancl V_max of ribulose 1,5-bisphosphate (RuBP) carboxylaseand its protein ratio to total soluble protein from Oryza speciesincluding cultivars (25 varieties) and wild types (11 species,21 strains) were surveyed. Their variabilities among cultivarsof O. sativa were very small. The averages of the K_m(CO₂) andV_max values and the ratio of carboxylase to soluble protein,and their standard errors were 10.2?1.0µM, 1.72?0.13units.mg^–1(pH 8.0 and 25?C) and 52?2%, respectively. However, some differencesseemed to exist based on genome constitution in the Oryza genus.RuBP carboxylases from the species with the A^gA^g genome, O.graberrima and O. breviligulate, exhibited low K_m(CO₂) values(8.0?0.8 µM). High V_max was associated with the CC genome,O. eichingeri and O. officinalis (2.08?0.15 units.mg^–1).A higher ratio of RuBP carboxylase protein to soluble proteinwas found for the AA genome, O. sativa and O. perennis. (Received September 24, 1986; Accepted April 15, 1987)     

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)     

Linearity and Functioning Forms in the Carboxylase Reaction of Spinach Ribulose 1,5-Bisphosphate Carboxylase/Oxygenase

The reaction of spinach RuBisCO activated with CO₂ and Mg²⁺proceeded in two phases, an initial burst for a few minutesand the subsequent linear phase, in the presence of saturatingconcentrations of CO₂, ribulose 1,5-bisphosphate (RuBP), andMg²⁺. The percentage of the activity in the linear phase tothat in the initial burst was 55% with RuBisCO prepared withpolyethylene glycol, and very close to the value with the enzymereleased immediately from isolated chloro-plasts. RuBisCO preparedwith ammonium sulfate had a much larger decrease of the activityin the linear phase. The Euglena enzyme had a linear courseof reaction with time for up to 20 minutes. The K_m for CO₂ of spinach RuBisCO activated beforehand was 20µM in the initial burst, and 28 µM in the linearphase. In the carboxylase reaction initiated with inactive enzyme,the activity was initially negligible, but in 5 minutes increasedto the level observed in the linear phase of the activated enzyme.The K_m for CO₂ in the linear phase of the pre-inactivated enzymewas 70 µM. The concentration of RuBP was the immediate cause of the two-phasiccourse of the carboxylase reaction of spinach RuBisCO. The curvatureof the time course was not observed below 35 µM RuBP.The enzyme required over 88 µM RuBP for the conventionaltwo-phasic course. Further increase of the concentration ofRuBP increased the extent of the curvature, but did not startthe curvature sooner after the start of the reaction. Even ifspinach RuBisCO was in the linear phase, dilution of RuBP orits consumption by the enzymatic reaction to less than 30 µMcaused the enzyme to show the resumed biphasic reaction courseafter addition of a high concentration of RuBP. ¹This paper is the twenty-fourth in a series on PhotosyntheticCarbon Metabolism in Euglena gracilis. (Received September 19, 1988; Accepted November 25, 1988)     

Purification, Properties and Phosphorylation of Anaerobically Induced Enolase in Echinochloa phyllopogon and E. crus-pavonis

Enolase (2-phospho-D-glycerate hydrolyase, EC 4.2.1.11 [EC] ) activityis differentially induced by anoxia in the flood-tolerant speciesE. phyllopogon (Stev.) Koss and the flood-intolerant speciesE. crus-pavonis (H.B.K.) Schult. To examine the regulation ofenolase at the protein level, we purified the enzyme from bothspecies to near homogeneity and compared their physico-chemicaland catalytic properties. Enolase purified from E. phyllopogonexhibits optimal activity at pH 7.0, a K_m of 80 µM for2-PGA, a Q₁₀ of 1.97 and an E_a of 12.3 kcal mol^-1. Similarly,enolase from E. crus-pavonis exhibits optimal activity at pH7.0, a K_m of 50 µM for 2-PGA, a Q₁₀ of 2.04 and an E_aof 12.9 kcal mol^-1. The enzyme from both species is thermostable(100% active after 15 min, 50°C) and is a homodimer of 52.5kDa subunits as resolved by SDS-PAGE and immunoblotting. E.phyllopogon enolase was phosphorylated in vitro using either[     

Purification and characterization of CMP-N-acetylneuraminic acid hydroxylase from pig submandibular glands

N-Glycoloylneuraminic acid (Neu5Gc) is synthesized as its CMP-giycosideby the action of CMPN-acetylneuramlnic acid (CMP-Neu5Ac) hydroxylase.This enzyme is a soluble cytochrome bs-dependent monooxygenaseand has been purified to apparent homogeneity from pig submandibularglands by precipitation with N-cetyN,N,N-trimethylam-moniumbromide and fractionation on Q-Sepharose, Cibacron Blue 3GA-Agarose,Reactive Brown 10-Agarose, Hexyl-Agarose and Superose S.12.This procedure resulted in an 8960-fold purification of thehydroxylase with a recovery of 0.8%. The molecular mass of thisprotein was shown to be 65 kDa on SDS-PAGE and 60 kDa as determinedby gel filtration on Superose S.12, which suggests that theenzyme is a monomer. The purified CMP-Neu5Ac hydroxylase isactivated by FeSO₄ and inhibited by iron-binding reagents suchas o-phenanthroline, KCN, Tiron and ferro-zine. An apparentKm of 11 µM was determined for the substrate CMP-Neu5Acusing purified hydroxylase in the presence of Triton X-100-solubilizedmicrosomes. In a reconstituted system consisting of purifiedhydroxylase, cytochrome b₅, cytochrome b₅ reductase and catalase,an apparent K_m of 3 µM was measured. The apparent K_mforcytochrome b₅ in this system was 0.24 µM. Immunizationof a rabbit with enriched and purified hydroxylase led to anantiserum that inhibited CMP-Neu5Ac hydroxylase activity andreacted with the purified 65 kDa protein on a Western blot afterSDS-PAGE. Antibodies specific for this 65 kDa protein were isolatedand showed a strong reaction with the purified CMP-Neu5Ac hydroxylasefrom mouse liver after immunoblotting. Initial experiments withthis monospecific antibody suggest that the activity of thehydroxylase in a particular tissue correlates with the amountof immuno-reactive protein. cytochrome b₅ N-glcoloylneuraminic acid hydroxylase pig submandibular gland mucin sialic acid