Studies on cutin-esterase II. Characteristics of cutin-esterase from Botrytis cinerea and its activity on tomato-cutin

The activity of cutin-esterase, cutinase, was detected in themycelial homogenate of Botrytis cinerea cultured in a peptone-sucrosemedium at 25°C for 7 days. The crude enzyme solution wasprepared from the homogenate by centrifugation at 106,600xg,treatment with (NH₄)₂SO₄ at 70% saturation, and dialysis against0.01 M phosphate buffer. The optimum pH, temperature and assayduration for enzyme activity were 5.0, 25°C and 18 hr, respectively.Specific activity was 255 mµmoles/mg protein/18 hr aspalmitate under optimum conditions. 83% of the activity waslost by heating the enzyme solution (pH 7.6) for 4 min at 95°C.Palmitic, stearic, oleic, 9, 10-dihydroxystearic or linoleic,dihydroxyeicosanoic and octadecanedioic acids were recognizedin the enzymic hydrolysate of tomato-cutin using gas-liquidchromatography. Among these fatty acids, palmitic, oleic andoctadecanedioic acids were readily liberated by the enzyme,but dihydroxyeicosanoic acid, the major component of tomato-cutin,was isolated only in small amounts. The enzyme is, therefore,an exo-type cutinase which hydrolyses minor side chains of fattyacids bound to the major structure of cutin. Cutinesterase mayfacilitate cuticular invasion by fungi as a result of reductionin mechanical strength of the cuticle by the enzyme ¹Biological Laboratory, Research Department, Nihon Noyaku Co.,Ltd., Kawachinagano, Osaka, Japan (Received June 16, 1970; )     

Lipid Mobilization During Dormancy Breakage in Oilseed of Corylus avellana

During the stratification necessary for the alleviation of dormancyin Corylus avellana L. there is a substantial reorganizationof metabolic processes. Changes in activities of some enzymesrelated to lipid mobilization were followed throughout a fruitstratification period at 5 °C and control treatment at 20°C. Significant increases in total lipase and isocitratelyase activities were found in both embryonic axes and cotyledonsof seeds from fruits stratified at 5 °C, whereas the activitiesremained consistently low in those held at 20 °C. Theseresults correlated with an earlier ultrastructural study whichshowed a reduction in storage lipid. The increased potentialfor lipolysis was concomitant with loss of dormancy as seenin germination tests. These findings suggest that lipid mobilizationduring stratification could be related to the overall patternof metabolic changes which are required for dormancy release. Corylus avellana L., hazel, lipid, catalase, isocitrate lyase, lipase, stratification     

A thiol-activated lipase from <Emphasis Type="Italic">Trichosporon asahii</Emphasis> MSR 54: detergent compatibility and presoak formulation for oil removal from soiled cloth at ambient temperature

An alkaline lipase from Trichosporon asahii MSR 54 was used to develop presoak formulation for removing oil stains at ambient temperature. The lipase was produced in a reactor followed by concentration by ultrafiltration and then it was dried with starch. The biochemical characteristics of enzyme showed that it was an alkaline lipase having pH activity in the range of pH 8.0–10.0 and temperature in the range of 25–50°C. The present lipase was active >80% at 25°C. The lipase was cystein activated with fourfold enhancement in presence of 5 mM cystein and likewise the activity was also stimulated in presence of papain hydrolysate which served as source of cystein. The presoak formulation consisted of two components A and B, component A was enzyme additive and B was a mixture of carbonate/bicarbonate source of alkali and papain hydrolysate as source of cystein. The results indicated that the presoaking in enzyme formulation followed by detergent washing was a better strategy for stain removal than direct washing with detergent in presence of lipase. Further, it was observed that 0.25% presoak component B in presence of 100 U enzyme component A (0.1 g) was the best formulation in removing maximum stain from mustard oil/triolein soiled clothes as indicated by increase in reflectance which was found equal to that of control cloth. The lipase action in presoaked formulation was clearly indicated by quantitated fatty acid release and also the TLC results of wash water, where oil hydrolytic products were visible only in presence of enzyme in the treatment. The wash performance carried at 25°C indicated that washing at 25°C was at par with that at 40°C as indicated by similar reflectance of the washed cloth piece though qualitative fatty acid release was higher at 40°C.     

Biochemical Changes in Seed of Pinus radiata D. Don during Stratification

Stratification at 0 °C accelerates subsequent germinationof seed of Pinus radiata D. Don when transferred to 25 °C;the effect of low temperature is on the megagametophyte, notthe embryo. Organic acids, sucrose, and organic phosphates accumulatein the seed during stratification but lipase and invertase havelow activities which do not increase during treatment at 0 °C.We conclude that this accumulation of metabolites underliesthe increase in rate of germination of stratified seed. Treatingseeds at 0 °C rather than 5 °C separates effects dueto stratification and growth.     

Purification of NADP-Malic Enzyme and Phosphoenolpyruvate Carboxylase from Sugar Cane Leaves

A procedure is described for the purification of phosphoenolpyruvatecarboxylase (EC 4.1.1.31 [EC] ) and NADP-dependent malic enzyme (EC1.1.1.40 [EC] ) from sugar cane leaves. Each enzyme was purified tohomogeneity as judged by sodium dodecyl sulfate-polyacrylamidegel electro-phoresis, with about 30% yield. Phosphoenolpyruvatecarboxylase was purified 54-fold. A molecular weight of 400,000and a homotetrameric structure were determined for the nativeenzyme. The purified carboxylase had a specific activity of20.0 {diaeresis}mol (mg protein)_–1 min_–1, and wasactivated by glucose-6-phosphate and inhibited by L-malate.K_m values at pH 8.0 for phosphoenolpyruvate and bicarbonatewere 0.25 and O.l0 mM, respectively. NADP-malic enzyme, 356-foldpurified, exhibited a specific activity of 71.2 {diaeresis}mol(mg protein)_–1 min_–1 and was characterized as ahomotetramer with native molecular weight of 250,000. Purifiedmalic enzyme showed an absolute specificity for NADP⁺ and requireda divalent metal ion for activity. K_m values of 0.33 and 0.008mM for L-malate and NADP⁺, respectively, were determined. Thisenzyme was inhibited by several organic acids, including ketoand amino acids; while succinate and citrate increased the enzymeactivity when assayed with 10{diaeresis}M L-malate. The effectsshown by amino acids and by citrate were dependent on pH, beinghigher at pH 8.0 than at pH 7.0. (Received October 26, 1988; Accepted February 3, 1989)     

Effects of Hydrostatic Pressure on Catalysis by Epaxial Muscle Phosphofructokinase from an Abyssal Fish

SYNOPSIS: Phosphofructokinase (PFK) extracted from muscle ofabyssal Coryphaenoides fishes common in the deep waters aroundthe Galápagos Archipelago is extremely unstable upondecompression and extraction and can be recovered only in lowactivities. Preliminary studies indicate that the pressure responsesof the enzyme are complex: At low pressures, the enzyme is activated;at moderate pressures, activity passes through a pressure optimum;at high pressures, maximum catalytic activity is decelerated.At low pressures, the volume change of activation is about –11cm³/mole at 3°C and increases to about –46 cm³/moleat 28°C. The homologous enzyme from a surface species (Oligoplitesmundus) appears to be more pressure sensitive at low temperatures.     

Lipase in the Lipid Bodies of Corn Scutella during Seedling Growth

In the scutella of corn (Zea mays), lipase activity is absent in ungerminated seeds and increases during seedling growth. At the peak stage of lipolysis, about 50% of the lipase activity is recovered in the lipid body fraction after flotation centrifugation. The lipase is tightly bound to the lipid bodies, and resists solubilization by repeated washing with buffers or NaCl solutions. Isolated lipid bodies undergo autolysis of internal triacylglycerols, resulting in the release of fatty acids. After the triacylglycerols in isolated lipid bodies have been extracted with diethyl ether, the lipase is recovered in the membrane fraction. The lipase has an optimal activity at pH 7.5 in the autolysis of lipid bodies, or on trilinolein or N-methylindoxylmyristate. Of the various acylglycerols examined, the enzyme is active only on acylglycerols of linoleic and oleic acids which are the major fatty acid constituents of corn oil. The activity is not greatly affected by NaCl, CaCl₂, or pretreatment of the enzyme with p-chloromercuribenzoate or mersalyl, and detergents abolish the activity. The enzyme hydrolyzes trilinolein completely to fatty acids; during the course of reaction, there is little accumulation of di- or mono-linolein.     

Corn leaf glutamate synthase: Purification and properties of the enzyme

An assay for ferredoxin-glutamate synthase is introduced thatuses an anion exchange resin to isolate the glutamate formedand subsequent determination with the ninhydrin procedure. Theenzyme was purified 200-fold from corn leaves by ammonium sulfatefractionation and chromatography on DEAE-cellulose, DEAE-Sephaceland ferredoxin- Sepharose. The purified enzyme had a specificactivity of 14 µmoles glutamate formed min^–1mg^–1protein. The enzyme has a molecular weight of 160,000. The pHoptimum for catalytic activity is 6.9. The isoelectric pointis at pH 4.2. The apparent Km values of the enzyme for L-glutamine,2-oxoglutarate and ferredoxin are 1,100, 240 and 1.7 µM.The enzyme has a high specificity toward these substrates witha stoichiometry between glutamate formation and glutamine consumption.Sulfhydryl reagents, bathophenanthroline, phthalein acids andazaserine produced strong inhibition of the enzyme activity. ¹Permanent address: Department of Agricultural Chemistry, KyotoUniversity, Kyoto 606, Japan. ²To whom inquiries should be addressed. (Received July 7, 1979; )     

Characteristics of Sugar, Amino Acid and Phosphate Release from the Seed Coat of Developing Seeds of Vicia faba and Pisum sativum

After removal of the embryo from developing seeds of Vicia fabaL. and Pisum sativum L., the ‘empty’ ovules werefilled with a standard solution (pH 5.5). Seed coat exudatesof both species were collected during relatively long experiments(up to about 12 h) and the concentration of sugar (mainly sucrose),amino acids and phosphate in the exudate measured. A discussionis presented on the amino acid/sugar ratio and the phosphate/sugarratio in the seed coat exudate. A pretreatment (15 min) withp-chloromercuribenzenesulphonic acid (PCMBS) reduced the releaseof sugar, amino acids and phosphate from broad bean seed coats.After excision of ‘empty’ ovules of Vicia faba andPisum sativum from the maternal plant, 2–4 h after thistreatment a strong difference became visible between sucroserelease from excised seed coats and sucrose release from attachedseed coats. Similarly, when the rate of phloem transport ofsucrose into an ‘empty’ ovule of Vicia faba or Pisumsativum was reduced by a sub-optimal mannitol concentrationin the solution, a reduced rate of sugar release from the seedcoat could be observed. Excision and treatment with a sub-optimalmannitol concentration reduced the release of amino acids toa lesser extent than for sucrose. These treatments did not reducethe rate of phosphate release from the seed coat. Key words: Seed development, Seed coat exudate, Phloem transport     

Development of Endopeptidase Activity in Cotyledons of Vigna mungo Seedlings: Effects of Exogenously Applied End-Products and Plant Hormones

The development of endopeptidase activity in cotyledons of Vignamungo seedlings was examined after application of exogenousamino acids, sugars and plant hormones. The endopeptidase activityin the cotyledons fell when germinating seeds were allowed toabsorb a solution of amino acids at high concentrations, andit was postulated that this effect might have been caused inpart by osmotic stress and in part by end-product repression.Protein immunoblotting with an antiserum against SH-EP, themajor cysteine endopeptidase occurring in the cotyledons, showedthat sugars and amino acids at high concentrations also delayedthe post-translational processing of SH-EP intermediates. Endopeptidaseactivity equivalent to nearly twice that in controls was observedwhen GA₃ was applied at 10 to 100 µM to cotyledons thathad been detached from the embryonic axis. In addition, naphthaleneaceticacid at 1 to 100 µM, kinetin at 1 to 10 µM and jasmonicacid at 1 to 10 µM also increased the activity to a limitedextent. Results of pulse-chase experiments suggested that theeffect of GA₁ on the endopeptidase activity in the detachedcotyledons was attributable to suppression of the degradationof the enzyme. Protein immunoblotting revealed the presenceof 34-kOa and 35-kDa intermediates of SH-EP in addition to previouslyreported 36-kDa and 43-kDa intermediates. (Received June 26, 1995; Accepted October 16, 1995)     

Structural and Kinetic Properties of NADP-Malic Enzyme from the Inducible Crassulacean Acid Metabolism Plant Mesembryanthemum crystallinum L.

NADP-malic enzyme (EC 1.1.1.40 [EC] ), which is involved in Crassulaceanacid metabolism (CAM), was purified to electrophoretic homogeneityfrom the leaves of the inducible CAM plant Mesembryanthemumcrystallinum. The NADP-malic enzyme, which was purified 1,146-fold,has a specific activity of 68.8 µmol (mg protein)^–1min^–1. The molecular weight of the subunits of the enzymewas 64 kDa. The native molecular weight of the enzyme was determinedby gel-filtration to be 390 kDa, indicating that the purifiedNADP-malic enzyme is a hexamer of identical subunits. The optimalpH for activity of the enzyme was around 7.2. Double-reciprocalplots of the enzymatic activity as a function of the concentrationof L-malate yielded straight lines both at pH 7.2 and at pH7.8 and did not reveal any evidence for cooperativity of bindingof L-malate. The K_m value for L-malate was 0.35 mM. Hill plotsof the activity as a function of the concentration of NADP⁺indicated positive cooperativity in the binding of NADP⁺ tothe enzyme with a Hill coefficient (nH) of 2.0. An S_0.5 value(the concentration giving half-maximal activity) of 9.9 µMfor NADP⁺ was obtained. Oxaloacetate inhibited the activityof the NADP-malic enzyme. Effects of succinate and NaHCO₃ onthe activity of NADP-malic enzyme were small. (Received October 30, 1991; Accepted May 1, 1992)     

Novel thermostable lipase from <Emphasis Type="Italic">Bacillus circulans</Emphasis> IIIB153: comparison with the mesostable homologue at sequence and structure level

Thermophilic Bacillus circulans IIIB153 isolated from hot springs of North West Himalayas, India, produced an extracellular lipase, which exhibited significant biofilm disruption property on the static biofilm disruption model with a single species of Actinomyces viscosous. The gene encoding the lipase was cloned and overexpressed in Escherichia coli. Recombinant Bacillus circulans lipase (BCL), a monomer with molecular mass of 43 kDa also exhibited significant biofilm disruption activity. The enzyme was optimally active at 60°C, pH 8.5 and retained >70% of its original activity after 1 h incubation at 60°C. 3D structure of BCL developed by homology modeling showed a typical α/β hydrolase fold, a characteristic feature of lipolytic enzymes. Comparison of thermostable BCL with mesostable lipase from Chromobacterium viscosum at the sequence and structure level showed distinct variations in the structural features, with the presence of a high content of proline residues, aromatic amino acids and salt bridges. These features along with the presence of zinc-binding site observed in BCL structure could have a potential role in thermal stability of the enzyme.     

Selective Enrichment of Omega-3 Fatty Acids in Oils by Phospholipase A1

Omega fatty acids are recognized as key nutrients for healthier ageing. Lipases are used to release ω-3 fatty acids from oils for preparing enriched ω-3 fatty acid supplements. However, use of lipases in enrichment of ω-3 fatty acids is limited due to their insufficient specificity for ω-3 fatty acids. In this study use of phospholipase A1 (PLA1), which possesses both sn-1 specific activity on phospholipids and lipase activity, was explored for hydrolysis of ω-3 fatty acids from anchovy oil. Substrate specificity of PLA1 from Thermomyces lenuginosus was initially tested with synthetic p-nitrophenyl esters along with a lipase from Bacillus subtilis (BSL), as a lipase control. Gas chromatographic characterization of the hydrolysate obtained upon treatment of anchovy oil with these enzymes indicated a selective retention of ω-3 fatty acids in the triglyceride fraction by PLA1 and not by BSL. ¹³C NMR spectroscopy based position analysis of fatty acids in enzyme treated and untreated samples indicated that PLA1 preferably retained ω-3 fatty acids in oil, while saturated fatty acids were hydrolysed irrespective of their position. Hydrolysis of structured triglyceride,1,3-dioleoyl-2-palmitoylglycerol, suggested that both the enzymes hydrolyse the fatty acids at both the positions. The observed discrimination against ω-3 fatty acids by PLA1 appears to be due to its fatty acid selectivity rather than positional specificity. These studies suggest that PLA1 could be used as a potential enzyme for selective concentrationof ω-3 fatty acids.     

Temperature differences for trans-glycosylation and hydrolysis reaction reveal an acceptor binding site in the catalytic mechanism of Trypanosoma cruzi trans-sialidase

Trypanosoma cruzi, the agent of Chagas disease, expresses onits surface a trans-sialidase that catalyzes preferentiallythe transference of -2,3-linked sialic acid to acceptors containingterminal β-galactosyl residues, instead of the typicalhydrolysis reaction, found in most sialidases. The trans-sialidaseis responsible for the acquisition of the host sialic acid bythis protozoan parasite, which does not synthesize sialic acids.Here, we have studied some kinetic properties of a recombinanttrans-sialidase expressed in Escherichia coli We found thatit has sequential-type kinetics for the transferase reaction,as shown for the parasite-derived enzyme. The rates of sialicacid transfer to water (hydrolysis), and to β-galactosylresidues have a unique behavior with respect to the reactiontemperature. While the hydrolysis rate of sialyUactose increasescontinuously up to 35°C, the temperature for the maximalrate of trans-glycosylation depends on the acceptor concentration.At low acceptor concentrations the rate of trans-glycosylationis maximal at 13°C and independent of the amount of sialicacid donors. With increasing acceptor concentrations, maximalrates of trans-glycosylation are shifted to higher temperatures.This finding is explained by an 8-fold increase in the K_m forthe acceptor from 13°C to 33°C. Differences in hydrolysisand transfer rates were also obtained by using 4-methyl-umbelliferyl-N-acetyl-neuraminicacid. However, its hydrolysis rate is much higher than the rateof transference to lactose, suggesting that a long-lived enzyme-sialosylintermediate is not formed. In addition, lactose does not increasethe rate of methyl-umbelliferone release at any temperature,indicating that the rate limiting step is the aglycon release.Based on these results we propose that trans-glycosylation inT.cruzi sialidase is favored by the existence of a binding sitefor β-galactosyl residues, which accepts the new glycosidicbond as sialic acid is released from the donor. With increasingtemperature the affinity for the acceptor decreases, resultingin a concomitant increase in the rate of transfer to water,which, in turn, can be suppressed by increasing the acceptorconcentration. Trypanosoma cruzi sialidase kinetics reaction mechanism temperature     

The Occurrence of 1,3-{beta}-Glucanase in Healthy and Verticillium-infected, Resistant and Susceptible Tomato Plants

Leaf, stem, and root extracts of near-isogenic tomato plantscv. Craigella, resistant and susceptible to Verticillium albo-atrum,showed constitutive 1,3-ß-glucanase activity whichincreased following inoculation with the pathogen. Partiallypurified enzyme extracts were obtained by dialysing a 30–80%ammonium sulphate fraction of the tissue brei. The enzyme hadpH and temperature optima of 5?5 and 44 ?C respectively, withhigh activity between 50 and 60 ?C. The response to laminarinconcentration was linear between 1?2 and 7?5 mg ml^–1.Root inoculation of susceptible plants with 10⁶ propagules ml^–1V. albo-atrum led to a umform 300 per cent increase in all steminternodes except the terminal one, which was 500 per cent ofthe controls. No spatial relationship of enzyme activity tothe localization of fungus within the stem was apparent. Petioles,leaves, and roots of susceptible infected plants similarly showedan increase in activity but less than that in stems. Changedlevels of stern enzyme activity at different times after inoculationwere associated with reductions in the number of vessels containinghyphae. Extracts of plants of the resistant isoline showed increasedglucanase activity over controls, but this was substantiallylower than that in susceptible plants and was associated withthe greatly reduced mycelial colonization in resistant plants. It is concluded that single gene resistance in tomato to Verticilliumis not associated with innately higher levels of 1,3-ß-glucanasein healthy plants. The increased activity in infected plantsis proportional to the overall quantity of pathogen in the plantor of pathogenic metabolites.     

Fungal Associations: III. The Role of Pectic Enzymes on the Synergistic Relation between Rhizoctonia solani Kuhn and Fusarium solani Snyder and Hansen, in the Rotting of Potato Tubers

The greatest activity of protopectinase obtained from the growthof Rhizoctonia solani and Fusarium solani on autoclaved potatoplugs occurred at pH 6.5, and greatest activity of the ‘lossof viscosity’ enzyme was found at 6–5 for Rhizoctonia,and between 6.5 and 8.3 for Fusarium. Protopectinase enzymeobtained from double infections of the Fusarium spp. with Rhizoctonia,or by mixing the enzymes of individual Fusarium spp. with Rhizoctoniaenzyme, were more active than the enzymes from single inoculations.Cylindrocarpon radicicola enzyme was more active when obtainedfrom a pure culture than from double infection. Similarly, mixingthis enzyme with the enzyme of Rhizoctonia reduced its activity.The evidence indicated that the protopectinase of Rhizoctoniawas similar to that of Cylindrocarpon and differed from thatof the Fusarium spp. Using paper partition chromatography, two bands from Rhizoctoniacrude enzyme had a stimulatory effect on Fusarium enzyme, whileonly one band from Fusarium enzyme stimulated Rhizoctonia enzyme. The purified enzyme of Rhizoctonia degraded pectin to galacturonicacid. Fusarium pure enzyme degraded pectin to an intermediatestage. A mixture of the two enzymes degraded pectin to galacturonicacid, without the intermediate stage formed by Fusarium alonebeing detected. The role played by pectic enzymes upon the synergistic relationof Rhizoctonia solani and Fusarium solani on rotting potatotubers is discussed.     

Growth Hormone Changes in Chrysanthemum morifolium: Effects of Environmental Factors Controlling Flowering

Simultaneous quantitative analyses have been made of the endogenouslevels of auxin- and gibberellin like substances, growth inhibitors,and auxin-oxidizing enzyme activity in the cold-requiring Chrysanthemummorifolium cv. Sunbeam subjected to different daylength, lightintensity and temperature regimes known to affect flowering.While little hormone or enzyme activity was found in extractsfrom unvernalized plants, a striking rise in auxin-oxidizingenzyme activity occurred rapidly after the end of cold treatment.Increased auxin activity was also recorded shortly after vernalization.At 28 °C both enzyme and auxin activity declined over aperiod of 3–4 weeks; at 20 °C this response was delayed.Gibberellin activity at 28 °C rose steeply about 2 weeksfrom vernalization and declined several weeks later; at 20 °Ca similar response was less marked. Low light intensity treatment,which may have increased endogenous auxin levels, or exogenousauxin application reduced gibberellin-like substance levelsand cause d devernalization.Phosphon D treatment also loweredgibberellin levels and prevented flowering. An extract fromvernalized plants containing gibberellin-like substances intensifiedthe flowering of partially vernalized test plants. Persistenceof high auxin activity in vernalized plants on long days wasassociated with failure to form normal flower buds. Stem elongationrates correlated in general with levels of endogenous auxin-and gibberellin-like substances. Significant amounts of an abscisin-likeinhibitor were found in extracts of flower buds. The mechanismof natural devernalization is discussed in relation to theseobservations.     

Fat Metabolism in Germinating Citrullus vulgaris

A column chromatographic technique, enabling identificationand quantitative estimation of fatty acids, has been employedto study fat metabolism in Citrullus during germination in thelight. This plant is characterized by an unusually rapid disappearanceof storage fat as the cotyledons expand and turn green. In spiteof the high catabolic activity there is no evidence for accumulationof free fatty acids or short-chain fatty acids at this stage.Information on this point derived from acid value or saponificationvalue of the oil is shown to be untrustworthy. Citrullus seed fat contains the following percentages of acids:linoleic 70·6, oleic 7·2, palmitic 10·1,stearic 11·2, and arachidic 0·6, and careful analysishas also revealed small amounts of octadecatrienoic acids, bothconjugated and non-conjugated. All the major acids are brokendown at rates proportionate to the quantities originally present,with the exception of oleic acid which is metabolized somewhatmore rapidly. ‘Linolenic’ acid is synthesized in the expandinggreen cotyledons and the fatty acid composition of the latter,in the late germination stages, resembles that of a green leafand is very different from that of the seed. The results suggest a rapid removal of storage fat from thecotyledons and concomitant formation in small quantity of atypial leaf fat as the new photo-synthetic function develops.     

Purification and properties of a nitrite reductase from Porphyra yezoensis Ueda

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

Synthesis of O-glycan core 3: Characterization of UDP-GlcNAc: GalNAc-R {beta}3-N-acetyl-glucosaminyltransferase activity from colonic mucosal tissues and lack of the activity in human cancer cell lines

UDP-GlcNAc: GalNAc-R ß3-GlcNAc-transferase (core 3ß3-GlcNAc-T, where GlcNAc is N-acetyl-D-glucosamine,GalNAc is N-acetyl-D-galactosamine and T is transferase) isexpressed in a tissue-specific fashion and is high in normalcolonic tissue, but downregulated in colon cancer. To furtherstudy the control of this enzyme, we examined the activity inpig, rat and human colonic tissues, and several human cancercell lines. The enzyme was difficult to solubilize by detergentsand was extremely unstable in the solubilized form. Using syntheticderivatives of the GalNAc-R substrate, we showed that the specificityof the enzyme in normal rat and human colonic mucosa requiresall the substituents of the GalNAc-sugar ring of substratesfor maximal activity. Core 3 ß3-GlcNAc-T was significantlyinfluenced by the structure of the aglycon group. None of theinactive substrate derivatives could inhibit the activity. N-Iodoacetamido-galactosamine