Chitinase levels in guinea pig blood are increased after systemic infection with Aspergillus fumigatus

The presence of chitinase activity in human serum has recentlybeen described by us. On that occasion we speculated on thepossible role of mammalian chitinases as a defense mechanismagainst chitin-containing pathogens. The results of the presentstudy substantiate our hypothesis. We demonstrate and partiallycharacterize the chitinase activities that are present in plasmaof guinea pigs and in homogenates of A.fumigatus with the aidof the substrates MU-[GlcNAc]_2,3 and also with glycol [³H]chitin.Upon infection with A.fumigatus the serum chitinase activitylevels in the circulation of pathogen-free guinea pigs increasedin a time-dependent manner. The increase was also dependenton the size of the infecting fungal inoculum. Antifungal treatmentdiminished the increases. The increased chitinase activity wasof guinea pig origin. The activity of ß-hexosaminidaseshowed a very slight increase subsequent to the infection. Theactivities of three other enzymes of lysosomal origin (     

{beta}1,4-Galactosyltransferase activity in B cells detected using a simple ELISA-based assay

Lymphocytic ß1,4-galactosyltransferase (ß1,4-GalTase,EC 2.4.1.38 [EC] ) activity was measured in B cells using a neoglycoprotein,N-acetylglucosamine-phenylisothlocyanate-bovine serum albumin(GlcNAc-pITC-BSA), as an acceptor substrate in a novel enzyme-linkedimmunosorbent assay (ELISA)-based method. This assay provedto be much simpler to use than the lengthy and expensive radiochemicalassays commonly used, and has the additional advantage thatit specifically detects the enzyme mediating transfer via theGalß1,4GlcNAc linkage. A F(ab')₂ antibody againstGalTase was able to specifically inhibit the reaction. Greatersensitivity for ß1,4-GalTase activity was obtainedusing GlcNAc-pITC-BSA as an acceptor substrate rather than ovalbumin.Low levels of ß-galactosidase activity were detectablein lymphocyte cell lysates at acidic pH, although such activitywas not detectable at the neutral pH used in the ß1,4-GalTaseactivity assay. Using this assay with the GlcNAc-pITC-BSA acceptor,similar ß1,4-GalTase activities were observed in CD19⁺B cells from patients with rheumatoid arthritis (RA) to thoseseen in normal control individuals. ELISA ß1,4-galactosyltransferase lymphocyte neoglycoprotein radiochemical     

Partial purification and further characterization of the novel endoglucosaminidase from human serum that hydrolyses 4-methylumbelliferyl-N-acetyl-β-d-chitotetraoside (MU-TACT hydrolase)

A novel endoglucosaminidase, originally described by Den Tandt et al. [Int. J. Biochem.20 (1988), 713–719] and bearing the provisional name MU-TACT hydrolase, was purified from human serum 56,000-fold by means of ammonium sulphate precipitation, anion-exchange chromatography, Con A-Sepharose chromatography and gel filtration on Sepharose CL-6B followed by Superose 12HR. Based on the latter technique the native apparent molecular weight of the enzyme appeared to be equal to that of myoglobin, being approx. 17 kD. The enzyme eluted clearly at a different volume than lysozyme. MU-TACT is a commercially available substrate for lysozyme. For unknown reasons two major peptides co-purify that give bands on SDS-PAGE of 55–60 and 31 kD, respectively.     

A novel goose-type lysozyme gene with chitinolytic activity from the moderately thermophilic bacterium <Emphasis Type="Italic">Ralstonia</Emphasis> sp. A-471: cloning,sequencing, and expression

In this study, we cloned the gene encoding goose-type (G-type) lysozyme with chitinase (Ra-ChiC) activity from Ralstonia sp. A-471 genomic DNA library. This is the first report of another type of chitinase after the previously reported chitinases ChiA (Ra-ChiA) and ChiB (Ra-ChiB) in the chitinase system of the moderately thermophilic bacterium, Ralstonia sp. A-471 and also the first such data in Ralstonia sp. G-type lysozyme gene. It consisted of 753 bp nucleotides, which encodes 251 amino acids including a putative signal peptide. This ORF was modular enzyme composed of a signal sequence, chitin-binding domain, linker, and catalytic domain. The catalytic domain of Ra-ChiC showed homologies to those of G-type lysozyme (glycoside hydrolases (GH) family 23, 16.8%) and lysozyme-like enzyme from Clostridium beijerincki (76.1%). Ra-ChiC had activities against ethylene glycol chitin, carboxyl methyl chitin, and soluble chitin but not against the cell wall of Micrococcus lysodeikticus. The enzyme produced α-anomer by hydrolyzing β-1,4-glycosidic linkage of the substrate, indicating that the enzyme catalyzes the hydrolysis through an inverting mechanism. When N-acetylglucosamine hexasaccharide [(GlcNAc)6] was hydrolyzed by the enzyme, the second and third glycosidic linkage from the non-reducing end were split producing (GlcNAc)2 + (GlcNAc)4 and (GlcNAc)3 + (GlcNAc)3 of almost the same concentration in the early stage of the reaction. The G-type lysozyme hydrolyzed (GlcNAc)6 in an endo-splitting manner, which produced (GlcNAc)3 + (GlcNAc)3 predominating over that to (GlcNAc)2 + (GlcNAc)4. Thus, Ra-ChiC was found to be a novel enzyme in its structural and functional properties. The sequence data reported in the present paper have been submitted to the DDBJ, EMBL, and NCBI databases under the accession number AB45458.     

Purification and characterization of avian {beta}1,4 galactosyltransferase: comparison with the mammalian enzyme

Avian ß1,4 galactosyltransferase (GalTase) was purifiedfrom chicken serum, partially characterized, and compared tomammalian GalTase using antibody cross-reactivity, North-ernblot hybridization and amino acid sequence analysis. The enzymewas purified to apparent homogeneity by lactalbumin(LA)-agaroseaffinity chromatography followed by preparative SDS-polyacrylamidegel electrophoresis, and identified as two proteins of apparentmolecular masses of 39 and 46 kD. Chicken serum GalTase hada K_m for UDPGal of 42 µM, for GlcNAc of 10 mM and hadoptimal activity in the presence of 10–20 mM MnCl₂ Substrateand linkage specificity analyses indicated that the purifiedenzyme behaves as a traditional Gal ß1,4 GlcNAc:GalTase,since: (i) the avian ß1,4 GalTase bound to -LA; (ii)terminal GlcNAc residues served as good acceptors for chickenserum GalTase; (iii) the enzyme was inhibited by high concentrationsof GlcNAc; (iv) the galactosylated product was sensitive toß1,4-specific ß-galactosidase. Finally,the disaccharide reaction product comigrated with authenticß1,4 N-acetyllactosamine standard. No other GalTaseactivities were detectable using a battery of defined glycosidesubstrates. Polyclonal antibodies raised against the two gel-purifiedGalTase proteins showed reactivity with avian GalTase by ELISAand immunoprecipitation assays. The antibodies also inhibitedGalTase activity toward both high mol. wt and monosaccharideacceptor substrates. Despite similar kinetics and substratespecificity, the avian and mammalian GalTases showed littleoverall structural similarity, since polyclonal anti-avian GalTaseIgG failed to react with mammalian GalTase purified from bovinemilk, and conversely anti-bovine milk GalTase IgG did not reactwith the avian enzyme. Furthermore, in Northern blot analysis,no hybridization was detected when chicken embryo liver poly(A)⁺RNA was probed with a mouse GalTase cDNA, even under conditionsof reduced stringency. Amino acid sequence analysis identifiedthree of five tryptic peptides that are homologous to the mammaliansequence within a putative substrate binding domain and thecarboxy terminal domain of the enzyme. Their overall structuraldisparity leads us to believe that regions of homology betweenthe avian and mammalian GalTases may represent active sitesof the enzyme. avian ß1,4 galactosyltransferase homology mammalian purification     

Elongation of the N-glycans of fowl plague virus hemagglutinin expressed in Spodoptera frugiperda (Sf9) cells by coexpression of human {beta}1,2-N-acetylglucosaminyltransferase I

Spodoptera frugiperda (Sf9)-cells differ markedly in their proteinglycosylation capacities from vertebrate cells in that theyare not able to generate complex type oligosaccharide side chains.In order to improve the oligosaccha ride processing propertiesof these cells we have used baculovirus vectors for expressionof human (ß1,2-N-acetylglucosaminyltransferase I (hGNT-I),the enzyme catalysing the crucial step in the pathway leadingto complex type N-glycans in vertebrate cells. One vector (Bac/GNT)was designed to express unmodified GNT-I protein, the secondvector (Bac/tagGNT) to express GNT-I protein with a tag epitopefused to its N-terminus. In Sf9-cells infected with Bac/tagGNT-virusa protein of about 50 kDa representing hGNT-I was detected withan antiserum directed against the tag epitope. HGNT-I activitywas increased at least threefold in lysates of infected cellswhen N-acetylglucosamine (GlcNAc)-free ovalbumine was used assubstrate. To monitor hGNT-I activity in intact Sf9-cells, theglycosylation of coexpressed fowl plague virus hemagglutinin(HA) was investigated employing a galactosylation assay andchromatographic analysis of isolated HA N-glycans. Coexpressionof hGNT-I resulted in an at least fourfold increase of HA carryingterminal GlcNAc-residues. The only structure detectable in thisfraction was GlcNAcMan₃GlcNAc₂. These results show that hGNT-Iis functionally active in Sf9-cells and that the N-glycans ofproteins expressed in the baculovirus/insect cell system areelongated by coexpression of glycosyltransferases of vertebrateorigin. Complete complex type oligosaccharide side chains werenot observed when hGNT-I was overexpressed, thus supportingthe concept that Sf9-cells do not contain glycosyltransferasesacting after hGNT-I. ß1,2-N-acetylglucosaminyltransferase I baculovirus expression of recombinant protiens N-glycosylation in Sf9-cells     

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     

A reducing-end-acting chitinase from <Emphasis Type="Italic">Vibrio proteolyticus</Emphasis> belonging to glycoside hydrolase family 19

A chitinase gene belonging to the glycoside hydrolase family 19 from Vibrio proteolyticus (chi19) was cloned. The recombinant enzyme (Chi19) showed weak activities against polymeric substrates and considerable activities against fully N-acetylated chitooligosaccharides, (GlcNAc) _n , whose degree of polymerization was greater than or equal to five. It hydrolyzed (GlcNAc) _n at the second linkage position from the reducing ends of the chitooligosaccharides. The hydrolytic products of colloidal chitin were mainly (GlcNAc)₂ from the initial stage of the reaction. The hydrolytic pattern of reduced colloidal chitin clearly suggested that the enzyme hydrolyzed the polymeric substrate from the reducing end.     

Human melanoma and Chinese hamster ovary cells galactosylate n-alkyl-{beta}-glucosides using UDP gal:GlcNAc {beta}1,4 galactosyltransferase

We previously showed that human melanoma, CHO and other cellscan convert ß-xylosides into structural analogs ofganglioside G_M3. We have investigated several potential acceptorsincluding a series of n-alkyl-ß-D-glucosides (n =6–9). All were labeled with ³H-galactose when incubatedwith human melanoma cells. Octyl-ß-D-glucoside (GlcßOctyl)was the best acceptor, whereas neither octyl--D-glucoside norN-octanoyl-methylglucamine (MEGA 8) were labeled. Analysis ofthe products by a combination of chromatographic methods andspecific enzyme digestions showed that the acceptors first receiveda single Galß1,4 residue followed by an 2,3 linkedsialic acid. Synthesis of these products did not affect cellviability, adherence, protein biosynthesis, or incorporationof radio-labeled precursors into glycoprotein, glycolipid orproteoglycans. To determine which ß1,4 galactosyltransferase synthesized Galß1,4GlcßOctyl,we analyzed similar incubations using CHO cells and a mutantCHO line (CHO 761) which lacks GAG-core specific ß1,4galactosyltransferase. The mutant cells showed the same levelof incorporation as the control, eliminating this enzyme asa candidate. Thermal inactivation kinetics using melanoma cellmicrosomes and rat liver Golgi to galactosylate GlcßOctylshowed the same half-life as UDP-Gal:GlcNAc ß1,4 galactosyltransferase,whereas LacCer synthase was inactivated at a much faster rate.We show that GlcßOctyl is a substrate for purifiedbovine milk UDP-Gal:GlcNAc ß1,4 galactosyltransferaseFurthermore, the galactosylation of GlcßOctyl by CHOcell microsomes can be competitively inhibited by GlcNAc orGlcNAcßMU . These results indicate that UDP-Gal:GlcNAcß1,4 galactosyltransferase is the enzyme used forthe synthesis of the alkyl lactosides when cells or rat liverGolgi are incubated with alkyl ß glucosides. alkylglucosides galactosyltransferase glycolipid artificial acceptors     

Flexibility in the donor substrate specificity of {beta} 1,4-galactosyltransferase: application in the synthesis of complex carbohydrates

Biosynthetically, bovine N-acetylglucosainine ß 1,4-galacto-syltransferase(GalT) catalyses the transfer of galactosyl residues from UDP-Galto the 4-position of GlcNAc units, resulting in the productionof N-acetyllactosamine sequences. UDP-Glc and UDP-GalNAc werealso found to act as donors for this enzyme, allowing the preparationof ßGlc(14)-ßGlcNAc and ßGalNAc(14)ßGlcNActerminating structures on the milligram scale. GalT could thusbe used to add ßGalNAc to ßGlcNAc(12)Manterminating structures, converting them to the ßGalNAc(14)ßGlcNAc(12)Mansequences found on glycoprotein hormones. GalT did not transferGlcNAc residues from UDP-GlcNAc, but it could utilize UDP-GlcNH₂as a donor. Synthesis of ßGlcNAc(14)ßGlcNAcsequences could therefore be accomplished by transfer of GlcNH₂from its UDP derivative, followed by N-acetylation of the productamino-disaccharide using acetic anhydride in methanol. The productsof the enzymatic reactions were characterized by ¹H-NMR-spectroscopyand fast-atom bombardment mass spectrometry. This work expandsthe scope of the combined chemical-enzymatic synthesis of complexcarbohydrates, using glycosyltrans-ferases, to the productionof oligosaccharides different from those for which these enzymeswere designed. These unnatural reactions should find applicationin glycoprotein and glycolipid remodelling. galactosyltransferase chemica1-enzymatic synthesis of oligosaccharides oligosaccharide analogues sugar-nucleotide analogues carbohydrate remodelling     

Glycosidases from Cotyledons of Pisum sativum L.

-Mannosidase and ß-N-acetylglucosaminidase were purifiedfrom extracts of cotyledons of germinating Pisum sativum L.A 13-fold purification of a-mannosidase free from ß-N-acetylglucosaminidaseactivity was achieved by precipitation in ammonium sulphate,column chromatography on DEAE-cellulose, and treatment with2 M pyridine. ß-N-Acetylglucosaminidase was purified200-fold by the use of (NH₄)₂SO₄, and chromatography on ConcanavalinA¹-Sepharose and Sephacryl-200. This preparation showed no measurablecontamination by -mannosidase activity. Both glycosidases appearto be glycoproteins and demonstrate optimal activity at pH valuesof 4.0–4.5. Both glycosidases appear to have very similarmolecular weights, with -mannosidase being slightly larger thanß-N-acetylglucosaminidase. An extensive search forthe activity of aspartylglycosylamine amido hydrolase in peacotyledons proved unsuccessful.     

Tritium labelling of amino sugars at C-2 by alkaline epimerization in tritiated water

N-Acetyl-D-[2-³H]glucosamine was synthesized from N-acetyl-D-mannosamineby alkaline 2-epimerization in pyridine containing ³H₂O andnickelous acetate. The reaction involves reversible formationof an enol intermediate and therefore also resulted in incorporationof tritium into N-acetylmannosamine. After completed reaction,the two N-acetylhexosamines were separated from other radioactiveproducts and Morgan-Elson chromogens by chromatography on acolumn of Sephadex G-10, which was eluted with 10% ethanol,and were then separated from each other by chromatography onSephadex G-15 in 0·27 M sodium borate (pH 7·8).The location of the incorporated tritium was established bytreatment of the N-acetylhexosamines with borate under the conditionsof the Morgan-Elson reaction, which converts the sugars to Kuhn'schromogen I with concomitant loss of the C-2 hydrogen. As expected,this treatment resulted in the formation of ³H₂O, indicatingthat the tritium was located at C-2. [2-³H]Glucosamine was preparedby acid hydrolysis of the labelled N-acetylglucosamine and wasconverted to [2-³H]glucosamine 6-phosphate by incubation withhexokinase and ATP. The sugar phosphate was used as a substratefor glucosamine 6-phosphate deaminase (isomerase, EC 5.3.1.10 [EC] )in a simple ³H₂O release assay. N-acetyl[2-³H]glucosamine N-acetyl[2-³H]mannosamine [2-³H]glucosamine glucosamine 6-phosphate deaminase [2-³H]mannosamine     

Non-identity of human plasma lysozyme and 4-methylumbelliferyl-tetra-N-acetyl-beta-D-chitotetraoside hydrolase

1. Using 4-methylumbelliferyl-tetra-N-acetyl-beta-D-chitotetraoside (MU-TACT) as substrate, it is possible to measure the activity of purified lysozyme and to demonstrate lysozyme activity in the urine of patients with acute monocytic leukemia, characterized by massive lysozymuria. 2. Notwithstanding this observation, we present evidence that in normal human plasma another acid endoglucosaminidase is hydrolyzing the substrate. 3. The following data support the hypothesis of the existence of a separate hydrolase: (a) Thermoinactivation is different for MU-TACT hydrolase and lysozyme. (b) In plasma and many other biological samples, the concentration of lysozyme is too low to be measured with the artificial substrate and there is no correlation between MU-TACT hydrolase and lysozyme. (c) Serum of lysozyme deficient rabbits has normal MU-TACT hydrolase activity. (d) On Sephadex G-200 and DEAE cellulose chromatography, lysozyme and MU-TACT hydrolase are eluted separately. (e) Immunoremoval of lysozyme from human plasma does not affect the activity towards MU-TACT. (f) The effect of N-acetylglucosamine and N-acetylmuramic acid on the activity of lysozyme and MU-TACT hydrolase is different.     

Chitinase activity from Candida albicans and its inhibition by allosamidin

Candida albicans chitinase isolated using the Dyno-Mill disruption technique was characterized using an improved radiometric assay procedure. The enzyme had apparent temperature and pH optima of 45 degrees C and 6.5, respectively. The preparation yielded an apparent Km of 3.9 mg chitin ml-1 [17.6 mM-N-acetylglucosamine (GlcNAc) equivalents] and V of 2.3 nmol GlcNAc formed min-1 (mg protein)-1. The potential of the streptomycete antibiotic allosamidin as an antifungal agent is discussed in view of its dose-dependent inhibition of C. albicans chitinase activity (IC50 = 0.3 microM). Allosamidin was a potent competitive inhibitor of enzyme activity (Ki = 0.23 microM).     

Partial Purification and Characterization of Gibberellin 3{beta}-hydroxylase from Immature Seeds of Phaseolus vulgaris L.

Gibberellin 3/ß-hydroxylase,a 2-oxoglutarate-dependentdioxygenase that catalyzes the hydroxylation of GA₂₀ to GA₁,was purified 313-fold from immature seeds of Phaseolus vulgarisL. The mol wt of the enzyme was estimated to be 42,000 by gelfiltration HPLC and SDS-polyacrylamide gel electrophoresis.The enzyme exhibited maximum activity at pH 7.7. The Km valuesfor [2,3-³H]GA20 and [2,3-³H]GA, were 0.29µu and 0.33µm, respectively. The enzyme requires 2-oxoglutarate asa cosubstrate; the K_m value for 2-oxoglutarate was 250µMusing [³H]- GA₂₀ as a substrate. Fe²⁺ and ascorbate significantlyactivated the enzyme at all purification steps, while catalaseand BSA activated the purified enzyme only. The enzyme was inhibitedby divalent cations Mn²⁺, Co²⁺, Ni²⁺, Cu²⁺, Zn²⁺, Cd²⁺ and Hg²⁺.3ß-Hydroxylation of [³H]- GA₂₀ was also inhibitedby non-radioactive GA₅, GA₉,GA₁₅, GA₂₀ and GA₄₄. The possiblesite of 3ß-hydroxylation in gibberellin biosynthesisis discussed in terms of the substrate specificity of partiallypurified gibberellin 3ß-hydroxylase. (Received February 29, 1988; Accepted June 3, 1988)     

Light and heavy lysosomes: characterization of N-acetyl-{beta}-D-hexosaminidase isolated from normal and I-cell disease lymphoblasts

We previously reported that I-cell disease lymphoblasts maintainnormal or near-normal intracellular levels of lysosomal enzymes,even though N-acetylglucosamine-1-phosphotransferase activityis severely depressed or absent (Little et al., Biochem. J.,248, 151–159, 1987). The present study, employing subcellularfractionation on colloidal silica gradients, indicates thatboth light and heavy lysosomes isolated from I-cell diseaseand pseudo-Hurler polydystrophy lymphoblasts possess normalspecific activity levels of N-acetyl-ß-D-hexosaminidase,-D-mannosidase and ß-D-glucuronidase. These currentfindings are in contrast to those of cultured fibroblasts fromthe same patients, where decreased intralysosomal enzyme activitiesare found. Column chromatography on Ricinus communis revealedthat N-acetyl-ß-D-hexosaminidase in both heavy andlight I-cell disease lysosomal fractions from lymphoblasts possessesan increased number of accessible galactose residues (30–50%)as compared to the enzyme from the corresponding normal controls.Endo-ß-N-acetylglucos-aminidase H treatment of N-acetyl-ß-D-hexosaminidasefrom the I-cell lysosomal fractions suggests that the majorityof newly synthesized high-mannose-type oligosaccharide chainsare modified to complex-type carbohydrates prior to being transportedto lysosomes. This result from lymphoblasts differs from previousfindings with fibroblasts, where N-acetyl-ß-D-hexosaminidasefrom I-cell disease and pseudo-Hurler polydystrophy lysosomesexhibited properties associated with predominantly high-mannose-typeoligosaccharide chains. The current results imply that differentcell types may modify the carbohydrate side chains of lysosomalenzymes in a differential manner, and that selected cell typesmay also employ mechanisms other than the mannose-6-phosphatepathway for targeting lysosomal enzymes to lysosomes. I-cell disease lymphoblasts lysosomes mannose-6-phosphate oligosaccharide chains pseudo-Hurler polydystrophy     

Isolation, characterization and inactivation of the mouse Mgat3 gene: the bisecting N-acetylglucosamine in asparagine-linked oligosaccharides appears dispensable for viability and reproduction

The biosynthesis of complex asparagine (N)-linked oligosaccharidesin vertebrates proceeds with the linkage of N-acetylglucosamine(GlcNAc) to the core mannose residues. UDP-N-acetylglucosamine:ß-D-mannosideß1–4 N-acetylglucosaminyltransferase III (GlcNAc-TIII,EC2.4.1.144) catalyzes the addition of GlcNAc to the mannosethat is itself ß1–4 linked to underlying N-acetylglucosamine.GlcNAc-TIII thereby produces what is known as a ‘bisecting’GlcNAc linkage which is found on various hybrid and complexN-glycans. GlcNAc-TIII can also play a regulatory role in N-glycanbiosynthesis as addition of the bisecting GlcNAc eliminatesthe potential for     

Effects of Deoxygibberellin C (DGC) and 16-Deoxo-DGC on Gibberellin 3{beta}-Hydroxylase and Plant Growth

Deoxygibberellin C (DGC), a C/D ring-rearranged isomer of GA₂₀,was shown to inhibit the conversion of [2,3-³H₂]GA₉ to [2-³H]GA₄by gibberellin 3ß-hydroxylase from immature seedsof Phaseolus vulgahs. Deoxygibberellin C inhibited the promotionof growth by exogenously applied GA₂₀ of rice (Oryza sativaL.) seedlings. Evidence is also presented that DGC is a competitiveinhibitor of the 3ß-hydroxylase from P. vulgaris.However, DGC only weakly inhibited the conversion catalyzedby the 3ß-hydroxylase from Cucurbita maxima at highconcentrations, and it did not inhibit the promotion of growthby exogenously applied GA₉ of cucumber (Cucumis sativus) seedlings.These results suggest that the 3ß-hydroxylases fromP. vulgaris and C. maxima have different structural requirementswith respect to their substrates. 16-Deoxo-DGC also inhibitedcatalysis of the same conversion by 3ß-hydroxylasefrom P. vulgaris, and it slightly inhibited the conversion catalyzedby the enzyme from C. maxima. Application of 16-deoxo-DGC causedthe promotion of the growth of seedlings of both rice and cucumber. ³ Present address: Genetic Engineering Center, Korea Instituteof Science and Technology, Daejeon 305–606, Korea ⁴ Present address: Department of Agricultural Chemistry, UtsunomiyaUniversity, Utsunomiya-shi, Tochigi, 321 Japan (Received September 25, 1990; Accepted December 17, 1990)     

Enzymatic characterization of the Plasmodium vivax chitinase, a potential malaria transmission-blocking target

The chitinase (EC 3.2.1.14) of the human malaria parasite Plasmodium falciparum, PfCHT1, has been validated as a malaria transmission-blocking vaccine (TBV). The present study aimed to delineate functional characteristics of the P. vivax chitinase PvCHT1, whose primary structure differs from that of PfCHT1 by having proenzyme and chitin-binding domains. The recombinant protein rPvCHT1 expressed with a wheat germ cell-free system hydrolyzed 4-methylumbelliferone (4MU) derivatives of chitin oligosaccharides (β-1,4-poly-N-acetyl glucosamine (GlcNAc)). An anti-rPvCHT1 polyclonal antiserum reacted with in vitro-obtained P. vivax ookinetes in anterior cytoplasm, showing uneven patchy distribution. Enzymatic activity of rPvCHT1 shared the exclusive endochitinase property with parallelly expressed rPfCHT1 as demonstrated by a marked substrate preference for 4MU-GlcNAc₃ compared to shorter GlcNAc substrates. While rPvCHT1 was found to be sensitive to the general family-18 chitinase inhibitor, allosamidin, its pH (maximal in neutral environment) and temperature (max. at ~ 25 °C) activity profiles and sensitivity to allosamidin (IC₅₀ = 6 µM) were different from rPfCHT1. The results in this first report of functional rPvCHT1 synthesis indicate that the P. vivax chitinase is enzymatically close to long form Plasmodium chitinases represented by P. gallinaceum PgCHT1.