Expression Patterns of PR proteins with Different Extract Methods during Germination of Rape Seed (<Emphasis Type="Italic">Brassica napus</Emphasis> L.)

This study was conducted to investigate the expression patterns of pathogenesis-related proteins (chitinase, β-1,3-glucanase and peroxidase) using activity staining of native-polyacrylamide gel electrophoresis (PAGE) and sodium dodecyl sulfate (SDS)-PAGE during germination of rape seed (Brassica napus L. cv. Saturnin). The crude enzymes were extracted by distilled water (DW, pH 6.0) and 100 mM K-PO₄ buffer (pH 7.0). The expression patterns of chitinase isozymes changed clearly on 10% native-PAGE gel with DW and K-PO₄ buffer extract and on 12% SDS-PAGE gel with K-PO₄ buffer extract, except for 12% SDS-PAGE conducted using DW during germination. The active bands of the chitinase isozymes were observed as four major bands (ch1, ch2, 86, and 78 kDa) and three minor bands (71, 60, and 54 kDa) on 10% native-PAGE gel conducted using DW and K-PO₄ buffer extract. The two active bands on the 12% (w/v) SDS-PAGE gel presented as 34 and 29 kDa with DW extract, whereas one active band of 34 kDa was observed when the K-PO₄ buffer extract was used. Active bands of β-1,3-glucanase isozymes changed slightly on 10% native-PAGE gel with DW and K-PO₄ buffer extract during germination. The active band of β-1,3-glucanase isozymes were shown to have a high molecular weight (G1 and G2) on native-PAGE gel with DW extract at 0, 1, 2, and 3 days after germination, but not at 4 and 5 days. One active band of β-1,3-glucanase presented as G1 in the K-PO₄ buffer extract. Active staining of peroxidase was stronger earlier in the DW extract than K-PO₄ buffer extract at 2 days. The active bands showed as P1 and P2 in both DW and K-PO₄ buffer extract at 5 days after germination.     

The extracellular constitutive production of chitin deacetylase in Metarhizium anisopliae: possible edge to entomopathogenic fungi in the biological control of insect pests

The possible contribution of extracellular constitutively produced chitin deacetylase by Metarhizium anisopliae in the process of insect pathogenesis has been evaluated. Chitin deacetylase converts chitin, a beta-1,4-linked N-acetylglucosamine polymer, into its deacetylated form chitosan, a glucosamine polymer. When grown in a yeast extract-peptone medium, M. anisopliae constitutively produced the enzymes protease, lipase, and two chitin-metabolizing enzymes, viz. chitin deacetylase (CDA) and chitosanase. Chitinase activity was induced in chitin-containing medium. Staining of 7.5% native polyacrylamide gels at pH 8.9 revealed CDA activity in three bands. SDS-PAGE showed that the apparent molecular masses of the three isoforms were 70, 37, and 26 kDa, respectively. Solubilized melanin (10microg) inhibited chitinase activity, whereas CDA was unaffected. Following germination of M. anisopliae conidia on isolated Helicoverpa armigera, cuticle revealed the presence of chitosan by staining with 3-methyl-2-benzothiazoline hydrazone. Blue patches of chitosan were observed on cuticle, indicating conversion of chitin to chitosan. Hydrolysis of chitin with constitutively produced enzymes of M. anisopliae suggested that CDA along with chitosanase contributed significantly to chitin hydrolysis. Thus, chitin deacetylase was important in initiating pathogenesis of M. anisopliae softening the insect cuticle to aid mycelial penetration. Evaluation of CDA and chitinase activities in other isolates of Metarhizium showed that those strains had low chitinase activity but high CDA activity. Chemical assays of M. anisopliae cell wall composition revealed the presence of chitosan. CDA may have a dual role in modifying the insect cuticular chitin for easy penetration as well as for altering its own cell walls for defense from insect chitinase.     

聚丙烯酰胺凝胶电泳配合荧光增白剂显色检测木霉几丁质酶同工酶谱

为提高木霉几丁质酶检测方法的准确性和灵敏度,建立一种快速检测几丁质酶同工酶的方法。采用活性凝胶电泳、变性凝胶电泳、原位显色凝胶电泳结合荧光增白剂（Calcofluor white M2R）显色从绿色木霉LTR-2发酵产物中检测几丁质酶同工酶。活性凝胶电泳在粗酶液浓缩5倍时显示两条活性谱带,变性凝胶电泳在浓缩10倍时显示一条活性谱带,原位显色凝胶电泳在浓缩20倍时显示两条不清晰的活性谱带,SDS-PAGE显示这两条活性谱带的分子量分别为65kDa和42kDa。结果表明活性聚丙烯酰胺凝胶电泳和Calcofluor white M2R显色相结合的方法在几丁质酶上样量为0.47U时具有较好的分辨能力,是检测木霉几丁质酶同工酶的有效的方法。     

Activity and Differential Induction of Chitinase Isozymes in Soybean Cultivars Resistant or Susceptible to Root-knot Nematodes

Host physiological events in relation to infestation by parasitic nematodes are not well documented. Soybean plant responses to Meloidogyne incognita infestation were compared to resistant (Bryan) and susceptible (Brim) cultivars at 0, 1, 3, 10, 20, and 34 days after infestation (DAI). The resistant cultivar had higher chitinase activity than the susceptible cultivar at every sample time beginning at 3 DAI. Results from isoelectric focusing gel electrophoresis analyses indicated that three acidic chitinase isozymes with isoelectric points (pIs) of 4.8, 4.4, and 4.2 accumulated to a greater extent in the resistant compared to the susceptible cultivar following challenge. SDS-PAGE analysis of root proteins revealed that two proteins with molecular weights of approximately 31 and 46 kD accumulated more rapidly and to a higher level in the resistant than in the susceptible cultivar. Additionally, three major protein bands (33, 22, and 20 kD) with chitinase activity were detected with a modified SDS-PAGE analysis in which glycolchitin was added into the gel matrix. These results indicate that higher chitinase activity and early induction of specific chitinase isozymes may be associated with resistance to root-knot nematode in soybean.     

Production and purification of extracellular chitinases from Penicillium aculeatum NRRL 2129 under solid-state fermentation

Fourteen Penicillium strains have been screened on wheat bran–crude chitin mixture medium for extracellular chitinase production in solid-state fermentation. Under the experimental conditions tested, Penicillium aculeatum NRRL 2129 (=ATCC 10409) was selected as the best enzyme producer. The optimum incubation period for chitinase production by the potent organism was found to be 72 h. Chromatofocusing was performed as the first step in the purification scheme, but high amount of contaminating proteins interfered with the method. Hence, ion-exchange chromatography experiments were carried out followed by gel filtration to separate and isolate chitinase isoenzymes. Four major chitinase peaks of molecular weight 82.7, 44.6, 28.2 and 26.9 kDa were observed after gel filtration chromatography while, on SDS-PAGE, three protein bands of molecular weights 82.6, 33.9 and 29.1 kDa were identified. The purified enzyme showed optimal temperature and pH at 50 and 5.5 °C, respectively.     

Production of an extracellular chitinolytic system byTalaromyces emersonii CBS 814.70.

Summary The thermophilic fungusTalaromyces emersonii CBS 814.70 produces a thermostable extracellular chitinolytic system when cultured on chitin containing media. The chitinolytic system consists of chitinase (EC 3.2.1.14) and N-acetylglucosaminidase (EC 3.2.1.30). Using fluorescent substrate analogues, in zymogram staining of polyacrylamide gradient and isoelectric focusing gels on which the chitinase system was electrophoresed and focused, respectively, it was found that a number of bands could be resolved. Using isoelectric focusing it was observed that at least 4 extracellular forms of chitinase activity are produced.     

Cytosolic and membrane-bound chitinases of two mucoraceous fungi: a comparative study.

Chitinases isolated from membrane and cytosolic fractions of two mucoraceous fungi, Choanephora cucurbitarum and Phascolomyces articulosus, were investigated. The membrane-bound chitinase was isolated by Bio-Gel P-100 and DEAE Bio-Gel A chromatographic techniques. On SDS-PAGE the chitinase from both fungi migrated as a single band of M(r) 66 kDa. The cytosolic chitinase from the mycelial extracts of these fungi was separated by heat treatment, ammonium sulphate precipitation, and by affinity chromatography with regenerated chitin. SDS-PAGE showed two bands for each fungus with M(r) of 69.5 and 55 kDa in C. cucurbitarum and M(r) 69.5 and 53 kDa in Ph. articulosus. Chitinases, membrane bound or cytosolic, hydrolyzed regenerated chitin, colloidal chitin, glycol chitin, N,N'-diacetylchitobiose, and N,N',N"-triacetylchitotriose. Heavy metals, inhibitors, and N-acetylglucosamine inhibited chitinase activity, whereas trypsin and an acid protease enhanced its activity. Chitinase preparations showed lysozyme activity that was inhibited by histamine but not by N-acetylglucosamine. There was no N-acetylglucosamanidase activity, but beta-1,3 glucanase activity was found in cytosolic preparations only. Despite slight differences in their molecular mass, both the membrane-bound and cytosolic chitinases showed similarities in substrate utilization, response to inhibitors, and activation by trypsin and acid protease; pH and temperature optima also were similar.     

Effects of carbon and nitrogen sources on the induction and repression of chitinase enzyme from<Emphasis Type="Italic">Metarhizium anisopliae</Emphasis> isolates

Metarhizium anisopliae, an entomopathogenic hyphomycete, is being used effectively in Integrated Pest Management (IPM) system. Foliar application of these fungi is quite satisfactory as it invades its host by adhering to insect cuticles and formation of penetration structures called appresoria, which produces various extracellular enzymes, including chitinase that causes the insect cuticle breaching. The induction and repression mechanism of chitinase activity is not entirely understood and activity of this enzyme is different in response to different carbon and nitrogen sources. This report illustrates the effect of two carbon sources viz. colloidal chitin and dextrose and a nitrogen source, yeast extract on the chitinase production of fourteenM. Anisopliae isolates. The chitinase activity varied among the isolates and the different media used. A high enzymatic activity was observed in the medium containing an extra nitrogen source (yeast extract) followed by the medium containing colloidal chitin as a sole source of carbon and nitrogen. The exochitinase activity and the chitinase activity gel were also studied for the isolates showing high chitinase enzyme production. An array of chitinase isozymes were observed on chitinase activity gel with a common 14.3 kDa enzyme for all the isolates.     

Transformants of Metarhizium anisopliae sf. anisopliae overexpressing chitinase from Metarhizium anisopliae sf. acridum show early induction of native chitinase but are not altered in pathogenicity to Manduca sexta

Extracellular chitinase activity has been implicated in the pathogenesis of several fungal infections. Following induction with chitin, the insect pathogens Metarhizium anisopliae sf. acridum ARSEF strain 324 and Metarhizium anisopliae sf. anisopliae ARSEF strain 2575 secrete 44-kDa basic and acidic isoforms of endochitinase, respectively. The gene from strain 324 (Chit1) was cloned and inserted into the genome of strain 2575 under the control of Aspergillus regulatory elements such that transgenic 2575 (2575-Chit(+)) expressed CHIT1 in a noninducing medium (i.e., not containing chitin). Isoelectric focusing followed by a zymogram technique revealed that neither wild-type 2575 nor 2575-Chit(+) produced significant amounts of the native 2575 acidic chitinase in a noninducing medium. However, in a chitin-containing medium, 2575-Chit(+) produced the native chitinase earlier than strain 2575, soon after secretion of CHIT1. We hypothesize that this is due to the production of soluble inducers following chitin hydrolysis by CHIT1 and that M. anisopliae uses enzymes expressed at low levels to sense the nature of the polymeric nutrient present in the immediate environment. However, the chitinase overproducers did not show altered virulence to caterpillars (Manduca sexta) compared to the wild-type fungus, suggesting that wild-type levels of chitinase are not limiting for cuticle penetration.     

Identification, effective purification and functional characterization of dextransucrase from Leuconostoc mesenteroides NRRL B-640

The extracellular dextransucrase from Leuconostoc mesenteroides NRRL B-640 was purified using polyethylene glycol fractionation (PEG) and gel-filtration. The cell free extract was subjected to fractionation by PEG-200, 400 and 1500. The 10% (w/v) PEG-1500 gave dextransucrase with maximum specific activity of 23 with 40 fold purification in a single step. The purified enzyme showed multiple molecular forms on SDS-PAGE, however the same sample showed a single band on non-denaturing native-PAGE. The purified dextransucrase fractions obtained from PEG-1500, confirmed the presence of dextran, when run on SDS-PAGE under non-denaturing gels for in situ activity detection by Periodic Acid Schiff's staining. The activity bands corresponded to the native and active form of the purified dextransucrase of approximately, 180kDa molecular size, that appeared on the denaturing gels stained with Coomassie Brilliant Blue. No bands appeared after staining the activity of dextransucrase on non denaturing SDS-PAGE gels with raffinose, which excluded the presence of fructosyltransferases. Further purification of 10% PEG-1500 purified dextransucrase by gel-filtration gave dextransucrase with specific activity of 35 with 61 fold purification.     

Purification, Characterization, and Antifungal Activity of Chitinase from Streptomyces venezuelae P10

Streptomyces venezuelae P₁₀ could produce extracellular chitinase in a medium containing 0.6% colloidal chitin that was fermented for 96 hours at 30°C. The enzyme was purified to apparent homogeneity with 80% saturation of ammonium sulfate as shown by chitin affinity chromatography and DEAE-cellulose anion-exchange chromatography. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) of the enzyme showed a molecular weight of 66 kDa. The chitinase was characterized, and antifungal activity was observed against phytopathogens. Also, the first 15 N-terminal amino-acid residues of the chitinase were determined. The chitin hydrolysed products were N-acetylglucosamine and N, N’-diacetylchitobiose.     

Bioconversion of shellfish chitin wastes for the production of Bacillus subtilis W-118 chitinase

Bacillus subtilis W-118, a strain that produces antifungal materials, excreted a chitinase when cultured in a medium containing shrimp- and crab-shell powder as the major carbon source. This chitinase, purified by sequential chromatography, had a molecular mass of 20,600 Da and a pI of 6. The optimum pH, optimum temperature, and pH stability of the chitinase were pH 6, 37 degrees C, and pH 5-7, respectively. The unique characteristics of the purified chitinase include low molecular mass and acidic pI. In the investigation of the inhibitory activity, it was found that the growth of Fusarium oxysporum was 100% inhibited after incubation for 1 day with sterilized W-118 chitinase solution (5.6 units/mL). The chitinase hydrolyzates of chitin with low degrees of polymerization (DP 1-6) were analyzed by HPLC. Longer reaction times led to the generation of chitin oligosaccharides with lower DP. The chitin oligosaccharides were examined for their inhibitory effects on F. oxysporum and human leukemia cell lines.     

Purification and Characteristics of an Autolytic Chitinase of Piromyces communis OTS1 from Culture Medium

An autolysis chitinase was purified from the cultural medium of the anaerobic fungus Piromyces communis OTS1 by ammonium sulfate precipitation, affinity chromatography with regenerated chitin, chromato-focusing, gel filtration, and chromato-focusing again. The optimal pH and temperature were 6.0 and 50°C, respectively, for a 20-min assay. The chitinase was stable from pH 6.0 to 8.0, but was unstable at 70°C for 20 min. The molecular mass of chitinase was estimated by SDS-PAGE to be 44.9 kDa, and its pI was 4.4. The enzyme activity, which was of the ‘endo’ type, was inhibited by Hg²⁺ and allosamidin. The chitinase hydrolyzes chitin powder and fungal cell walls at a higher rate than an artificial chitin substrate. It can be concluded that extracellular chitinase is similar to cytosolic chitinase, but they are not the same protein. Received: 3 December 1996 / Accepted: 28 January 1997     

Production of chitinolytic enzymes from a novel species ofAeromonas

A bacterial strain secreting potent chitinolytic activity was isolated from shrimp-pond water by enrichment culture using colloidal crab-shell chitin as the major carbon source. The isolated bacterium, designated asAeromonas sp No. 16 exhibited a rod-like morphology with a polar flagellum. Under optimal culture conditions in 500-ml shaker flasks, it produced a chitinolytic activity of 1.4 U ml^–1. A slightly higher enzymatic activity of 1.5 U ml^–1 was obtained when cultivation was carried out in a 5-liter jar fermentor using a medium containing crystalline chitin as the carbon source. The secretion of the enzyme(s) was stimulated by several organic nitrogenous supplements. Most carbon sources tested (glucose, maltose, N-acetylglucosamine, etc) enhanced cell growth, but they slightly inhibited enzyme secretion. Glucosamine (0.5% w/v) severely inhibited cell growth (16% of the control), but it did not significantly affect enzyme secretion. The production of chitinolytic enzymes was pH sensitive and was enhanced by increasing the concentration of colloidal chitin to 1.5%. The observed chitinolytic activity could be attributed to the presence of -N-acetylglucosaminidase and chitinase. Chitinase was purified by ammonium sulfate fractionation and preparative gel electrophoresis to three major bands on SDS-PAGE. An in-gel enzymatic activity assay indicated that all three bands possessed chitinase activity. Analysis of the enzymatic products indicated that the purified enzyme(s) hydrolyzed colloidal chitin predominantly to N,N-diacetyl-chitobiose and, to a much lesser extent, the mono-, tri, and tetramer of N-acetylglucosamine, suggesting that they are mainly endochitinases.     

Assignment of human plasma methylumbelliferyl-tetra-N-acetylchitotetraoside hydrolase or chitinase to chromosome 1q by a linkage study

Plasma methylumbelliferyl tetra-N-acetylchitotetraoside hydrolase or chitinase (CHIT) might play a role in degrading the chitin wall of some microorganisms. In about 6% of Caucasian people the enzyme shows pseudodeficiency (defined as very low activity without apparent symptoms). We have mapped this locus by linkage analysis to the marker D1S306 (z = 4.00 at θ _{M = F} = 0.0) on chromosome 1q between the flanking markers D1S191 and D1S245 in the area of 1q31–1qter. Received: 13 December 1996 / Accepted: 8 July 1997     

Purification,characterization, and molecular cloning of chitinases from the stomach of the threeline grunt Parapristipoma trilineatum

Two chitinase isozymes, PtChiA and PtChiB, were purified from the stomach of the threeline grunt, Parapristipoma trilineatum. The molecular masses of PtChiA and PtChiB were estimated to be 50 and 60 kDa by SDS-PAGE, respectively. Both chitinases were stable at pH 3.0–6.0 (acidic) and showed the optimum pH toward both short and long substrates in the acidic region (pH 2.5–5.0). PtChiA and PtChiB preferentially degraded the second and third glycosidic bonds from the non-reducing end of N-acetylchitooligosaccharides, respectively. PtChiA and PtChiB exhibited wide substrate specificities toward crystalline chitin. Moreover, 2 cDNAs encoding PtChiA and PtChiB, PtChi-1 and PtChi-2, respectively, were cloned. The deduced amino acid sequences of both chitinase cDNAs comprised N-terminal signal peptides, glycoside hydrolase 18 catalytic domains, linker regions, and C-terminal chitin-binding domains. Phylogenetic tree analysis of vertebrate chitinases revealed that fish stomach chitinases including PtChi-1 and PtChi-2 form unique chitinase groups, acidic fish chitinase-1 (AFCase-1) and acidic fish chitinase-2 (AFCase-2), which differ from the acidic mammalian chitinase (AMCase) group. The present results suggest that fish have a chitin-degrading enzymatic system in which 2 different chitinases, AFCase-1 and AFCase-2, with different degradation patterns are expressed in the stomach.     

Purification and characterization of chitinases from <Emphasis Type="Italic">Paecilomyces</Emphasis><Emphasis Type="Italic">variotii</Emphasis> DG-3 parasitizing on <Emphasis Type="Italic">Meloidogyne incognita</Emphasis> eggs

Two extracellular chitinases were purified from Paecilomyces variotii DG-3, a chitinase producer and a nematode egg-parasitic fungus, to homogeneity by DEAE Sephadex A-50 and Sephadex G-100 chromatography. The purified enzymes were a monomer with an apparent molecular mass of 32 kDa (Chi32) and 46 kDa (Chi46), respectively, and showed chitinase activity bands with 0.01% glycol chitin as a substrate after SDS-PAGE. The first 20 and 15 N-terminal amino acid sequences of Chi32 and Chi46 were determined to be Asp-Pro-Typ-Gln-Thr-Asn-Val-Val-Tyr-Thr-Gly-Gln-Asp-Phe-Val-Ser-Pro-Asp-Leu-Phe and Asp-Ala-X-X-Tyr-Arg-Ser-Val-Ala-Tyr-Phe-Val-Asn-Trp-Ala, respectively. Optimal temperature and pH of the Chi32 and Chi46 were found to be both 60°C, and 2.5 and 3.0, respectively. Chi32 was almost inhibited by metal ions Ag⁺ and Hg²⁺ while Chi46 by Hg²⁺ and Pb²⁺ at a 10 mM concentration but both enzymes were enhanced by 1 mM concentration of Co²⁺. On analyzing the hydrolyzates of chitin oligomers [(GlcNAc) _n , n = 2–6)], it was considered that Chi32 degraded chitin oligomers as an exo-type chitinase while Chi46 as an endo-type chitinase.     

Chitinase system of Bacillus circulans WL-12 and importance of chitinase A1 in chitin degradation.

Bacillus circulans WL-12, isolated as a yeast cell wall-lytic bacterium, secretes a variety of polysaccharide-degrading enzymes into culture medium. When chitinases of the bacterium were induced with chitin, six distinct chitinase molecules were detected in the culture supernatant. These chitinases (A1, A2, B1, B2, C, and D) showed the following distinct sizes and isoelectric points: Mr 74,000, pI 4.7 (A1); Mr 69,000, pI 4.5 (A2); Mr 38,000, pI 6.6 (B1); Mr 38,000, pI 5.9 (B2); Mr 39,000, pI 8.5 (C); and Mr 52,000, pI 5.2 (D). Among these chitinases, A1 and A2 had the highest colloidal-chitin-hydrolyzing activities. Chitinase A1 showed a strong affinity to insoluble substrate chitin. Purified chitinase A1 released predominantly chitobiose [(GlcNAc)2] and a trace amount of N-acetylglucosamine (GlcNAc) from colloidal chitin. N-terminal amino acid sequence analysis of chitinases A1 and A2 indicated that chitinase A2 was generated from chitinase A1, presumably by proteolytic removal of a C-terminal portion of chitinase A1. Since chitinase A2 did not have the ability to bind to chitin, the importance of the C-terminal region of chitinase A1 to the strong affinity of chitinase A1 to substrate chitin was suggested. Strong affinity of the chitinase seemed to be required for complete degradation of insoluble substrate chitin. From these results, it was concluded that chitinase A1 is the key enzyme in the chitinase system of this bacterium.     

Characterization of the eggshell of Haemonchus contortus--I. Structural components.

1. By transmission electron microscopy, the eggshell of Haemonchus contortus was seen to be similar to previously studied nematodes, with an outer vitelline layer bounded by a trilaminate membrane, a broad medial region, containing chitin, and an electron dense basal region, containing lipid and protein. 2. Exposure of Haemonchus contortus eggs to proteases resulted in disruption of the shell with removal of components of the outer, medial and basal regions. Exposure to chitinase depleted fibrillar components of the medial region of the shell, while collagenase had no effect. 3. Chloroform/methanol extraction of fresh eggshells caused a minor condensation of the outer, vitelline layer and some depletion of the basal layer. 4. After normal hatching, shells appeared similar to those treated with protease and chitinase, but also lacked the basal, lipid layer. 5. Extracts of isolated unhatched eggshells and hatched eggshells, and extracts of biotin-labelled whole fresh eggs showed three major protein bands when run on sodium dodecyl sulphate-polyacrylamide gels indicating that these three proteins are most likely structural in nature and do not participate in the release of the larva from the eggshell. 6. Biotin-labelled protein bands were degraded by proteases and chitinase, but not collagenase or lipase.     

Leuconostoc mesenteroides B-1355 Mutants Producing Alternansucrases Exhibiting Decreases in Apparent Molecular Mass

Mutants of Leuconostoc mesenteroides B-1355 exhibiting decreases in the apparent molecular mass of alternansucrase on sodium dodecyl sulfate (SDS)-polyacrylamide gels stained for enzyme activity were isolated after mutagenizing strain R15 with N-methyl-N(prm1)-nitro-N-nitrosoguanidine. Strain R15 was a UV mutant of strain B-1355 which was enriched for production of alternansucrase. All strains produced principal and minor alternansucrase bands on SDS gels when cultures were subjected to SDS-polyacrylamide gel electrophoresis (SDS-PAGE). The patterns of the principal and minor activity bands on our SDS gels did not result from dextran-enzyme complexes, because mutants constitutive for synthesis of glucosyltransferases (GTFs) on sugars other than sucrose produced activity bands after growth in glucose medium that were the same as those produced after growth in sucrose medium. Dextransucrase, which had been inactivated by heating at 45(deg)C, was reactivated when subjected to SDS-PAGE, showing that our SDS-PAGE conditions were reversibly denaturing. Thermal denaturation at 45(deg)C did not involve a dispersal of GTFs into subunits. Densitometry measurements showed a roughly linear relationship between enzyme activity and band intensity over a loading range of 0.2 to 0.8 mU per sample well. We concluded that SDS-PAGE followed by activity staining was a reliable method for estimating numbers and ratios of GTFs produced by Leuconostoc sp. in media containing sucrose.