Purification and characterization of two types of fumarase from Escherichia coli

Two distinct types of fumarase were purified to homogeneity from aerobically grown Escherichia coli W cells. The amino acid sequences of their NH2-terminals suggest that the two enzymes are the products of the fumA gene (FUMA) and fumC gene (FUMC), respectively. FUMA was separated from FUMC by chromatography on a Q-Sepharose column, and was further purified to homogeneity on Alkyl-Superose, Mono Q, and Superose 12 columns. FUMA is a dimer composed of identical subunits (Mr = 60,000). Although the activity of FUMA rapidly decreased during storage, reactivation was attained by anaerobic incubation with Fe2+ and thiols. Studies on the inactivation and reactivation of FUMA suggested that oxidation and the concomitant release of iron inactivated the enzyme in a reversible manner. While the inactivated FUMA was EPR-detectable, through a signal with g perpendicular = 2.02 and g = 2.00, the active enzyme was EPR-silent. These results suggested FUMA is a member of the 4Fe-4S hydratases represented by aconitase. After the separation of FUMC from FUMA, purification of the former enzyme was accomplished by chromatography on Phenyl-Superose and Matrex Gel Red A columns. FUMC was stable, Fe-independent and quite similar to mammalian fumarases in enzymatic properties.     

Continuous production ofL-malic acid by immobilizedBrevibacterium ammoniagenes cells

Summary Continuous production ofL-malic acid from fumaric acid using immobilized microbial cells was investigated. Several microorganisms having fumarase activity were immobilized into a polyacrylamide gel lattice. Among the microorganisms tested, immobilizedBrevibacterium ammoniagenes IAM 1645 showed the highest enzyme activity, but produced an unwanted by-product, succinic acid. Conditions for suppression of this side reaction were investigated, and bile extract treatment of immobilized cells was found to be effective.The bile extract treatment of immobilized cells also resulted in a marked increase of reaction rate forL-malic acid formation.No difference was observed between the native enzyme and immobilized cells in optimal pH and temperature of the enzyme reaction.The effect of temperature on the reaction rate and the stability of fumarase activity of an immobilized cell column were investigated under conditions of continuous enzyme reaction. The decay of enzyme activity during continuous enzyme reaction was expressed by an exponential relationship. Half-life of the fumarase activity of the immobilized cell column at 37°C was calculated to be 52.5 days.Presented at the Annual Meeting of the Society of Fermentation Technology, Japan, Osaka, Japan, October 30, 1975.     

Immobilization of Brevibacterium flavum with carrageenan and its application for continuous production of l-malic acid

Extensive experiments were carried out to improve the productivity ofl-malic acid from fumaric acid using Brevibacterium flavum immobilized with carrageenan. The most favourable preparation for the continuous production ofl-malic acid was obtained when 16 g of B. flavum cells was entrapped in 100 ml 3.4% carrageenan gel. However, the immobilized cells produced an unwanted by-product, succinic acid. Treatment of the immobilized cells with 0.6% bile extract suppressed the side reaction and gave the highest operational stability of fumarase activity. By the immobilization of intact cells, the optimal temperature of the enzyme reaction shifted to 10°C higher, the optimal pH became broader, and the operational stability of fumarase activity increased. The effect of temperature on the stability of fumarase activity in the immobilized cell column was investigated under conditions of continuous enzyme reaction. The decay of fumarase activity during continuous enzyme reaction was expressed by an exponential relationship. The productivity of the immobilized B. flavum using carrageenan was as high as 5.2 times that of the conventional immobilized B. ammoniagenes using polyacrylamide.     

Physiological roles of two enzymes with fumarase activity in two Pseudomonads

Effects of Fe2+ ions on the levels of two enzymes (fumarase and mesaconase) with fumarase activity in two Pseudomonads grown under various nutritional conditions were investigated. Fe2+ ions decreased fumarase but increased mesaconase. A high level of mesaconase was found in Ps. arvilla which was unable to metabolize itaconate. The level of mesaconase in the itaconate-grown cells of Ps. fluorescens was almost the same as that in the glucose-grown cells. This suggests that mesaconase is not an enzyme involved in the metabolism of C5-branched-chain dicarboxylates but presumably, taking the place of fumarase, plays a role in the operation of the tricarboxylic acid cycle in the cells grown in the medium containing Fe2+ ions more than 10 nmol/ml.     

Two biochemically distinct classes of fumarase in Escherichia coli

Biochemical studies with strains of Escherichia coli that are amplified for the products of the three fumarase genes, fumA (FUMA), fumB (FUMB) and fumC (FUMC), have shown that there are two distinct classes of fumarase. The Class I enzymes include FUMA, FUMB, and the immunologically related fumarase of Euglena gracilis. These are characteristically thermolabile dimeric enzymes containing identical subunits of Mr 60,000. FUMA and FUMB are differentially regulated enzymes that function in the citric acid cycle (FUMA) or to provide fumarate as an anaerobic electron acceptor (FUMB), and their affinities for fumarate and L-malate are consistent with these roles. The Class II enzymes include FUMC, and the fumarases of Bacillus subtilis, Saccharomyces cerevisiae and mammalian sources. They are thermostable tetrameric enzymes containing identical subunits Mr 48,000-50,000. The Class II fumarases share a high degree of sequence identity with each other (approx. 60%) and with aspartase (approx. 38%) and argininosuccinase (approx. 15%), and it would appear that these are all members of a family of structurally related enzymes. It is also suggested that the Class I enzymes may belong to a wider family of iron-dependent carboxylic acid hydro-lyases that includes maleate dehydratase and aconitase. Apart from one region containing a Gly-Ser-X-X-Met-X-X-Lys-X-Asn consensus sequence, no significant homology was detected between the Class I and Class II fumarases.     

Purification, characterization, and immunological properties of fumarase from Euglena gracilis var. bacillaris.

A rapid three-step procedure utilizing heat treatment, ammonium sulfate fractionation, and affinity chromatography on Matrex gel Orange A purified fumarase (EC 4.2.1.2) 632-fold with an 18% yield from crude extracts of Euglena gracilis var. bacillaris. The apparent molecular weight of the native enzyme was 120,000 as determined by gel filtration on Sephacryl S-300. The preparation was over 95% pure, and the subunit molecular weight was 60,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, indicating that the enzyme is a dimer composed of two identical subunits. The pH optimum for E. gracilis fumarase was 8.4. The Km values for malate and fumarate were 1.4 and 0.031 mM, respectively. Preparative two-dimensional gel electrophoresis was used to further purify the enzyme for antibody production. On Ouchterlony double-immunodiffusion gels, the antifumarase serum gave a sharp precipitin line against total E. gracilis protein and purified E. gracilis fumarase. It did not cross-react with purified pig heart fumarase. On immunoblots of purified E. gracilis fumarase and crude cell extracts of E. gracilis, the antibody recognized a single polypeptide with a molecular weight of approximately 60,000, indicating that the antibody is monospecific. This polypeptide was found in E. gracilis mitochondria. The antibody cross-reacted with an Escherichia coli protein whose molecular weight was approximately 60,000, the reported molecular weight of the fumA gene product of E. coli, but it failed to cross-react with proteins found in crude mouse cell extracts, Bacillus subtilis extracts, or purified pig heart fumarase.     

Kinetcs and decay of fumarase activity of immobilized Brevibacterium ammoniagenes cells for continuous production of L-malic acid

The kinetics of the reversible fumarase reaction of immobilized Brevibacterium ammoniagenes cells and the decay behavior of enzyme activity were investigated in a plug flow system. The time course of the reaction in the immobilized cell column was well explained by the time-conversion equation including the apparent kinetic constants of the immobilized cell enzyme. The decay rate of fumarase activity was faster in the upper sections of the column (inlet side of the substrate solution) compared with the lower sections when 1M sodium fumarate (pH 7.0) was continuously passed through the column at 37°C. It was shown that the decay rate of the fumarase activity in the immobilized cell column depends on the flow rate of the substrate solution. The effect of flow rate on the decay rate of enzyme activity was considered to be related to the rate of contamination of enzyme with poisonous substances derived from the substrate solution or to the rate of leakage of enzyme stabilizers and/or enzyme itself from the immobilized cells.     

Copper deprivation potentiates oxidative stress in HL-60 cell mitochondria.

Cytochrome-c oxidase is the copper-dependent terminal respiratory complex (complex IV) of the mitochondrial electron transport chain whose activity in a variety of tissues is lowered by copper deficiency. Because inhibition of respiratory complexes increases the production of reactive oxygen species by mitochondria, it is possible that copper deficiency increases oxidative stress in mitochondria as a consequence of suppressed cytochrome-c oxidase activity. In this study, the activities of respiratory complex I + III, assayed as NADH:cytochrome-c reductase, complex II + III, assayed as succinate:cytochrome-c reductase, complex IV, assayed as cytochrome-c oxidase, and fumarase were measured in mitochondria from HL-60 cells that were grown for seven passages in serum-free medium that was either unsupplemented or supplemented with 50 n M CuSO4. Fumarase activity was not affected by copper supplementation, but the complex I + III:fumarase and complex IV:fumarase ratios were reduced 30% and 50%, respectively, in mitochondria from cells grown in the absence of supplemental copper. This indicates that copper deprivation suppressed the electron transfer activity of copper-independent complex I + III as well as copper-dependent complex IV. Manganese superoxide dismutase (MnSOD) content was also increased 49% overall in the cells grown in the absence of supplemental copper. Furthermore, protein carbonyl groups, indicative of oxidative modification, were present in 100-kDa and 90-kDa proteins of mitochondria from copper-deprived cells. These findings indicate that in cells grown under conditions of copper deprivation that suppress cytochrome-c oxidase activity, oxidative stress in mitochondria is increased sufficiently to induce MnSOD, potentiate protein oxidation, and possibly cause the oxidative inactivation of complex I.     

Synthesis and secretion of lecithin-cholesterol acyltransferase by the human hepatoma cell line HepG2

The human liver cell line HepG2 was investigated for its synthesis and secretion of lecithin-cholesterol acyltransferase. The cells were grown to confluency in Eagle's minimal essential medium plus 10% fetal bovine serum. At the onset of the study, fetal bovine serum was removed and cells were grown in minimal essential medium only. At 6, 12, 24, and 48 h the cells were harvested, and the culture medium collected at each time point was assayed for lecithin-cholesterol acyltransferase mass and activity, cholesterol esterification rate, and apolipoprotein A-I mass. The rate of the enzyme secretion measured by both mass and activity was linear over 24 h of culture. The enzyme mass by radioimmunoassay was 1.7, 4.1, 7.9 and 13.7 ng/ml culture medium (or 8.3, 19.9, 38.5 and 66.7 ng/mg cell protein), respectively, and enzyme activity using an exogenous source of phosphatidylcholine/cholesterol liposomes containing apolipoprotein A-I as substrate was 85, 170, 315, and 402 pmol cholesterol esterified/h per ml culture medium (or 414, 828, 1534 and 1957 pmol cholesterol esterified/h per mg cell protein) for 6, 12, 24, and 48 h of culture, respectively. The endogenous cholesterol esterification rate of the culture medium was 47, 104, 224 and 330 pmol/h per ml and apolipoprotein A-I mass was 305, 720, 2400 and 3940 ng/ml culture medium over the same time frame. In contrast to culture medium, low levels of enzyme activity (approximately 10% of that in culture medium at 24 and 48 h) were observed in the extracts of HepG2 cells. The enzyme secreted by HepG2 was found to be similarly activated by apolipoprotein A-I, apolipoprotein E, or apolipoprotein A-IV, and was similarly inhibited by phenylmethylsulfonyl fluoride, dithiobisnitrobenzoate, p-hydroxymercuribenzoate, or iodoacetate as compared to human plasma enzyme. High-performance gel filtration of the culture medium revealed that the HepG2-secreted enzyme was associated with a fraction having a mean apparent molecular weight of approximately 200,000. We concluded that human hepatoma HepG2 cells synthesize and secrete lecithin-cholesterol acyltransferase, which is functionally homologous to the human plasma enzyme.     

Hormone-responsive alkaline proteinase in rat skeletal muscle is not a mast cell-derived enzyme

Proteinase activity was determined in myofibrils from intact rat skeletal muscle and from skeletal muscle myocytes grown in culture. In vivo administration of the mast cell degranulator compound 48/80 abolished the alkaline proteinase activity in myofibrils obtained from normal or streptozotocin-diabetic rats. Exposure of myocytes to compound 48/80 in cell cultures had no effect on their myofibrillar proteinase activity, nor did it affect the rate of overall protein degradation in these cells. Co-incubation of cultured mast cells (line P815Y) with myocytes followed by sonication of the cell mixture resulted in a marked reduction of the proteinase activity in the pellet fraction, suggesting that the mast cells contain inhibitor(s) of myofibrillar proteinase activity. It is suggested that the myofibril-bound alkaline proteinase activity is not a mast cell-derived enzyme but a genuine component of muscle cells. The in vivo 48/80-induced reduction of muscle myofibrillar proteinase activity appears to be due to release of a soluble inhibitory activity rather than removal of mast cell proteinase from the tissue by degranulation.     

A Deuterium Effect in Insect Attraction

Brevibacterium flavum cells obtained from different growth phases were immobilized with κ-carrageenan and the stability of the fumarase activity was investigated. The stability of fumarase activity of the immobilized preparation of cells of the stationary growth phase was highest. The highest stability of the immobilized cells seemed to be correlated to the high stability of fumarase activity in free cells of the stationary phase. High rigidity of the cell wall and membrane of B. flavum cells of the stationary phase and firm binding of fumarase protein to the cell membrane were suggested from several lines of evidence obtained on treatment of the cells with lysozyme and detergents or sonication of the cells. Electronmicrographs showed that the cells of the stationary phase retained the original shape after repeated batch reactions. Solubilized fumarase prepared from cells of the stationary phase showed the highest stability. Experiments using the partially purified enzyme strongly suggested the existence of fumarase-stabilizing components in the cells.     

Nucleotide sequence of the FNR-regulated fumarase gene (fumB) of Escherichia coli K-12

The nucleotide sequence of a 3,162-base-pair (bp) segment of DNA containing the FNR-regulated fumB gene, which encodes the anaerobic class I fumarase (FUMB) of Escherichia coli, was determined. The structural gene was found to comprise 1,641 bp, 547 codons (excluding the initiation and termination codons), and the gene product had a predicted Mr of 59,956. The amino acid sequence of FUMB contained the same number of residues as did that of the aerobic class I fumarase (FUMA), and there were identical amino acids at all but 56 positions (89.8% identity). There was no significant similarity between the class I fumarases and the class II enzyme (FUMC) except in one region containing the following consensus: Gly-Ser-Xxx-Ile-Met-Xxx-Xxx-Lys-Xxx-Asn. Some of the 56 amino acid substitutions must be responsible for the functional preferences of the enzymes for malate dehydration (FUMB) and fumarate hydration (FUMA). Significant similarities between the cysteine-containing sequence of the class I fumarases (FUMA and FUMB) and the mammalian aconitases were detected, and this finding further supports the view that these enzymes are all members of a family of iron-containing hydrolyases. The nucleotide sequence of a 1,142-bp distal sequence of an unidentified gene (genF) located upstream of fumB was also defined and found to encode a product that is homologous to the product of another unidentified gene (genA), located downstream of the neighboring aspartase gene (aspA).     

Thermostability of enzymes of the tricarboxylic acid cycle ofBacillus coagulans

The thermostability of four enzymes of the tricarboxylic acid cycle has been studied in the facultative thermophile,Bacillus coagulans. Although isocitrate dehydrogenase appeared to be more temperature-sensitive in whole-cell extracts of cultures grown at 30°C compared with that in cultures grown at 55°C, this difference could be largely eliminated by the removal of cell-wall material. The specific activity of each of the enzymes examined was approximately threefold higher in cultures grown at 55°C than in those grown at 30°C. The maximum temperature, Arrhenius plot and effect of stabilizing agents for each enzyme were examined and found to be independent of growth temperature. Sodium chloride (10% w/v) was an effective protective agent for fumarase, aconitase and malate dehydrogenase. Protection from thermal denaturation of isocitrate dehydrogenase, aconitase and fumarase but not malate dehydrogenase was also given when the enzymes were heated in the presence of their substrates. These results are discussed in light of the generalized theories of facultative thermophily which have been proposed.     

Intracellular distribution of fumarase in various animals

The subcellular distribution of fumarase was investigated in the liver of various animals and in several tissues of the rat. In the rat liver, fumarase was predominantly located in the cytosolic and mitochondrial fractions, but not in the peroxisomal fraction. The amount of fumarase associated with the microsomes was less than 5% of the total enzyme activity. The investigation of the intracellular distribution of hepatic fumarase of the rat, mouse, rabbit, dog, chicken, snake, frog, and carp revealed that the amount of the enzyme located in the cytosol was comparable to that in the mitochondria of all these animals. The subcellular distribution of the enzyme in the kidney, brain, heart, and skeletal muscle of rat, and in hepatoma cells (AH-109A) was also investigated. Among these tissues, the brain was the only exception, having no fumarase activity in the cytosolic fraction, and the other tissues showed a bimodal distribution of fumarase in the cytosol and the mitochondria. The mitochondrial fumarase was predominantly located in the matrix. About 10% of the total fumarase was found in the outer and inner membrane, although it was unclear whether this fumarase was originally located in these fractions. No fumarase activity was detected in the intermembranous space.     

Nutritional regulation of organelle biogenesis inEuglena: Photo- and metabolite induction of mitochondria

Exposure of dark-grown restingEuglena gracilis Klebs var.bacillaris Cori to light, ethanol, or malate produced an increase in the specific activity of fumarase (EC. 4.2.1.2) and succinate dehydrogenase (EC. 1.3.99.1) during the first 8–12 h of exposure to inducer, followed by a decrease in the specific activity of both mitochondrial enzymes between 12 and 72 h. The increased specific activity represented a net increase in the level of active enzyme, and it was dependent upon cytoplasmic protein synthesis. The photoinduction of fumarase required continuous illumination while the subsequent decrease in fumarase specific activity was independent of light. Light had little effect on the ethanol and malate induction of fumarase and succinate dehydrogenase. In the mutant W₃BUL, which has no detectable protochlorophyll(ide) and chloroplast DNA, light induced both mitochondrial enzymes and the kinetics of enzyme induction were similar to the induction kinetics in wild-type cells. The induction of mitochondrial enzymes appears to be controlled by a non-chloroplast photoreceptor. Dark-grown resting cells of the plastidless mutant W₁₀SmL have lost the ability to regulate fumarase levels. In this mutant, the specific activity of fumarase fluctuated and light had little effect on these fluctuations, indicating that fumarase synthesis was uncoupled from the nonchloroplast photoreceptor. Ethanol addition produced transient changes in fumarase specific activity in W₁₀SmL indicating that in this mutant, mitochondrial enzymes are still inductible by metabolites. Fumarase synthesis in wild-type cells was not induced in the dark by levulinic acid, a chemical inducer of the breakdown ofEuglena storage carbohydrates. Taken together, our results indicate that the photoinduction of mitochondrial enzyme synthesis is not a result of the photoinduction of carbohydrate breakdown. The mechanisms by which light and organic carbon induce the synthesis ofEuglena mitochondria may differ.     

Induction of fumarase in resting Euglena

Exposure of dark-grown resting (carbon deficient) Euglena to light, ethanol or malate produced a transient increase in the specific activity of fumarase. Fumarase levels decreased 8–12 h after the start of induction and this decrease could not be prevented by additional inducer. During the period of fumarase accumulation, cycloheximide prevented further fumarase synthesis and enzyme levels decreased at a rate comparable to the rate of decline normally observed 8–12 h after the start of induction. Although the addition of ethanol to ethanol-induced cultures or malate to malate-induced cultures 12 or 24 h after the initial induction failed to maintain or induce additional fumarase synthesis, the addition of organic carbon to photoinduced cells 8 or 24 h after light exposure induced additional enzyme synthesis. Additional enzyme synthesis was not induced when ethanol- or malate-induced cells were exposed to light 12 or 24 h after organic carbon addition. Light exposure or ethanol addition failed to induce fumarase synthesis during balanced growth indicating that fumarse inducibility is a property of resting cells.     

Partial purification and properties of a beta-glucosidase from Erwinia herbicola Y46.

A constitutive beta-glucosidase of Erwinia herbicola Y46 was studied as a prerequisite to an assessment of its significance in the release of bacteriotoxic aglycones from plant beta-glucosides, and the possible effects of the aglycones on the course of such plant diseases as "fire-blight". The enzyme was purified 86.5-fold from crude extracts of cells grown on yeast beef broth. Ammonium sulfate precipitation, DEAE-cellulose fractionation, and gel filtration through Sephadex G-100 resulted in a preparation having one peak of activity on isoelectrofocussing, on gel filtration through Sephadex G-200, and on polyacrylamide gel electrophoresis. The latter techniques demonstrated, in addition to the major protein band associated with activity, a single minor impurity. The enzyme was active against p-nitrophenyl-beta-glucoside (p-NPG) and phloridzin, but showed only very slight activity against salicin and arbutin, and no detectable activity against beta-methyl-D-glucoside, cellobiose, lactose, and esculin. The production of beta-glucosidase was maximum at the late log phase of growth on yeast beef broth medium and declined somewhat thereafter. The incorporation of inducers (carbohydrates) in defined basal medium resulted in only small variations in specific activity in the resulting cells; The activity (p-NPG substrate) was not inhibited by D-glucose, phloretin, esculin, salicin, arbutin, lactose, or cellobiose, but was slightly inhibited by 1.0 mM phloridzin. Slight inhibition was observed in the presence of sulfhydryl reagents (iodoacetamide, p-chloromercuribenzoate), but sodium azide, ethylene-diaminetetraacetic acid, Cu2+, and Zn2+ ions produced no effect. The activity was stable, in both crude and purified preparations, over the pH ranges 6.0-7.5 (100% activity) and 4.5-greater than 8.5 (50% activity). The enzyme retained 80% activity after 30 min at 50 degrees C, but only 25% after 30 min at 60 degrees C. The enzyme had a mean K-m value (phloridzin) of 1.35 times 10-4 M, an isoelectric point of 4.75, a molecular weight, determined by Sephadex G-200 gel filtration, of about 122 000, and an optimum pH for activity of 6.5-7.0.     

Staining of argininosuccinate lyase activity in polyacrylamide gel.

A procedure for the direct staining of argininosuccinate lyase activity in polyacrylamide gel is described. The method was based on coupling one of the enzymatic products fumarate with fumarase and malic enzyme catalyzed reactions. Fumarate was first converted to L-malate by fumarase. Malic enzyme then catalyzed the oxidative decarboxylation of L-malate to give CO2 and pyruvate with concomitant reduction of NADP+ to NADPH. Finally the reducing power of NADPH was coupled to phenazine methosulfate and in turn to nitroblue tetrazolium yielding a deeply colored insoluble formazan which may be quantitized or semiquantitized by densitometer.     

Mitochondrial import of human and yeast fumarase in live mammalian cells: retrograde translocation of the yeast enzyme is mainly caused by its poor targeting sequence

Studies on yeast fumarase provide the main evidence for dual localization of a protein in mitochondria and cytosol by means of retrograde translocation. We have examined the subcellular targeting of yeast and human fumarase in live cells to identify factors responsible for this. The cDNAs for mature yeast or human fumarase were fused to the gene for enhanced green fluorescent protein (eGFP) and they contained, at their N-terminus, a mitochondrial targeting sequence (MTS) derived from either yeast fumarase, human fumarase, or cytochrome c oxidase subunit VIII (COX) protein. Two nuclear localization sequences (2x NLS) were also added to these constructs to facilitate detection of any cytosolic protein by its targeting to nucleus. In Cos-1 cells transfected with these constructs, human fumarase with either the native or COX MTSs was detected exclusively in mitochondria in >98% of the cells, while the remainder 1-2% of the cells showed varying amounts of nuclear labeling. In contrast, when human fumarase was fused to the yeast MTS, >50% of the cells showed nuclear labeling. Similar studies with yeast fumarase showed that with its native MTS, nuclear labeling was seen in 80-85% of the cells, but upon fusion to either human or COX MTS, nuclear labeling was observed in only 10-15% of the cells. These results provide evidence that extramitochondrial presence of yeast fumarase is mainly caused by the poor mitochondrial targeting characteristics of its MTS (but also affected by its primary sequence), and that the retrograde translocation mechanism does not play a significant role in the extramitochondrial presence of mammalian fumarase.     

Activity staining of blotted enzymes by reaction coupling with transfer membrane-immobilized auxiliary enzymes

A blotting method is described to detect enzymes that do not normally yield a colored product. The method can be used for dot blotting as well as blotting after gel electrophoresis of many enzymes if the reactions they catalyze can be coupled to an oxidase or a dehydrogenase. The latter, designated "auxiliary enzymes," are preimmobilized on membranes of nitrocellulose or positively charged nylon and the reaction they catalyze is coupled with reduction of tetrazolium salt to yield colored formazan on areas of the transfer membrane occupied by the blotted enzymes. In the examples reported here, preimmobilized glucose oxidase, L-amino acid oxidase, xanthine oxidase, malate dehydrogenase, and a mixture of hexokinase and glucose-6-phosphate dehydrogenase were used as auxiliary enzymes to detect blotted invertase, leucine aminopeptidase, purine nucleoside phosphorylase, fumarase, and adenylate kinase, respectively. Detection limits varied, but never exceeded 100 ng for these enzymes. After blotting from polyacrylamide gels, the fumarase assay was the most sensitive of those investigated, detecting 10 ng of enzyme used for electrophoresis. Invertase, a glycoprotein, was detected with higher sensitivity on nitrocellulose membranes when concanavalin A was present on the membrane in addition to the auxiliary enzyme, glucose oxidase. On blots from isoelectric focusing gels, the assay detected two isozymes of purine nucleoside phosphorylase in a sample from calf spleen and at least five isozymes of this enzyme in lysates from human red cells.