Enzymatic characterization of Babesia bovis

Agarose gel electrophoresis was used to identify metabolic enzymes in Babesia bovis and B. bigemina. Glutamate dehydrogenase, lactate dehydrogenase, glucose phosphate isomerase, and hexokinase were identified in B. bovis- and B. bigemina-infected erythrocytes and B. bovis merozoite preparations. A specific electrophoretic mobility was observed for each enzyme. Malate dehydrogenase, glucose 6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, and adenylate kinase were only detected in normal erythrocyte preparations. Inter-species, but not intra-species, variation was noted when comparing electrophoretograms of both species. Kinin-activating activity was not detected in B. bovis-infected erythrocyte or merozoite preparations at pH 4.2 or 7.6.     

Concentration and Enzyme Content of In Vitro-Cultured Babesia bigemina-Infected Erythrocytes1

ABSTRACT. Clones of in vitro-cultured Babesia bigemina-infected erythrocytes were concentrated by several density gradient procedures. The density range of infected erythrocytes containing pairs of parasites was 1.077 to 1.089 g/ml, whereas the density range of infected erythrocytes containing single parasites was 1.092 to 1.100 g/ml. Three enzymes-lactate dehydrogenase, glucose-phosphate isomerase, and glutamate dehydrogenase-were found associated with infected erythrocytes. The parasite-specific enzyme and/or isoenzymes were shown to have different mobility patterns in starch gel electrophoresis from those found in the normal bovine erythrocytes. The enzyme 6-phosphogluconate dehydrogenase was not detected as a parasite-specific enzyme in B. bigemina-infected erythrocytes.     

Molecular Characterization of a New Babesia bovis Thrombospondin-Related Anonymous Protein (BbTRAP2)

A gene encoding a Babesia bovis protein that shares significant degree of similarity to other apicomplexan thrombospondin-related anonymous proteins (TRAPs) was found in the genomic database and designated as BbTRAP2. Recombinant protein containing a conserved region of BbTRAP2 was produced in E. coli. A high antigenicity of recombinant BbTRAP2 (rBbTRAP2) was observed with field B. bovis-infected bovine sera collected from geographically different regions of the world. Moreover, antiserum against rBbTRAP2 specifically reacted with the authentic protein by Western blot analysis and an indirect fluorescent antibody test. Three bands corresponding to 104-, 76-, and 44-kDa proteins were identified in the parasite lysates and two bands of 76- and 44-kDa proteins were detected in the supernatant of cultivated parasites, indicating that BbTRAP2 was proteolytically processed and shed into the culture. Apical and surface localizations of BbTRAP2 were observed in the intracellular and extracellular parasites, respectively, by confocal laser microscopic examination. Moreover, native BbTRAP2 was precipitated by bovine erythrocytes, suggesting its role in the attachment to erythrocytes. Furthermore, the specific antibody to rBbTRAP2 inhibited the growth of B. bovis in a concentration-dependent manner. Consistently, pre-incubation of the free merozoites with the antibody to rBbTRAP2 resulted in an inhibition of the parasite invasion into host erythrocytes. Interestingly, the antibody to rBbTRAP2 was the most inhibitive for the parasite’s growth as compared to those of a set of antisera produced against different recombinant proteins, including merozoite surface antigen 2c (BbMSA-2c), rhoptry-associated protein 1 C-terminal (BbRAP-1CT), and spherical body protein 1 (BbSBP-1). These results suggest that BbTRAP2 might be a potential candidate for development of a subunit vaccine against B. bovis infection.     

Distribution of erythrocytic allozymes in two sibling species of greater galago [Galago crassicaudatus E. Geoffroy 1812 and G. garnettii (Ogilby 1838)]

This study investigated the use of erythrocyte enzymes as indicators of the presence or absence of gene flow between the sibling species G. crassicaudatus and G. garnettii. Fifty-five animals deriving from 14 different source populations were included in the analyses. In addition to hemoglobin, eight enzyme systems were examined: acid phosphatase, adenylate kinase, carbonic anhydrase II, esterase D, glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, peptidase A, and peptidase B. of these, adenylate kinase, glucose-6-phosphate dehydrogenase, hemoglobin, peptidase A, and peptidase B showed no interspecific or intraspecific variation. Esterase D was polymorphic in certain populations of G. crassicaudatus but not in others or in G. garnettii. Acid phosphatase and 6-phosphogluconate dehydrogenase were polymorphic in G. garnettii but monomorphic in all G. crassicaudatus populations. The taxa showed fixation for different alleles at the carbonic anhydrase II locus, indicating a lack of gene exchange between the taxa. We suggest that acid phosphatase, 6-phosphogluconate dehydrogenase, and carbonic anhydrase II may be used as genetic markers in the identification of these two taxa.     

Recombinant Escherichia coli engineered for production of L-lactic acid from hexose and pentose sugars

Recombinant Escherichia coli have been constructed for the conversion of glucose as well as pentose sugars into L-lactic acid. The strains carry the lactate dehydrogenase gene from Streptococcus bovis on a low copy number plasmid for production of L-lactate. Three E. coli strains were transformed with the plasmid for producing L-lactic acid. Strains FBR9 and FBR11 were serially transferred 10 times in anaerobic cultures in sugar-limited medium containing glucose or xylose without selective antibiotic. An average of 96% of both FBR9 and FBR11 cells maintained pVALDH1 in anaerobic cultures. The fermentation performances of FBR9, FBR10, and FBR11 were compared in pH-controlled batch fermentations with medium containing 10% w/v glucose. Fermentation results were superior for FBR11, an E. coli B strain, compared to those observed for FBR9 or FBR10. FBR11 exhausted the glucose within 30 h, and the maximum lactic acid concentration (7.32% w/v) was 93% of the theoretical maximum. The other side-products detected were cell mass and succinic acid (0.5 g/l). Journal of Industrial Microbiology & Biotechnology (2001) 27, 259–264. Received 05 November 2000/ Accepted in revised form 03 July 2001     

Gliding motility of Babesia bovis merozoites visualized by time-lapse video microscopy

Background

Babesia bovis is an apicomplexan intraerythrocytic protozoan parasite that induces babesiosis in cattle after transmission by ticks. During specific stages of the apicomplexan parasite lifecycle, such as the sporozoites of Plasmodium falciparum and tachyzoites of Toxoplasma gondii, host cells are targeted for invasion using a unique, active process termed “gliding motility”. However, it is not thoroughly understood how the merozoites of B. bovis target and invade host red blood cells (RBCs), and gliding motility has so far not been observed in the parasite.

Methodology/Principal Findings

Gliding motility of B. bovis merozoites was revealed by time-lapse video microscopy. The recorded images revealed that the process included egress of the merozoites from the infected RBC, gliding motility, and subsequent invasion into new RBCs. The gliding motility of B. bovis merozoites was similar to the helical gliding of Toxoplasma tachyzoites. The trails left by the merozoites were detected by indirect immunofluorescence assay using antiserum against B. bovis merozoite surface antigen 1. Inhibition of gliding motility by actin filament polymerization or depolymerization indicated that the gliding motility was driven by actomyosin dependent process. In addition, we revealed the timing of breakdown of the parasitophorous vacuole. Time-lapse image analysis of membrane-stained bovine RBCs showed formation and breakdown of the parasitophorous vacuole within ten minutes of invasion.

Conclusions/Significance

This is the first report of the gliding motility of B. bovis. Since merozoites of Plasmodium parasites do not glide on a substrate, the gliding motility of B. bovis merozoites is a notable finding.     

Immunity against Boophilus annulatus induced by the Bm86 (Tick-GARD) vaccine

Friesian cattle were immunized with two inoculations of anti-tick Bm86 (Tick-GARD) vaccine and were challenged 30 or 90 d later with Boophilus annulatus larvae derived from 1.2 g of eggs. No nymphs or adult ticks were found on the immunized cattle during four weeks after challenge. Repeated infestations (2 to 4) with larvae on three other calves during a period of 160 and 390 d after the immunization did not result in development of nymphal and adult stages. In control, non-immunized cattle infested with corresponding batches of larvae 1380 to 4653 replete adult female ticks were collected. Larvae issued from Babesia bovis-infected female ticks transmitted the infection to Bm86-immunized cattle, but the progeny of B. bigemina-infected females did not. Since B. bigemina is transmitted exclusively by nymphal stages of Bo. annulatus these results support the observation that immunity induced by Bm86 affects the larval stage of this tick.     

Deoxyribonuclease Activity in Selenomonas ruminantium, Streptococcus bovis, and Bacteroides ovatus

Six Selenomonas ruminantium strains (132c, JW13, SRK1, 179f, 5521c1, and 5934e), Streptococcus bovis JB1, and Bacteroides ovatus V975 were examined for nuclease activity as well as the ability to utilize nucleic acids, ribose, and 2-deoxyribose. Nuclease activity was detected in sonicated cells and culture supernatants for all bacteria except S. ruminantium JW13 and 179f sonicated cells. S. ruminantium strains were able to utilize several deoxyribonucleosides, while S. bovis JB1 and B. ovatus V975 showed little or no growth on all deoxyribonucleosides. When S. ruminantium strains 5934e, 132c, JW13, and SRK1 were incubated in medium that contained 15 mm ribose, the major end products were acetate, propionate, and lactate. S. ruminantium 5521c1 and S. bovis JB1 did not grow on ribose, and none of the S. ruminantium strains or S. bovis JB1 grew on 15 mm 2-deoxyribose. In contrast, B. ovatus V975 was able to grow on ribose and 2-deoxyribose. In conclusion, all S. ruminantium strains, S. bovis JB1, and B. ovatus V975 had nuclease activity. However, not all bacteria were able to utilize deoxyribonucleosides, ribose, or 2-deoxyribose. Received: 9 February 2000 / Accepted: 27 March 2000     

Comparison of the Surface Proteins and Glycoproteins On Erythrocytes of Calves Before and During Infection With Babesia Bovis

The surface proteins and glycoproteins on red cells from normal and Babesia bovis-infected calf blood have been compared. Several radiolabeling probes were used to label specifically external membrane molecules which were then separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and identified by autoradiography or fluorography. No differences were observed among the Coomassie Blue-stained membrane proteins of erythrocytes from individual uninfected calves. Comparison of red cells from these animals also indicated no qualitative differences in the surface proteins with accessible tyrosyl residues labeled by lactoperoxidase-catalyzed radioiodnation, although some quantitative variation in the uptake of radioactivity into particular proteins was observed. the major radioiodinated bands on normal bovine erythrocytes had Mr of 165, 130, 90, and 45 kiloDaltons. However, labeling of surface glycoproteins by the periodate/[³H]NaBH₄ and galactose oxidase (± neuraminidase)/[³H]NaBH₄ methods showed significant differences in the surface proteins of red cells from individual uninfected calves. of 14 animals tested, 5 had major labeled glycoproteins of unique Mr. No changes were observed in radioiodinated surface proteins of total red cell samples from infected calves with 0.5-6% parasitemia. Radioiodination of concentrated infected red cells from the same samples (concentrated by selective hypotonic lysis of uninfected erythrocytes in KC1) resulted in the labeling of 3 new surface proteins, with Mr of 118, 115, and 60 kiloDaltons. the same new ¹²⁵I-labeled bands were identified on infected cells from 3 avirulent strains of B. bovis used in vaccine production. Furthermore, in concentrated infected cells there was very poor radiolabeling of major bands strongly labeled on uninfected cells (Mr 165, 130, and 90 kiloDaltons), suggesting parasite-induced loss of these proteins. Although there were some differences in ³H-labeled surface glycoproteins of red cells from normal and. B. bovis -infected blood, they were restricted to minor labeled bands and were not seen consistently. the labeled surface glycoproteins of concentrated infected cells were very similar to those of the uninfected red blood cells from infected blood.     

Glucose 6-phosphate dehydrogenase activity in membranes of erythrocytes from normal individuals and subjects with Mediterranean G6PD deficiency

Unsealed, hemoglobin-free erythrocyte ghosts contain low yet significant levels of Glucose 6-phosphate dehydrogenase (G6PD) activity. This activity is comparable in erythrocyte ghosts obtained from normal individuals and from G6PD-deficient subjects (of Mediterranean type), in spite of the marked differences found in the corresponding cytosolic compartments. The membrane preparations can bind purified human G6PD (type B) to their cytoplasmic surface according to patterns of positive cooperativity. 2.4 × 10⁴ and 1.6 × 10⁴ G6PD-binding sites are present on the inner surface of each ghost obtained from normal and from G6PD-deficient erythrocytes, respectively, the relevant association constants being 2.8 × 10⁶ M^?1 and 0.82 × 10⁶ M^?1. The interaction of G6PD with the ghosts is unaffected by different ionic strengths or by metabolites such as glucose 6-phosphate, NADP and NADPH.     

Artemisone inhibits in vitro and in vivo propagation of Babesia bovis and B. bigemina parasites

Artemisone was evaluated, in in vitro and in vivo, for control of bovine babesiosis caused by Babesia bigemina and Babesiabovis parasites. In vitro, artemisone reduced parasitemia in a dose-dependent manner: the inhibitory effects increased gradually, reaching a maximum inhibition of 99.6% and 86.4% for B. bigemina and B. bovis, respectively 72 h after initiation of treatment with initial parasitemia of 0.5%. In calves infected with either B. bigemina or B. bovis artemisone treatment was well tolerated and prevented development of acute babesiosis in all animals except for one B. bovis-infected calf. The treatment did not eliminate all blood parasites, and recovered animals carried a persistent low-level infection. Treatment with artemisone may be useful as an alternative drug for preventing the pathology that results from babesiosis, without interfering with acquired immune protection following recovery from an acute babesiosis infection or vaccination.     

Factors Affecting the Antibacterial Activity of the Ruminal Bacterium,Streptococcus bovis HC5

Streptococcus bovis HC5 inhibits a variety of S. bovis strains and other Gram-positive bacteria, but factors affecting this activity had not been defined. Batch culture studies indicated that S. bovis HC5 did not inhibit S. bovis JB1 (a non-bacteriocin-producing strain) until glucose was depleted and cells were entering stationary phase, but slow-dilution-rate, continuous cultures (0.2 h⁻¹) had as much antibacterial activity as stationary-phase batch cultures. Because the activity of continuous cultures (0.2–1.2 h⁻¹) was inversely related to the glucose consumption rate, it appeared that the antibacterial activity was being catabolite repressed by glucose. When the pH of continuous cultures (0.2 h⁻¹) was decreased from 6.7 to 5.4, antibacterial activity doubled, but this activity declined at pH values less than 5.0. Continuous cultures (0.2 h⁻¹) that had only ammonia as a nitrogen source had antibacterial activity, and large amounts of Trypticase (10 mg ml⁻¹) caused only a 2.0-fold decline in the amount of HC5 cell-associated protein that was needed to prevent S. bovis JB1 growth. Because S. bovis HC5 was able to produce antibacterial activity over a wide range of culture conditions, there is an increased likelihood that this activity could have commercial application. Received: 6 February 2002 / Accepted: 27 March 2002     

Genetic regulation of glucose 6-phosphate dehydrogenase activity in the inbred mouse

The erythrocyte glucose 6-phosphate dehydrogenase activity characteristic of each of 16 inbred mouse strains falls into one of three distinct classes. Strains C57L/J and C57BR/cdJ represent the low activity class: strains A/J and A/HeJ represent the high activity class; other strains have intermediate activities. There is no evidence that structural variation is responsible for the variation in G6PD activity, since partially purified enzyme from each class has the same thermal stability, pH-activity profile, Michaelis constants for G6P and NADP, electrophoretic mobility, and activity using 2-deoxy d-glucose 6-phosphate as substrate. The activities of 6-phosphogluconate dehydrogenase and glucose phosphate isomerase do not differ in erythrocytes of the three G6PD activity classes. Young red cells have higher G6PD activities than old red cells and there is evidence that the intracellular stability of the enzyme is reduced in red cells of strain C57L/J. G6PD activities in kidney and skeletal and cardiac muscle from animals with low red cell G6PD are slightly lower than the activities in kidney and muscle from animals with high red cell G6PD activity. The quantitative differences in red cell G6PD activity are not regulated by X-linked genes, but by alleles at two or more autosomal loci. A simple genetic model is proposed in which alleles at two unlinked, autosomal loci, called Gdr-1 and Gdr-2 regulate G6PD activity in the mouse erythrocyte.     

Studies on cell fusion between Babesia rodhaini-infected mouse erythrocytes and baby hamster kidney cells.

Heterokaryons were formed by fusion of B. rodhaini-infected mouse erythrocytes and baby hamster kidney (BHK) cells, using Sendai virus. The erythrocyte membrane rapidly lysed inside the BHK cell cytoplasm releasing free parasites. There was no evidence that parasite multiplication occurred inside the BHK cells, nor that parasitized BHK cells were infective for mice.Transient erythrocyte homokaryons were observed in some preparations.The approach indicates a possible method for the in vitro cultivation of Babesia.     

Vaccination against babesiosis using recombinant GPI-anchored proteins

The increase in human babesiosis is of major concern to health authorities. In the USA, most of these cases are due to infections with Babesia microti, whereas in Europe B. divergens is the major cause of clinical disease in humans. Here we review the immunological and biological literature of glycosylphosphatidylinositol (GPI)-anchored merozoite proteins of human Babesia parasites with emphasis on their role in immunity, and provide some new bioinformatical information on B. microti GPI-Anchored Proteins (GPI-AP). Cattle can be vaccinated with soluble parasite antigens (SPA) of Babesia divergens that are released by the parasite during proliferation. The major component in SPA preparations appeared to be a 37?kDa merozoite surface protein that is anchored in the merozoite membrane by a GPI anchor. Animals could be protected by vaccination with the recombinant 37?kDa protein expressed in Escherichia coli, provided the protein had a hydrophobic terminal sequence. Based on this knowledge, a recombinant vaccine was developed against Babesia canis infection in dogs, successfully. In order to identify similar GPI-AP in B. microti, the genome was analysed. Here it is shown that B. microti encodes all proteins necessary for GPI assembly and its subsequent protein transfer. In addition, in total 21 genes encoding for GPI-AP were detected, some of which reacted particularly strongly with sera from B. microti-infected human patients. Reactivity of antibodies with GPI-anchored merozoite proteins appears to be dependent on the structural conformation of the molecule. It is suggested that the three-dimensional structure of the protein that is anchored in the membrane is different from that of the protein that has been shed from the merozoite surface. The significance of this protein’s dynamics in parasite biology and immune evasion is discussed. Finally, we discuss developments in tick and Babesia vaccine research, and the role such vaccines could play in the control of human babesiosis.     

Effects of Some Antibiotics on Human Erythrocyte 6-Phosphogluconate Dehydrogenase: An in vitro and in vivo Study

The in vitro and in vivo effects of some antibiotics on human erythrocyte 6-phosphogluconate dehydrogenase were investigated. Human erythrocyte 6-phosphogluconate dehydrogenase was purified with ammonium sulphate precipitation, 2′,5′ ADP-Sepharose 4B affinity and gel filtration chromatography. Some antibiotics (netilmicin sulphate, cefepime, amikacin, isepamycin, chloramphenicol, ceftazidim, teicoplanin, ampicillin, ofloxacin, levofloxacin, cefotaxime, penicillin G, gentamicin sulphate, ciprofloxacin) inhibited enzyme activity in vitro but others (cefozin, decefin, streptomycin, combisid, and meronem) were devoid of inhibitory effects. For the drugs having low IC50 values (netilmicin sulphate and cefepime), in vivo studies were performed in rats. Netilmicin sulphate at 15-mg/kg inhibited enzyme activity significantly (p < 0.001) 1h, 2h, and 3h after dosing and cefepime at 200-mg/kg very significantly (p < 0.001) inhibited the enzyme 1 h and 2 h after dosing. Netilmicin sulphate and cefepime inhibited rat erythrocyte 6-phosphogluconate dehydrogenase both in vivo and in-vitro.     

Identification of Surface Proteins on Viable Plasmodium knowlesi Merozoites

Viable merozoites of Plasmodium knowlesi were isolated and the proteins that were labeled on intact merozoites by lactoperoxidase-catalyzed radioiodination were identified. Sodium dodecyl sulfate polyacrylamide gel electrophoresis and autoradiography of Triton soluble extracts of labeled merozoites demonstrated eight major bands ranging in apparent molecular weight from 150,000 D to 22,000 D. Exposure of intact merozoites to trypsin (10 μg/ml) for 10 min resulted in the loss of the two highest molecular weight proteins (150,000 D and 105,000 D) and the appearance of two new bands at 70,000 D and 62,000 D. Trypsin treatment under these conditions also removed the receptor(s) for merozoite attachment to erythrocytes. Therefore, these high molecular weight proteins are candidates for the merozoite component that attaches to erythrocytes. There was no evidence that the labeled membrane components were serum or erythrocyte membrane components, two potential contaminants in the preparation. Anti-rhesus erythrocyte antibody did not precipitate labeled merozoite proteins. Furthermore, the immunoprecipitation of labeled merozoite proteins by rhesus anti-merozoite serum was not inhibited by erythrocyte ghosts.     

Effect of astaxanthin and aluminum chloride on erythrocyte G6PD and 6PGD enzyme activities in vivo and on erythrocyte G6PD in vitro in rats

In this study, we investigated the effect of astaxanthin (Ast) and aluminum (Al) on the erythrocyte glucose‐6‐phosphate dehydrogenase (G6PD) and 6‐phosphogluconate dehydrogenase (6PGD) enzymes activities in vivo and on G6PD enzyme in vitro in rats. For in vitro studies, G6PD enzyme was purified from rat erythrocyte by using 2′,5′‐ADP‐Sepharose 4B affinity gel. The effects of Ast and Al³⁺ ion were investigated on the purified enzyme. It was determined that Ast increased the enzyme activity, whereas Al³⁺ inhibited the enzyme activity noncompetitively (IC₅₀ values; 0.679 mM, K_i values 1.32 mM). For in vivo studies, the rats were divided into the groups: control (Cont.), Al, Ast, and Al + Ast. The last three groups were compared with the control group. In Al group, a significant degree of inhibition was observed in the activity of G6PD and 6PGD enzymes when compared with the control group (P < 0.05), whereas there was an increase in the activities of G6PD and 6PGD enzymes in Ast and Al + Ast groups (P < 0.05).     

Properties and Role of Glyceraldehyde-3-Phosphate Dehydrogenase in the Control of Fermentation Pattern and Growth in a Ruminal Bacterium, <Emphasis Type="Italic">Streptococcus bovis</Emphasis>

To clarify the control of glycolysis and the fermentation pattern in Streptococcus bovis, the molecular and enzymatic properties of NAD⁺-specific glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were examined. The GAPDH gene (gapA) was found to cluster with several others, including those that encode phosphoglycerate kinase and translation elongation factor G, however, gapA was transcribed in a monocistronic fashion. Since biochemical properties, such as optimal pH and affinity for glyceraldehyde-3-phosphate (GAP), were not very different between GAPDH- and NADP⁺-specific glyceraldehyde-3-phosphate dehydrogenase (GAPN), the flux from GAP may be greatly influenced by the relative amounts of these two enzymes. Using S. bovis JB1 as a parent, JB1gapA and JB1ldh, which overproduce GAPDH and lactate dehydrogenase (LDH), respectively, were constructed to examine the control of the glycolytic flux and lactate production. There were no significant differences in growth rates and formate-to-lactate ratios among JB1, JB1gapA, and JB1ldh grown on glucose. When grown on lactose, JB1ldh showed a much lower formate-to-lactate ratio than JB1gapA, which showed the highest NADH-to-NAD⁺ ratio. However, growth rates did not differ among JB1, JB1gapA, and JB1ldh. These results suggest that GAPDH is not involved in the control of the glycolytic flux and that lactate production is mainly controlled by LDH activity.     

Circadian Variations in the Activities of 6‐Phosphogluconate Dehydrogenase and Glucose‐6‐Phosphate Dehydrogenase in the Liver of Conrol and Streptozotocin‐Induced Diabetic Rats

The aim of this study was to examine: the 24 h variation of 6‐phosphogluconate dehydrogenase and glucose‐6‐phosphate dehydrogenase activities, key enzymes for the maintenance of intracellular NADPH concentration, in rat liver in control and streptozotocin‐induced diabetic animals. Adult male rats were fed ad libitum and synchronized on a 12:12 h light‐dark cycle (lights on 08:00 h). One group of animals was treated with streptozotocin (STZ, 55 mg/kg, intraperitoneal) to induce experimental diabetes. Eight weeks after STZ injection, the animals were sacrificed at six different times of day—1, 5, 9, 13, 17 and 21 Hours After Lights On (HALO)—and livers were obtained. Enzyme activities were determined spectrophotometrically in triplicate in liver homogenates and expressed as units per mg protein. 6‐phosphogluconate dehydrogenase activity was measured by substituting 6‐phosphogluconate as substrate. Glucose‐6‐phosphate dehydrogenase activity was determined by monitoring NADPH production. Treatment, circadian time, and interaction between treatment and circadian time factors were tested by either one or two way analysis of variance (ANOVA). Two‐way ANOVA revealed that 6‐phosphogluconate dehydrogenase activity significantly depended on both the treatment and time of sacrifice. 6‐phosphogluconate dehydrogenase activity was higher in control than diabetic animals; whereas, glucose‐6‐phosphate dehydrogenase activity did not vary over the 24 h in animals made diabetic by STZ treatment. Circadian variation in the activity of 6‐phosphogluconate dehydrogenase was also detected in both the control and STZ treatment groups (one‐way ANOVA). Time‐dependent variation in glucose‐6‐phosphate dehydrogenase activity during the 24 h was detected in control but not in diabetic rats. No significant interaction was detected between STZ‐treatment and time of sacrifice for both hepatic enzyme activities. These results suggest that the activities of NADPH‐generating enzymes exhibit 24 h variation, which is not influenced by diabetes.