Enzydmatic Characterization of Babesia bovis1

ABSTRACT. 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.     

Babesia bovis: gene isolation and characterization using a mung bean nuclease-derived expression library

Genomic DNA prepared from erythrocyte cultures of Babesia bovis merozoites was digested with mung bean nuclease and used to construct a lambda gt11 expression library of B. bovis recombinants. Immunoscreening with two polyclonal antibody probes detected multiple recombinants from which two, designated Bb-1 and Bb-3, were chosen for further analysis. Monospecific immunoglobulins isolated from the screening sera using nitrocellulose-bound fusion proteins were employed to determine the native molecular weight and the intracellular location of the babesial proteins encoded by the recombinants. Clone Bb-1 encodes an antigen of 77,000 Da located at the apical end of the intraerythrocytic parasite. A protein of 75,000 Da encoded by clone Bb-3 is associated with the infected red blood cell cytoplasm and/or membrane but not with the merozoite.     

Phosphoenolpyruvate-dependent phosphorylation of hexoses by ruminal bacteria: evidence for the phosphotransferase transport system.

Six species of ruminal bacteria were surveyed for the phosphoenolpyruvate (PEP)-dependent phosphorylation of glucose. Selenomonas ruminantium HD4, Streptococcus bovis JB1, and Megasphaera elsdenii B159 all showed significant activity, but Butyrivibrio fibrisolvens 49, Bacteroides succinogenes S85, and Bacteroides ruminicola B1(4) showed low rates of PEP-dependent phosphorylation and much higher rates in the presence of ATP. S. ruminantium HD4, S. bovis JB1, and M. elsdenii B159 also used PEP to phosphorylate the nonmetabolizable glucose analog 2-deoxy-D-glucose (2-DG). Rates of 2-DG phosphorylation with ATP were negligible for S. bovis JB1 and M. elsdenii B159, but toluene-treated cells of S. ruminantium HD4 phosphorylated 2-DG in the presence of ATP as well as PEP. Cell-free extracts of S. ruminantium HD4 used ATP but not PEP to phosphorylate glucose and 2-DG. Since PEP could serve as a phosphoryl donor in toluene-treated cells but not in cell-free extracts, there was evidence for membrane and hence phosphotransferase system involvement in the PEP-dependent activity. The ATP-dependent phosphorylating enzymes from S. ruminantium HD4 and S. bovis JB1 had molecular weights of approximately 48,000 and were not inhibited by glucose 6-phosphate. Based on these criteria, they were glucokinases rather than hexokinases. The S. ruminantium HD4 glucokinase was competitively inhibited by 2-DG and mannose, sugars that differ from glucose in the C-2 position. Since 2-DG was a competitive inhibitor of glucose, the same enzyme probably phosphorylates both sugars. The S. bovis JB1 glucokinase was not inhibited by either 2-DG or mannose and had a higher Km and Vmax for glucose.     

Phosphoenolpyruvate-dependent phosphorylation of hexoses by ruminal bacteria: evidence for the phosphotransferase transport system

Six species of ruminal bacteria were surveyed for the phosphoenolpyruvate (PEP)-dependent phosphorylation of glucose. Selenomonas ruminantium HD4, Streptococcus bovis JB1, and Megasphaera elsdenii B159 all showed significant activity, but Butyrivibrio fibrisolvens 49, Bacteroides succinogenes S85, and Bacteroides ruminicola B1(4) showed low rates of PEP-dependent phosphorylation and much higher rates in the presence of ATP. S. ruminantium HD4, S. bovis JB1, and M. elsdenii B159 also used PEP to phosphorylate the nonmetabolizable glucose analog 2-deoxy-D-glucose (2-DG). Rates of 2-DG phosphorylation with ATP were negligible for S. bovis JB1 and M. elsdenii B159, but toluene-treated cells of S. ruminantium HD4 phosphorylated 2-DG in the presence of ATP as well as PEP. Cell-free extracts of S. ruminantium HD4 used ATP but not PEP to phosphorylate glucose and 2-DG. Since PEP could serve as a phosphoryl donor in toluene-treated cells but not in cell-free extracts, there was evidence for membrane and hence phosphotransferase system involvement in the PEP-dependent activity. The ATP-dependent phosphorylating enzymes from S. ruminantium HD4 and S. bovis JB1 had molecular weights of approximately 48,000 and were not inhibited by glucose 6-phosphate. Based on these criteria, they were glucokinases rather than hexokinases. The S. ruminantium HD4 glucokinase was competitively inhibited by 2-DG and mannose, sugars that differ from glucose in the C-2 position. Since 2-DG was a competitive inhibitor of glucose, the same enzyme probably phosphorylates both sugars. The S. bovis JB1 glucokinase was not inhibited by either 2-DG or mannose and had a higher Km and Vmax for glucose.     

Glucose metabolism in the mucosa of the small intestine. Glycolysis in subcellular preparations from the cat and rat

1. Lactic acid formation in supernatant fractions of homogenates of cat or rat small-intestinal mucosa was measured under optimum conditions with glucose, fructose, glucose 6-phosphate, fructose 1,6-diphosphate or 3-phosphoglycerate as substrate. 2. Between 80 and 107% of the glycolytic activity of the homogenate was recovered in these particle-free preparations when glucose, fructose, glucose 6-phosphate or fructose 1,6-diphosphate was used as substrate. 3. Evidence was obtained that hexokinase and phosphofructokinase were the rate-limiting enzymes in the initial sequence of glycolytic reactions. The limitation of rate by hexokinase was much more pronounced in preparations from the cat than in those from the rat. 4. With subcellular preparations from cat or rat small intestine lactic acid was also formed from ribose 5-phosphate and at rates similar to those observed with glucose. 5. A higher rate of glycolysis was observed with glucose 6-phosphate as substrate with preparations from the proximal half of the small intestine of the rat as compared with the distal half. 6. Mucosal preparations from rats starved for 24-48hr. exhibited only about one-quarter of the glycolytic activity of those of fed control groups. The decreased rate of formation of lactic acid from either glucose or fructose was mainly due to a decrease in the activity of hexokinase(s). The activities of glucose 6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase and a number of other enzymes were not significantly decreased by starvation. 7. The results are discussed in relation to metabolic control of glycolysis in other mammalian tissues.     

Lipid metabolism by rat lung in vitro. Effect of starvation and re-feeding on utilization of [U-14C]glucose by lung slices

1. The incorporation of [U-(14)C]glucose into several lipid components of lung and liver slices, and the activities of glucose 6-phosphate dehydrogenase (EC 1.1.1.49), 6-phosphogluconate dehydrogenase (EC 1.1.1.44), ;malic' enzyme (EC 1.1.1.40) and NADP-isocitrate dehydrogenase (EC 1.1.1.42) of the cell cytosol were examined in normal, starved and re-fed rats. 2. Lipogenesis and the activities of these enzymes in liver were decreased markedly in rats starved for 72h. Re-feeding starved rats on a fat-free diet for 72h resulted in the well documented hyperlipogenic response in liver, particularly in its ability to convert glucose into neutral lipid, and increased activities of glucose 6-phosphate dehydrogenase, ;malic' enzyme and 6-phosphogluconate dehydrogenase to values approx. 700, 470 and 250% of controls respectively. 3. Approx. 70% of the total label in lung lipids was present in the phospholipid fraction. Hydrolysis of lung phospholipids revealed that lipogenesis from glucose was considerable, with approx. 40% of the total phospholipid radioactivity present in the fatty acid fraction. 4. Incorporation of glucose into total lung lipids was decreased by approx. 40% in lung slices of starved rats and was returned to control values on re-feeding. Although phospholipid synthesis from glucose was decreased in lung slices of starved rats, the decrease proportionally was greater for the fatty acid fraction (approx. 50%) as compared with the glycerol fraction (approx. 25%). 5. The activities of lung glucose 6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase and NADP-isocitrate dehydrogenase were not affected by the dietary alterations. ;Malic' enzyme activity was not detected in lung cytosol preparations. 6. The results are discussed in relation to the surface-active lining layer (surfactant) of the lung.     

Expression of Babesia equi merozoite antigen-2 by recombinant baculovirus and its use in the ELISA

In this study, the gene encoding Babesia equi merozoite antigen-2 was inserted into a baculovirus transfer vector, and a recombinant virus expressing B. equi merozoite antigen-2 was isolated. Two B. equi merozoite antigen-2-related recombinant baculovirus-expressed peptides of 25 and 30 kDa were detected with a murine anti serum against B. equi merozoite antigen-2; these corresponded to the native B. equi merozoite antigen-2 and were secreted into the supernatants of insect cell cultures. Recombinant B. equi merozoite antigen-2 was not effected by tunicamycin treatment, indicating that B. equi merozoite antigen-2 was not glycosylated. The potential of recombinant B. equi merozoite antigen-2 for use in the ELISA was evaluated by measuring the antibody response to B. equi merozoite antigen-2 in horses experimentally infected with B. equi.     

DNA probes distinguish geographical isolates and identify a novel DNA molecule of Babesia bovis

A genomic DNA library of Babesia bovis was screened to identify DNA probe candidates for direct detection of the parasite. Two sequences, Bo6 and Bo25, had the highest sensitivity and further analysis revealed unique characteristics of each of these. Neither sequence hybridized detectably to bovine DNA. Bo6 detected 100 pg of both a Mexican and an Australian isolate of B. bovis, but Bo6 also detected 1.0 ng of Babesia bigemina DNA under identical conditions. A unique characteristic of Bo6 is that it hybridizes to an apparent 7.4-kilobase DNA in undigested genomic DNA of both B. bovis and B. bigemina. The sequence is well conserved between the 2 geographic isolates of B. bovis, but it is apparently divergent in B. bigemina. Bo25 did not hybridize detectably to bovine or B. bigemina DNA. This sequence detected 100 pg of homologous B. bovis Mexican isolate DNA, but the sensitivity was reduced to 1 ng for the Australian isolate DNA. The restriction enzyme profile of the Bo25 sequence in genomic DNA differed markedly in the number, size, and intensity of bands between the 2 B. bovis geographic isolates tested. Thus, the Bo25 sequence can distinguish geographic isolates of B. bovis.     

Evidence for a complex of three beta-oxidation enzymes in Escherichia coli: induction and localization.

The enzymes for beta-oxidation of fatty acids in inducible and constitutive strains of Escherichia coli were assayed in soluble and membrane fractions of disrupted cells by using fatty acid and acyl-coenzyme A (CoA) substrates containing either 4 or 16 carbon atoms in the acyl moieties. Cell fractionation was monitored, using succinic dehydrogenase as a membrane marker and glucose 6-phosphate dehydrogenase as a soluble marker. Acyl-CoA synthetase activity was detected exclusively in the membrane fraction, whereas acyl-CoA dehydrogenase, 3-hydroxyacyl-CoA dehydrogenase, enoyl-CoA hydratase, and 3-ketoacyl-CoA thiolase activities that utilized both C4 and C16 acyl-CoA substrates were isolated from the soluble fraction. 3-Hydroxyacyl-CoA dehydrogenase, enoyl-CoA hydratase, and 3-ketoacyl-CoA thiolase activities assayed with both C4 and C16 acyl-CoA substrates co-chromatographed on gel filtration and ion-exchange columns and cosedimented in glycerol gradients. The data show that these three enzyme activities of the fad regulon can be isolated as a multienzyme complex. This complex dissociates in very dilute preparations; however, in those preparations where the three activities are separated, the fractionated species retain activity with both C4 and C16 acyl-CoA substrates.     

Proteins and antigens of merozoites and sporozoites of Eimeria bovis (Apicomplexa)

Proteins and antigens of first-generation merozoites and sporozoites of Eimeria bovis were examined using standard sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), Western blotting and lactoperoxidase iodination procedures. SDS-PAGE gels revealed both common and unique protein bands in merozoite and sporozoite extracts, ranging in molecular weight (Mr) from 15,000 to 215,000. Nitrocellulose immunoblots of separated proteins, when probed with sera obtained from immunized calves, revealed numerous IgG-binding antigens of Mr 18,000 to 180,000 in merozoites and Mr 28,000 to approximately 118,000 in sporozoites. Although merozoite and sporozoite preparations each contained antigens of different molecular weights, 4 antigens had the same migratory distance in both preparations (Mr 58,000, 70,000, 83,000, 98,000). Of 3 types of immune sera used to probe immunoblots, serum taken from a calf that had been inoculated with oocysts of E. bovis and boosted 10 wk later by subcutaneous injection with 2 X 10(7) live merozoites emulsified in Freund's complete adjuvant consistently identified and reacted more intensely with more antigens of merozoites and sporozoites than the other immune sera tested. Autoradiographic analysis of radioiodinated parasites revealed major surface proteins on merozoites of between 15,000 and 18,000 Mr and 3 surface proteins on sporozoites of Mr 28,000, 77,000, and 183,000. All but the 183,000 protein elicited an IgG antibody response in the host.     

Mannitol metabolism in Eimeria tenella.

, and 1992. Mannitol metabolism in Eimeria tenella. International Journal for Parasitology 22: 1157–1163. Unsporulated oocysts of Eimeria tenella contain large quantities of carbohydrates, namely amylopectin, mannitol and glucose. Analysis of the carbohydrate content of sporulating oocysts revealed that mannitol content increased markedly during early stages of sporogony (first 4–6 h) but slowly diminished during the next 40 h of sporulation. Accumulation of mannitol was accompanied by a rapid decrease in amylopectin and free glucose, suggesting that mannitol might be synthesized from glucose released from amylopectin. Mannitol was also detected in sporozoite and merozoite extracts. All four mannitol cycle enzymes were detected in oocysts. Sporozoites excysted in vitro had lower activities of all four enzymes. Mannitol-1 -phosphatase and mannitol dehydrogenase activity was also detected in merozoites obtained from the second stage schizonts. Sporozoites incubated with ¹⁴C-glucose accumulated radioactively labelled precursor continuously for over 12 h and some of the ¹⁴C-glucose was converted into ¹⁴C-mannitol. These results indicate that mannitol plays an important role in the metabolism and development of the intracellular stages of the parasite.     

Inhibition of glucose metabolism by n-hexadecane in Cladosporium (Amorphotheca) resinae.

When Cladosporium resinae is provided with n-hexadecane and glucose, n-hexadecane is used preferentially. Studies using [14C]glucose indicated that n-hexadecane did not inhibit glucose uptake but did retard oxidation of glucose to CO2 and assimilation of glucose carbon into trichloroacetic acid-insoluble material. Glucose could be recovered quantitatively from hydrocarbon-grown cells that had been transferred to glucose. Four enzymes that may be involved in glucose metabolism, hexokinase, glucose-6-phosphate dehydrogenase, glucose-phosphate isomerase, and succinate dehydrogenase, were not detected in cells grown on hexadecane but were present in cells grown on glucose. Addition of hexadecane to extracts of glucose-grown cells resulted in immediate loss of activity for each of the four enzymes, but two other enzymes did not directly involved in glucose metabolism, adenosine triphosphatase and alanine-ketoacid aminotransferase, were not inhibited by hexadecane in vitro. Cells grown on hexadecane and transferred to glucose metabolize intracellular hexadecane; after 1 day, activity of hexokinase, glucose-6-phosphate dehydrogenase, glucosephosphate isomerase, and succinate dehydrogenase could be detected and 22% of the intracellular hydrocarbon had been metabolized. Hexadecane-grown cells transferred to glucose plus cycloheximide showed the same level of activity of all the four enzymes as cells transferred to glucose alone. Thus, intracellular n-hexadecane or a metabolite of hexadecane can inthesis of those enzymes is not inhibited.     

Fermentation of pectin and glucose,and activity of pectin-degrading enzymes in the rabbit caecal bacterium Bifidobacterium pseudolongum

AIMS: In a rabbit caecal bacterium Bifidobacterium pseudolongum, metabolites of pectin and glucose, and activities of enzymes involved in the degradation of pectin were assayed. Simultaneously, activities of these enzymes were assayed in a rumen pectinolytic strain of Streptococcus bovis. METHODS AND RESULTS: A strain B. pseudolongum P6 which grew best on pectin was selected among bifidobacteria isolated from the rabbit caecum. Cultures of B. pseudolongum P6 grown on pectin produced significantly less formate, lactate and ethanol, and more acetate and succinate than cultures grown on glucose. No CO2 production on pectin was observed. Pectin macromolecule was degraded by extracellular pectinase (EC 3.2.1.15). Cell extracts possessed the activity of 2-keto-3-deoxy-6-phosphogluconate (KDPG) aldolase (EC 4.1.2.14). Streptococcus bovis X4, possessed activity of exopectate lyase and pectinase, but not that of KDPG aldolase. CONCLUSIONS: Our results are consistent with the assumption that in B. pseudolongum P6 acidic products of pectin degradation are catabolized via a modified Entner-Doudoroff pathway, as shown previously in rumen pectin-utilizing bacteria. The missing KDPG aldolase activity in Strep. bovis X4 seems to be the reason for the absence of growth of this bacterium on pectin. SIGNIFICANCE AND IMPACT OF THE STUDY: Information on polysaccharide metabolism in bifidobacteria is fragmentary. This study extends the knowledge on pectin metabolism in intestinal bacteria.     

Oxidative Enzymes and Pathways of Hexose and Triose Metabolism in Chlorella

Cell-free preparations of Chlorella pyrenoidosa Chick, van Niel's strain, were assayed for oxidative enzymes, utilizing isotopic and spectrophotometric techniques. The enzyme activity of heterotrophic and autotrophic cells was compared. The study was divided into categories, one concerned with the spectrophotometric detection of enzymes involved in the initial reactions of glycolysis and the hexose monophosphate shunt, and the other with the direct oxidation of glucose as compared with that oxidized via glycolysis. The reduction of pyridine nucleotides in crude extracts was studied with glucose, glucose-6-phosphate, 6-phosphogluconate, and fructose-1-6-diphosphate as substrates. Enzymes detected in both heterotrophic and autotrophic cells were hexokinase, fructose-diphosphate-aldolase, NAD-linked 3-phosphoglyceraldchyde dehydrogenase, glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, and a NADP-linked 3-phosphoglyceraldchyde dehydrogenase. In addition to isotopic studies designed to make an appraisal of the hexose monophosphate shunt, a comparison of the rate of reduction of NADP by glucose-6-phosphate and 6-phosphogluconate in relation to the reduction of NAD by 3-phosphoglyceraldehyde was made in light- and dark-grown cells. The rate of reduction of NADP appeared to be lowered in the light-grown cells, suggesting, as did also the isotopic studies, that the hexose monophosphate shunt is less active in autotrophic metabolism than in heterotrophic metabolism.     

The effect of lead on the calcium-handling capacity of rat heart mitochondria.

1. Glucose 6-phosphate dehydrogenase (D-glucose 6-phosphate-NADP+ oxidoreductase, EC 1.1.1.49) from baker's yeast (Saccharomyces cerevisiae) was immobilized on CNBr-activated Sepharose 4B with retention of about 3% of enzyme activity. This uncharged preparation was stable for up to 4 months when stored in borate buffer, pH7.6, at 4 degrees C. 2. Stable enzyme preparations with negative or positive overall charge were made by adding valine or ethylenediamine to the CNBr-activated Sepharose 4B 30min after addition of the enzyme. 3. These three immobilized enzyme preparations retained 40-60% of their activity after 15 min at 50 degrees C. The soluble enzyme is inactivated by these conditions. 4. The soluble enzyme lost 45 and 100% of its activity on incubation for 3h at pH6 and 10 respectively. The three immobilized-enzyme preparations were completely stable over this entire pH range. 5. The pH optimum of the positively and negatively charged immobilized-enzyme preparations were about 8 and 9 respectively. The soluble enzyme and the uncharged immobilized enzyme had an optimum pH at about 8.5 6. Glucose 6-phosphate dehydrogenase immobilized on CNBr-activated Sephadex G-25 was unstable, as was enzyme attached to CNBr-activated Sepharose 4B to which glycine, asparitic acid, valine or ethylenediamine was added at the same time as the enzyme.     

Glycolytic enzymes and a GLUT-1 glucose transporter in the outer segments of rod and cone photoreceptor cells

The presence of glycolytic enzymes and a GLUT-1-type glucose transporter in rod and cone outer segments was determined by enzyme activity assays, glucose uptake measurements, Western blotting, and immunofluorescence microscopy. Enzyme activities of six glycolytic enzymes including hexokinase, phosphofructokinase, aldolase, glyceraldehyde-3-phosphate dehydrogenase, phosphoglycerate kinase, pyruvate kinase, and lactate dehydrogenase, were found to be present in purified rod outer segment (ROS) preparations. Immunofluorescence microscopy of bovine and chicken retina sections labeled with monoclonal antibodies against glyceraldehyde-3-phosphate dehydrogenase, phosphoglycerate kinase, and lactate dehydrogenase have confirmed that these enzymes are present in rod and cone outer segments and not simply contaminants from the inner segments or other cells. Rod outer segments were also found to contain glucose transport activity as detected by 3-O-[14C]methylglucose uptake and exchange. The glucose transporter had a Km of 6.3 mM and a Vmax of 0.15 nmol of 3-O-methylglucose/s/mg of ROS membrane protein for net uptake and a Km of 29 mM and a Vmax of 1.06 nmol of 3-O-methylglucose/s/mg of ROS membrane protein for equilibrium exchange. These Km values for net uptake and equilibrium exchange are similar to values obtained for human red blood cells and are characteristic of GLUT-1-type glucose transporter. The transport was inhibited by both cytochalasin B and phloretin. Western blot analysis and immunofluorescence microscopy using type-specific glucose transporter antibodies indicated that both rod and cone outer segment plasma membranes have a GLUT-1 glucose transporter of Mr 45K as found in red blood cells and brain microsomal membranes. Solid-phase radioimmune competitive inhibition studies indicated that rod outer segment plasma membranes contained 15% the number of glucose transporters found in human red blood cell membranes and had an estimated density of 400 glucose transporter per micron2 of plasma membrane. These studies support the view that outer segments can generate energy in the form of ATP and GTP by anaerobic glycolysis to supply at least some of the energy requirements for phototransduction and other metabolic processes.     

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.     

Interaction of ruminal bacteria in the production and utilization of maltooligosaccharides from starch.

The degradation and utilization of starch by three amylolytic and one nonamylolytic species of ruminal bacteria were studied. Pure cultures of Streptococcus bovis JB1, Butyrivibrio fibrisolvens 49, and Bacteroides ruminicola D31d rapidly hydrolyzed starch and maltooligosaccharides accumulated. The major starch hydrolytic products detected in S. bovis cultures were glucose, maltose, maltotriose, and maltotetraose. In addition to these oligosaccharides, B. fibrisolvens cultures produced maltopentaose. The products of starch hydrolysis by B. ruminicola were even more complex, yielding glucose through maltotetraose, maltohexaose, and maltoheptaose but little maltopentaose. Selenomonas ruminantium HD4 grew poorly on starch, digested only a small portion of the available substrate, and generated no detectable oligosaccharides as a result of cultivation in starch containing medium. S. ruminantium was able to grow on a mixture of maltooligosaccharides and utilize those of lower degree (less than 10) of polymerization. A coculture system containing S. ruminantium as a dextrin-utilizing species and each of the three amylolytic bacteria was developed to test whether the products of starch hydrolysis were available for crossfeeding to another ruminal bacterium. Cocultures of S. ruminantium and S. bovis contained large numbers of S. bovis but relatively few S. ruminantium and exhibited little change in the pattern of maltooligosaccharides observed for pure cultures of S. bovis. In contrast, S. ruminantium was able to compete with B. fibrisolvens and B. ruminicola for these growth substrates. When grown with B. fibrisolvens, S. ruminantium grew to high numbers and maltooligosaccharides accumulated to a much lesser degree than in cultures of B. fibrisolvens alone. S. ruminantium-B. ruminicola cultures contained large numbers of both species, and maltooligosaccharides never accumulated in these cocultures.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inactivation of glucose 6-phosphate dehydrogenase during germination and outgrowth of Bacillus cereus T endospores.

The specific activity and total activity of glucose 6-phosphate dehydrogenase (EC 1.1.1.49) under conditions of complete cell breakage fall 10-20-fold during a 3h period of spore germination and outgrowth. The spores must germinate (lose refractility), but do not have to undergo outgrowth, for the loss of activity to occur. Glucose 6-phosphate dehydrogenase activity from cells as any stage of development is completely stable in extracts at 4 degrees C or 30 degrees C. All of the enzyme activity is found in a soluble (50000g supernatant) fraction and remains completely soluble throughout development. Soluble protein and total cellular protein remain constant for about 2h. Proteinases could not be detected or protein turnover demonstrated during the morphogenetic process. Phenylmethanesuophony fluoride and o-phenanthroline, inhibitors of proteolytic enzymes, do not prevent glucose 6-phosphate dehydrogenase inactivation when added to whole cells. Mixing experiments show no inhibitor of glucose 6-phosphate dehydrogenase to be present in late-stage cells. The enzyme is not excreted into the culture medium. Chloramphenicol and rifampicine immediately stop protein synthesis and development but not the inactivation of glucose 6-phosphate dehydrogenase. NaN3, 2,4-dinitrophenol or anaerobiosis immediately stop development and prevent the loss of enzyme activity. A requirement for metabolic energy is therefore probable. Extracts of spores pre-labelled with L[14C]leucine were made at various stages of morphogenesis and subjected to polyacrylamide-gel electrophoresis. Glucose 6-phosphate dehydrogenase, which was identified by a specific stain, did not lose 14C label, and therefore may not be degraded during the inactivation process.