Multiple forms of glucosephosphate isomerase in maize

Three apparently different glucosephosphate isomerases are found in the developing seeds of maize (Zea mays L.). Glucosephosphate isomerase I is found in both the endosperm and embryo. It is separable by column chromatography from glucosephosphate isomerase II of the developing endosperm and glucosephosphate isomerase III of the developing embryo and is further distinguished from them by heat stability, temperature activation, and relative insensitivity to the presence of zinc ions in the reaction mixture. Glucosephosphate isomerases II and III elute in the same fractions from diethylaminoethyl cellulose columns but are distinguished by electrophoretic mobility and reaction to the presence of adenosine 5′-triphosphate in the reaction mixture. All three isomerases give multiple banding patterns on electrophoresis. An extensive investigation of the conditions generating additional electrophoretic species and chromatographically separable minor activity peak (Ia) from glucosephosphate isomerase I has shown that these transformations are enhanced by dialysis, column chromatography, ammonium sulfate fractionation, and treatment with urea. The transformations are retarded by the presence of mercaptoethanol during these operations. We concluded that the multiple banding pattern seen on electrophoresis of glucosephosphate isomerase I prepared by certain procedures is artifactual. In germinating seeds of maize, glucosephosphate isomerases I and III are detectable, but II is not. It is possible that glucosephosphate isomerase II specifically catalyzes a step in starch biosynthesis.     

Isolation of rabbit muscle glucosephosphate isomerase by a single-step substrate elution.

A simple method has been developed for the rapid isolation of crystalline glucosephosphate isomerase (EC 5.3.1.9) from rabbit muscle. The enzyme is first bound to cellulose phosphate by adding the ion exchanger to a solution of the crude tissue extract. After filtering and washing the cellulose with buffer, the isomerase is specifically eluted in a batch process by its substrate, glucose 6-phosphate. The entire procedure is very rapid and results in a good recovery (at least 50%) of the enzyme with specific activity of approximately 900 units per mg. The enzyme is homogeneous by polyacrylamide gel electrophoresis in the presence of absence of sodium dodecyl sulfate and by analytical ultracentrifugation.     

Biochemical identification of Moxostoma rhothoecum and M. hamiltoni

The acid phosphatase, glycerol-3-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, and glucosephosphate isomerase enzyme systems of Moxostoma rhothoecum and M. hamiltoni have been analyzed by means of starch gel electrophoresis. The Roanoke River population has been biochemically identified as M. rhothoecum. Glycerol-3-phosphate dehydrogenase and glucosephosphate isomerase polymorphisms are described in M. rhothoecum.     

Differences in tissue expressions of enzyme activities in interspecific sunfish (Centrarchidae) hybrids and their backcross progeny

The extent of naturally occurring variations of enzyme locus expression was determined for three tissues (liver, muscle, and eye) in two species of sunfish (Centrarchidae), the green sunfish (Lepomis cyanellus) and the redear sunfish (L. microlophus). The genetic basis for species differences in tissue enzyme specific activities of malate dehydrogenase (EC 1.1.1.37), lactate dehydrogenase (EC 1.1.1.27), phosphoglucomutase (EC 2.7.5.1), and glucosephosphate isomerase (EC 5.3.1.9) was investigated by determining enzyme specific activities in the tissues of the reciprocal F1 hybrids and of their backcross progenies. The specific activities for most enzymes in hybrids were intermediate between those of the parental species. Significant differences in enzyme specific activity were detected among the F1 progeny as well as those of backcrosses. Variations in specific activity levels in one tissue were often independent of variations in specific activities in a different tissue. However, the changes in the specific activities of different enzymes within the same tissue were often positively correlated. The tissue glucosephosphate isomerase activity differences appear not to be due to different functional contributions of the glucosephosphate isomerase allelic isozymes. Cluster analysis of distributions of specific activities revealed no simple Mendelian pattern of inheritance for control of tissue enzyme activity. Our results suggest a polygenic control of tissue enzyme specific activity levels.     

A biochemical comparison of glucosephosphate isomerase isozymes from Trypanosoma cruzi

The glucosephosphate isomerase (D-glucose-6-phosphate Ketol-isomerase, EC 5.3.1.9) isozymes of Trypanosoma cruzi were characterized with respect to their native and subunit molecular size, isoelectric point and in vitro thermostability. The molecular weight data are consistent with a dimeric enzyme structure. The apparent native and subunit size homogeneity and differences in pI values imply that the electrophoretic mobility differences of isozymes in native gels are determined by their molecular charge. Minor differences in peptide maps indicate the existence of some heterogeneity in the primary structure of the isozymes. The stability of triple-banded glucosephosphate isomerase electrophoretic profiles was confirmed, supporting the view that these phenotypes represent non-interconvertible enzyme species.     

Comparative and genetic studies of isozymes in resynthesized and cultivated Brassica napus L., B. campestris L. and B. alboglabra Bailey

Summary Enzyme electrophoresis was used to compare newly resynthesized Brassica napus with its actual parental diploid species, B. campestris and B. alboglabra. Comparisons were also made with cultivated B. napus. Of the eight enzyme systems assayed, four were monomorphic (hexokinase, malate dehydrogenase, mannose phosphate isomerase and peroxidase), whereas the remaining four were polymorphic (glucosephosphate isomerase, leucine aminopeptidase, phosphoglucomutase and shikimate dehydrogenase), when comparisons were made within or between species. The polymorphic enzyme patterns observed in the newly resynthesized B. napus disclosed that the homoeologous loci contributed by the parental species were expressed in the amphiploid. Analysis of the glucosephosphate isomerase enzyme in a breeding line (Sv 02372) of B. napus indicated that, in this case, the gene originating from B. campestris was switched off whereas that of B. oleracea was expressed. Duplicated enzyme loci were observed in B. campestris and B. alboglabra, thus providing additional evidence to support the hypothesis that these species are actually secondary polyploids derived from an unknown archetype of x=6.     

Identification of glycosomes and metabolic end products in pathogenic and nonpathogenic strains of Cryptobia salmositica (Kinetoplastida: Bodonidae)

Whole cell lysates of pathogenic and nonpathogenic strains of Cryptobia salmositica were subjected to subcellular fractionation using differential and isopycnic centrifugation in sucrose. The glycolytic enzymes hexokinase, fructose-1,6-biphosphate aldolase, triosephosphate isomerase, glucosephosphate isomerase and glyceraldehyde-3-phosphate-dehydrogenase and the peroxisomal enzyme catalase were associated with a microbody that had a buoyant density in sucrose of 1.21 g cm-3. Lactate dehydrogenase was detected in whole cell lysates, but not in purified organelles. A microbody with a positive reaction for catalase was detected in electron microscope sections of the pathogenic and nonpathogenic strains. These catalase-containing microbodies fused with lipid bodies and vacuoles, arose by division from pre-existing microbodies and expelled their contents into the cytoplasm of the cell. Both strains also modified the catalase content in their microbodies. Under aerobic conditions, they metabolized glucose to pyruvate and lactate. We conclude that part of the glycolytic pathway in C. salmositica is compartmentalized in a microbody called the glycosome.     

A spectrophotometric assay of ATP synthesized by sarcoplasmic reticulum.

The problems encountered with a coupled enzyme assay for ATP using glucose, hexokinase and glucose-6-phosphate dehydrogenase are discussed and a modification where fructose and glucosephosphate isomerase were substituted for glucose is described. This modified assay was used successfully to measure the ATP synthesized by reversal of the sarcoplasmic reticulum ATPase. ATP synthesized by adenylate kinase contaminating the sarcoplasmic reticulum was easily corrected for by a subtraction procedure.     

Enzyme proteins of different classes of northern red-backed and large-toothed red-backed voles]

Electrophoretic characters of 9 enzyme systems from different tissue homogenates supposedly controlled by 17 loci are described. Polymorphism detected in loci for glutamate-oxalate transaminase, adenylate kinase, esterase D. glucosephosphate isomerase was thoroughly investigated. Methodic characteristics of enzyme electrophoresis and population-genetic characters of the species studied are under discussion.     

The mutant gene "wal" is active in cells of mouse hair follicles

In order to determine the place of action of the mutant gene waved alopecia (wal), we have obtained chimeric wal/wal c/c Gpi-1aa<-->+/+ C/C Gpi-1bb animals by aggregation of eight-cellular embryos of BALB/c-wal/wal mice and CBA (+/+) mice. The presence or absence of the chimeric structure was determined from the mosaic nature of fur color and hair structure, as well as on the basis of the presence of electrophoretically distinct variants of glucosephosphate isomerase in blood. Chimeras had alternating transverse patches of different lengths and widths consisting of curly (genotype wal/wal) or straight (genotype +/+) hairs. The percentage of cells with wal/wal mutant genotype in chimeras established on the basis of glucosephosphate isomerase isozymes varied from 10 to 80%. A higher percentage of the parental wal/wal component in chimeras correlated with the number of patches having wavy hairs. Analysis of the fur pattern represented by the alternation of transverse patches of wavy or straight hairs in chimeric wal/wal (+/+ mice has shown that mutant gene wal acts in ectodermal cells of hair follicles.     

Schistosoma mansoni: Inhibition of glucosephosphate isomerase and glycolysis by sugar phosphates

Glucosephosphate isomerase (EC 5.3.1.9) of Schistosoma mansoni is inhibited competitively by a number of tetrose, pentose, and hexose phosphates with inhibitor constant (K_i) values in the range of 0.5 to 400 μM. The most potent inhibitor is 5-phospho-d-arabinonate which resembles the cis-enediolate transition state intermediate of the reaction. These analogs were also found to be effective inhibitors of the production of lactate from glucose by suitably supplemented worm homogenates. The rank order of potency of inhibition of glycolysis was inversely related to the magnitudes of the K_i values for glucosephosphate isomerase. These K_i values were similar to those previously reported for mammalian glucosephosphate isomerase, suggesting similarities in the steric and electronic characteristics of the active sites of these isofunctional enzymes. This conclusion was further supported by the observed pH dependence of the inhibition by 5-phospho-d-arabinonate. Although glucosephosphate isomerase is not a rate-limiting enzyme of glycolysis, in the conventional sense, its selective inhibition could be of chemotherapeutic importance, in part because of the accumulation in glycolyzing systems of glucose 6-phosphate which is a potent feedback inhibitor of hexokinase.     

A technique for detection and relative quantitative analysis of glucosephosphate isomerase isozymes from nanogram tissue samples

An improved method for detecting and measuring the enzyme glucosephosphate isomerase after starch gel electrophoresis is described. Nitrocellulose filters are used in a gel overlay procedure which increases the sensitivity of the staining reaction and provides a simple means for accurate quantitation of the isozyme pattern. This staining technique may have wider application with other gel media and also with other enzymes.This work was supported by the M.R.C. Group in Developmental Neurobiology, McMaster University, Canada.     

Deficiencies of glycolytic enzymes as a possible cause of hemolytic anemia

The critical minimum values of Na,K-ATPase and glycolytic enzyme activities at which the erythrocyte viability is lost were calculated using the mathematical model of the erythrocyte, which included all reactions of glycolysis, adenylate metabolism, ionic balance, and osmotic regulation of erythrocyte volume. The criterion for cell death was an increase in its volume to the level at which it is sequestrated from the circulation or is lysed. In hemolytic anemia associated with hexokinase or pyruvate kinase deficiency, activities of these enzymes measured in patient erythrocytes appeared to be close to the calculated critical values. By contrast, in hemolytic anemia associated with phosphofructokinase, glucosephosphate isomerase, triosephosphate isomerase, or phosphoglycerate kinase deficiency, activities of these enzymes measured in patient erythrocytes were significantly greater than the calculated critical values. In this case, if the deficient enzyme were stable, i.e. its activity in the cell were low, but constant in time, the deficiency observed would not account for the erythrocyte destruction observed and the development of hemolytic anemia. It was shown, however, that in phosphofructokinase, glucosephosphate isomerase, triosephosphate isomerase, or phosphoglycerate kinase deficiency, hemolytic anemia can arise because of the instability of these enzymes in time.     

Use of nicotinamide adenine dinucleotide (NAD)-dependent glucose-6-phosphate dehydrogenase in enzyme staining procedures

Substitution of nicotinamide adenine dinucleotide dependent glucose-6-phosphate dehydrogenase for the nicotinamide adenine dinucleotide phosphate dependent enzyme has produced identical results in a number of enzyme-linked electrophoretic staining procedures. This substitution significantly reduces the cost of staining for adenylate kinase, creatine kinase, glucosephosphate isomerase, mannosephosphate isomerase, phosphoglucomutase, and pyruvate kinase activity by utilizing NAD rather than the more expensive NADP.     

The de novo and salvage pathways of GDP-mannose biosynthesis are both sufficient for the growth of bloodstream-form Trypanosoma brucei

The sugar nucleotide GDP-mannose is essential for Trypanosoma brucei. Phosphomannose isomerase occupies a key position on the de novo pathway to GDP-mannose from glucose, just before intersection with the salvage pathway from free mannose. We identified the parasite phosphomannose isomerase gene, confirmed that it encodes phosphomannose isomerase activity and localized the endogenous enzyme to the glycosome. We also created a bloodstream-form conditional null mutant of phosphomannose isomerase to assess the relative roles of the de novo and salvage pathways of GDP-mannose biosynthesis. Phosphomannose isomerase was found to be essential for parasite growth. However, supplementation of the medium with low concentrations of mannose, including that found in human plasma, relieved this dependence. Therefore, we do not consider phosphomannose isomerase to be a viable drug target. We further established culture conditions where we can control glucose and mannose concentrations and perform steady-state [U-(13) C]-D-glucose labelling. Analysis of the isotopic sugar composition of the parasites variant surface glycoprotein synthesized in cells incubated in 5 mM [U-(13) C]-D-glucose in the presence and absence of unlabelled mannose showed that, under physiological conditions, about 80% of GDP-mannose synthesis comes from the de novo pathway and 20% from the salvage pathway.     

Use of Nicotinamide Adenine Dinucleotide (NAD)-Dependent Glucose-6-Phosphate Dehydrogenase in Enzyme Staining Procedures

Substitution of nicotinamide adenine dinucleotide dependent glucose-6-phosphate dehydrogenase for the nicotinamide adenine dinucleotide phosphate dependent enzyme has produced identical results in a number of enzyme-linked electrophoretic staining procedures. This substitution significantly reduces the cost of staining for adenylate kinase, creatine kinase, glucosephosphate isomerase, mannosephosphate isomerase, phosphoglucomutase, and pyruvate kinase activity by utilizing NAD rather than the more expensive NADP.     

Enzymes of glycolysis and the pentose phosphate pathway during development of the rabbit stomach worm Obeliscoides cuniculi

The activities of glycolytic and other enzymes of carbohydrate metabolism were measured in free-living and parasitic stages of the rabbit stomach worm Obeliscoides cuniculi. Glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, phosphoglucomutase, hexokinase, glucosephosphate isomerase, phosphofructokinase, aldolase, triosephosphate isomerase, α-glycerophosphatase, glyceraldehyde-3-phosphate dehydrogenase, phosphoglycerate kinase, phosphoglycerate mutase, enolase, pyruvate kinase, phosphoenol pyruvate carboxykinase, lactate dehydrogenase, alcohol dehydrogenase, and glucose-6-phosphatase activities were present in worms recovered 14, 20 and 190 days postinfection.The presence of glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase, and glucose-6-phosphatase indicates the possible function of a pentose phosphate pathway and a capacity for gluconeogenesis, respectively, in these worms.The ratio of pyruvate kinase (PK) to phosphoenol pyruvate carboxykinase (PEPCK) less than I in parasitic stages suggests that their most active pathway is that fixing CO₂ into phosphoenol pyruvate to produce oxaloacetate.Low levels of glucose-6-phosphate dehydrogenase, triosephosphate isomerase, PEPCK and PK were recorded in infective third-stage larvae stored at 5°C for 5 and 12 mos. The ratio of PK to PEPCK greater than 1 indicates that infective larvae preferentially utilize a different terminal pathway than the parasitic stages.     

Specific cleavage of glucosephosphate isomerases at cysteinyl residues using 2-nitro-5-thiocyanobenzoic acid: analyses of peptides eluted from polyacrylamide gels and localization of active site histidyl and lysyl residues

Specific chemical cleavage of human placental and porcine muscle glucosephosphate isomerases at three amino peptide bonds of cysteinyl residues with 2-nitro-5-thiocyanobenzoic acid was achieved. Four primary peptides were generated from the cyanylated human glucosephosphate isomerase, indicating the quantitative cleavage of this enzyme. Four primary plus six overlap peptides were obtained from the cleavage of the swine muscle enzyme. The peptides were separated by SDS-polyacrylamide gel electrophoresis and eluted from the gels. Amino acid and carboxyl terminal analyses of the eluted peptides have permitted the alignment of these peptides with respect to the native polypeptide chain. The analysis of the enzyme which had been specifically covalently labeled at the essential lysine and histidine residues of the active center revealed that the active-site histidine and lysine residues are located on two distinct peptides with molecular weights of 27,500 and 14,000, respectively.     

A fast electrophoretic isoenzyme technique for the identification of invasive and non-invasive Entamoeba histolytica and "E. histolytica-like" organisms

Cellulose acetate electrophoresis (CAE) was used to separate glucosephosphate isomerase, hexokinase, malic enzyme, and phosphoglucomutase extracted from invasive and non-invasive Entamoeba histolytica and "E. histolytica-like" organisms. Each of these morphologically similar organisms possessed a unique CAE isoenzyme profile that can be used as an aid in their identification. The CAE technique used to obtain these isoenzyme profiles is rapid, simple, and economical, and it requires neither specialized training nor elaborate equipment.     

Studies on glucose isomerase from a Streptomyces species.

Production and properties of glucose isomerase from a Co2+-sensitive Streptomyces species were studied. After 4 days of shaking cultivation at 30 degrees C and 200 rpm, a maximum of 1.1 enzyme units per ml of broth was obtained. Cell-free glucose isomerase, obtained from mycelia heat-treated in the presence of 0.5 mM Co2+, showed a 3.5-fold increase in specific activity over enzyme obtained from untreated mycelia. The optimum pH and temperature for the glucose isomerase were 7 to 8 and 80 degrees C, respectively. The Michaelis constant for fructose was 0.40 M. Mg2+ was found to enhance the glucose isomerase activity, whereas the effect of Co2+ on enzyme activity depended on the manner in which the enzyme was prepared. This glucose isomerase was quite heat stable, with a half-life of 120 h at 70 degrees C.