Phosphoglucomutase Isoenzymes Research Articles

Genetics of Zoarces populations. I. Three loci determining the phosphoglucomutase isoenzymes in brain tissue.

Horizontal starch-gel electrophoresis has revealed three autosomal loci for phosphoglucomutase in the teleostean marine fish Z. viviparus L. In two natural populations, two of the loci are polymorphic for two common codominant alleles but a rare allele was also found in both. The third locus is dominated by a single very common allele but at least one rare allele occurs also. The genotypes of the three loci were found to be randomly combined in the populations and a segregation analysis of litters from double-heterozygous females shows that the two polymorphic loci recombine freely. Three different paternal alleles are required to account for the segregation found in a single litter; thus the mother must have mated with at least two different males. Segregations not complying with Mendelian expectations in litters from homozygous females indicate that an appreciable fraction of the females have mated with at least two males per brood.

Genetic characterization of new varieties and hybrids of citrus table fruit through isoenzymes

The aim of this study was to establish isoenzymatic patterns to genetically characterize the orange varieties ‘Lane Late’, ‘Navalete’, ‘Navelina’ and ‘Salustiana’, mandarin varieties ‘Clemenules’, ‘Marisol’ and ‘Satsuma Okitsu’, and the hybrids ‘Nova’ and ‘Ortanique’. The following isoenzymatic systems were utilized: alcohol dehydrogenase (ADH), esterase (EST), acid phosphatase (APS), phosphoglucoisomerase (PGI), phosphoglucomutase (PGM), 6-phosphogluconate dehydrogenase (6-PGDH), glutamate oxaloacetate transaminase (GOT), isocitrate dehydrogenase (IDH), leucine aminopeptidase (LAP), malate dehydrogenase (MDH), peroxidase (PRX), sorbitol dehydrogenase (SDH), superoxidase dismutase (SOD) and shikimate dehydrogenase (SKDH). Proteins were extracted from leaves and the isoenzymatic polymorphisms were analyzed by polyacrylamide gel electrophoresis. The PGM isoenzyme showed the highest differentiation among the genetic materials tested. The banding profiles observed for PGM, SKDH and EST were satisfactory to characterize the mandarin varieties and hybrids studied. However, no polymorphic bands were found in sweet oranges with the 14 isoenzymes tested.

Molecular and biochemical characterization of phosphoglucomutases from Entamoeba histolytica and Entamoeba dispar

Entamoeba histolytica and Entamoeba dispar have only recently been defined as two separate species. E. histolytica, the pathogenic species, is the microorganism causing invasive intestinal amoebiasis and/or liver abscess, while the morphologically similar E. dispar is nonpathogenic and noninvasive. The gold standard for the distinction of the two species has been the isoenzyme electrophoresis of phosphoglucomutases (EC 5.4.2.2) and hexokinases (EC 2.7.1.1), but there had also been a controversy about the possibility of a conversion of isoenzyme patterns. In this study, we cloned the phosphoglucomutase (PGM) cDNAs from the pathogenic and the nonpathogenic species. The deduced amino acid sequences were only 2.4% different. The cDNAs were expressed in Escherichia coli under the control of a T7 RNA polymerase promoter. The recombinant polypeptides displayed strong phosphoglucomutase activity, each of the recombinant enzymes comigrated with its natural counterpart from E. histolytica and E. dispar in the starch gel electrophoresis. Our results give a biochemical interpretation of the PGM isoenzyme pattern and support the clear distinction between the two species.

Epidemiological and genetic observations on human schistosomiasis in Kinshasa, Zaire

A survey for Schistosoma intercalatum conducted in Kinshasa, Zaire, in September 1994 revealed a prevalence of 3·6% ( n = 167). Three isolates of schistosomes were made by exposing Bulinus wrighti to miracidia hatched from eggs collected from 2 infected children. Characterization of the isolates by biochemical (isoenzymes of phosphoglucomutase), molecular (restriction fragment length polymorphism and randomly amplified polymorphic deoxyribonucleic acid analysis) and morphological (egg measurements) techniques confirmed the existence of an autochthonous transmission focus of S. intercalatum in Kinshasa. The study also provided evidence of the occurrence of natural hybridization between S. intercalatum and S. haematobium. No potential snail host for either species was found in the 2 rivers examined. Apart from Bu. globosus from Zambia and Bu. wrighti, snail infection experiments showed an incompatible relationship between the parasite isolates and snails belonging to the Bu. forskalii group, the Bu. truncatus/Bu. tropicus complex, and the Bu. africanus group.

A family of hexosephosphate mutases in Saccharomyces cerevisiae.

The Saccharomyces cerevisiae PGM1 and PGM2 genes encoding two phosphoglucomutase isoenzymes have been isolated and sequenced. The derived protein sequences are closely related to one another and show distinct sequence similarities to the human and rabbit phosphoglucomutases, especially in the region supposed to constitute the active site. PGM1 and PGM2 are located on chromosomes XI and XIII, respectively, just upstream of the known genes YPK1 and YKR2 coding for a pair of closely related putative protein kinases. These observations suggest that an extended region of DNA arose by the process of gene duplication. Cells deleted for both, PGM1 and PGM2, could not grow on galactose. No residual phosphoglucomutase activity could be measured in crude extracts or in permeabilized cells of pgm1/2 double mutants. Unexpectedly, growth with glucose was not impaired and the mutant cells were still able to accumulate trehalose and glycogen, although at a reduced level. Two further genes could be isolated and characterized which when over-expressed on a multi-copy plasmid could restore growth on galactose of the pgm1/2 double deletion mutant. Multi-copy complementation was due to a sharply increased level of phosphoglucomutase activity. Partial sequencing and characterization of the two genes revealed one of them to be SEC53 encoding phosphomannomutase. No extended sequence similarities could be found in the databases for the second gene. However, part of the derived amino acid sequence contained a region of high similarity to the active-site consensus sequence of hexosephosphate mutases from different organism. Further investigations suggest that a complex network of mutases exist in yeast which interact and can partially substitute for each other.

Phosphoglucomutase isoenzymes in pollen grains

The possibility that contaminating pollen grains interfere with the electrophoretic phosphoglucomutase phenotyping of human body fluid stained samples was investigated. Pollen grains from forty-two different species were examined. Forty showed detectable amounts of activities of isoenzymes of phosphoglucomutase following starch gel electro~horesis. while 23 showed discernible isoenzymes followihg ultrathin isoelectric focussing. In several cases, phosphoglucomutases extracted from pollen grains comigrated with human types. Phosphoglucomutase isoenzymes of pollen grains of Sambucus nigra (common elder), in particular, interfered with phosphoglucomutase phenotyping using starch gel electrophoresis and isoelectric focussing. These results suggest that it may be necessary to examine some forensic specimens for contamination with pollen prior to the electrophoretic separation of phosphoglucomutase isoenzymes.

Change of isoenzyme pattern during long-term polyxenic cultivation ofEntamoeba histolytica

Isoenzymes of phosphoglucomutase and hexokinase were repeatedly evaluated using starch gel electrophoresis in polyxenic cultures of Entamoeba histolytica. In two out of 18 strains spontaneous changes of isoenzyme patterns were recorded. While originally they were categorized into virulent group of zymodemes, following isoenzyme analysis classified them as non-virulent. The relation between virulence and isoenzyme pattern is questionable.

Differentiation of clinical isolates of Entamoeba histolytica by using specific DNA probes

Most individuals infected with Entamoeba histolytica are reported to be clinically asymptomatic. On the basis of the electrophoretic migration of hexokinase and phosphoglucomutase isoenzymes, two groups of E. histolytica isolates have been classified. Those derived from symptomatic cases were found to have fast-migrating hexokinase bands and were labeled pathogenic. The others, isolated from cyst passers, had (in most cases) slow-migrating bands and were called nonpathogenic. Differences between these two groups of E. histolytica were found recently at the DNA level. Two sets of different DNA probes derived from tandemly repeated sequences present in extrachromosomal circular DNA elements in each group of E. histolytica were characterized. Using these probes with procedures for direct hybridization of trophozoites on nylon membranes, we could correctly correlate hexokinase electromobility with the DNA hybridization signal of 81 different isolates of E. histolytica. The advantages of using DNA probes lie in their sensitivity (fewer than 200 trophozoites can be detected) and specificity. The probes hybridized only with amebae from the E. histolytica species and not with other enteric protozoa and can be useful as a diagnostic tool.

Isoenzymes of Phosphoglucomutase from Human Red Blood Cells: Isolation and Kinetic Properties

A procedure has been developed for the purification of phosphoglucomutase from human red cell (phenotype PGM1 a1 or a3) lysates. It yields homogeneous isoenzyme preparations of the products ("primary" and "secondary") of the two PGM1 and PGM2 loci with distinctive pI (from 6.07 to 5.29). There are substantial differences between PGM1 and PGM2 isoenzymes, having single polypeptide chains of 58,500 and 69,000 Mr respectively and showing different thermostability. The kinetic properties of all the isoenzymes for the phosphoglucomutase reaction are essentially the same (apart from the specific activity of 1089-1263 units/mg for PGM1 forms vs 37-42 units/mg for PGM2 forms), but there are striking differences in substrate specificity. In fact the products of PGM1 locus are "true" phosphoglucomutases, being specific to mutate glucose monophosphates, whereas the PGM2 forms also display phosphoribomutase and glucose 1,6-bisphosphate synthetic activities. Some kinetic properties of these "side activities" are also reported.

Phenotyping of phosphoglucomutase (PGM1) isoenzymes--a new method for the follow-up of chimerism after bone marrow transplantation. Follow-up of chimerism after bone marrow transplantation by phenotyping of phosphoglucomutase (PGM1) isoenzymes.

In this study, phenotyping of phosphoglucomutase isoenzymes (PGM1, EC 2.7.5.1) in erythrocytes of bone marrow donors and hosts was shown to present a simple and exact method for the follow-up of chimerism after bone marrow transplantation, aimed at a better assessment of the transplant state and function.

Isoenzyme patterns as a tool in taxonomy of chlorococcal algae.

From isolated lichen algae of the genus Trebouxia, De Puymaly isoenzymes of phosphoglucomutase, malate dehydrogenase and superoxide dismutase were studied by isoelectric focusing in hybrid immobilized pH gradients. Phosphoglucomutase has proven to be a suitable marker since it is highly variable whereas malate dehydrogenase and superoxide dismutase are less variable. The focusing patterns are species specific and provide a basis for a reevaluation of species classification of Trebouxia.

Isolation and partial characterization of the hexokinase isoenzymes from pathogenic and non-pathogenic strains of Entamoeba histolytica

Isoenzyme electrophoretic patterns (zymodemes) are increasingly used to distinguish between pathogenic and non-pathogenic strains of Entamoeba histolytica. Isolates of E. histolytica from asymptomatic and symptomatic cases have been shown to differ in the electrophoretic mobility of their hexokinase and phosphoglucomutase isoenzymes. The hexokinase isoenzymes from a non-pathogenic strain and from a pathogenic strain of E. histolytica were purified by fast protein liquid chromatography in several steps, which included a separation by size, chromatofocusing, and anion exchange chromatography. The isoenzymes differed in their isoelectric points, which ranged from pH 4.8–5.4, but had very similar kinetic properties and almost identical apparent molecular weights (48 000) in sodium dodecyl sulfate polyacrylamide gels, as well as on gel filtration columns. Comparison of tryptic peptide analysis of each of the isoenzymes indicated considerable homology between the non-pathogenic and pathogenic forms. Antibodies produced against each of the two pathogenic hexokinase isoenzymes inhibited their enzymatic activity. The antibodies also inhibited the activity of the isoenzymes of the non-pathogenic strain. Our findings suggest that the isoenzymes have structural similarities, and that the pathogenic ones differ from the non-pathogenic ones in their electromobility due to post-translational modifications.

Phosphoglucomutase in Helianthus debilis: a polymorphism for isoenzyme number

Helianthus debilis is an annual, outcrossing sunflower species comprising five subspecies. Subspecies cucumerifolius and silvestris are characterized by two chloroplastic and one cytosolic phosphoglucomutase (PGM) isoenzymes, whereas the remaining three subspecies, debilis, tradiflorus. and vestitus exhibit two chloroplastic and two cystosolic PGM isoenzymes. Polymorphism for isoenzyme number within a plant species has rarely been reported.

Changes in isoenzyme patterns of a cloned culture of nonpathogenic Entamoeba histolytica during axenization.

The axenization of an Entamoeba histolytica isolate with a nonpathogenic isoenzyme electrophoretic pattern (zymodeme) was recently achieved for the first time (15). Forty days after the cells were transferred to the medium used for axenic cultivation, the amebae developed virulence properties, and the zymodeme converted to a pathogenic pattern. To exclude the possibility that the original isolate consisted of two zymodeme populations and that conditions of growth selected for a particular population, the experiment was repeated with a cloned culture of a nonpathogenic (zymodeme III) strain, E. histolytica SAW 1734R clAR, isolated by and obtained from P. G. Sargeaunt. Axenization was accomplished, as before, by transferring trophozoites to TYI-S-33 medium containing a mixture of antibiotics to suppress the growth of the associated bacterial flora and a nutritional supplement consisting of gamma-irradiated bacteria. A change in the hexokinase and phosphoglucomutase isoenzyme pattern was observed 21 days after the amebae had been transferred to the axenic medium but before complete axenization of the amebae had occurred. The change in zymodeme was accompanied by an increase in virulence, as evidenced by the ability of fewer amebae to induce hepatic abscesses in hamsters. A reverse conversion to a nonpathogenic zymodeme was also accomplished by reassociating and subculturing the newly converted pathogenic trophozoites of strain SAW 1734R clAR with the bacterial flora that accompanied this ameba in the original xenic culture. The electromobilities of the hexokinase isoenzymes changed back to their original pattern 7 days after the amebae were returned to xenic growth conditions. Our in vitro results demonstrate that culture conditions and bacterial flora can cause changes in the zymodeme and virulence of a cloned ameba isolate and raise the concern that this could happen also in vivo. Thus, the finding of a particular zymodeme in a culture of E. histolytica isolated from a carrier should not be used to predict a clinical condition or serve as a basis for the recommendation of therapy.

Identification of Kentucky Bluegrass Cultivars with Esterase and Phosphoglucomutase Isoenzyme Markers1

Starch gel electrophoresis was used to further extend the identification of Kentucky bluegrass cultivars. A total of 24 cultivars were examined for isoenzyme variation in esterase (EST), phosphoglucomutase (PGM), phosphoglucoisomerase (PGI), and glutamate‐oxaloacetate transaminase (GOT) using starch gel‐electrophoresis. Esterase and phosphoglucomutase patterns were varible among the 24 cultivars. ‘Baron’, ‘Fylking’, ‘Merion’, and ‘Newport’ were examined for isoenzyme pattern differences between seed and seedling materials. Differences of esterase isoenzyme patterns were found between the two kinds of tissues. Seeds of ‘Adelphi’ and ‘Glade’ harvested from different fields and years were examined for isoenzyme consistency and within seed lot variation. Variation within seed lots differed from seed lot to seed lot. If seedlings from a seed lot were mixed, a constant pattern of both EST and PGM isoenzyme systems was obtained for different seed lots of the same cultivar. Therefore the process of mixing seedlings allows the finger‐printing characterization for Kentucky bluegrass cultivars even if there is occasional segregation from sexual reproduction.

Isoelectric Points (pI) of the Isoenzymes of Phosphoglucomutase (PGM 1 and PGM 2) Observed by Isoelectric Focusing, and Some Theoretical Considerations

This report describes experiments designed to ascertain the pI values of the various PGM 1 and PGM 2 isoenzymes from the human red cell as observed by isoelectric focusing in a 2% ampholine gradient (pH5–pH7).

The PGM isoenzyme distribution among negroid people of Zimbabwe

The value of the phosphoglucomutase isoenzyme system as a method for typing negroid blood in Zimbabwe has been evaluated. Due to the high frequency of the 1 phenotype (70%) a significant improvement in discriminating power is obtained when isoelectric focussing is used.

Detectability of Selected Genetic Markers in Dried Blood on Aging

Abstract The detectability of selected blood genetic markers aged up to six months deposited on six substrata, glass, wool, nylon, and three types of cotton (plain cotton, permanent press, and denim), was investigated. The resulting dried blood specimens were aged at ambient temperature at 20 and 66% relative humidities; a few samples were aged at —20°C Analyses were performed on the samples kept blind for the investigators at 1-, 2-, 4-, 13-, and 26-week aging periods. Red cell antigen systems selected for this study were ABO, MN, Rh, Kidd, Duffy, and Kell. The most stable antigens were A, B, and O of ABO; M, N, and s of MN; and D of the Rh system. These variants were identified in specimens aged for 26 weeks at both low and high relative humidities. The least stable antigens, Jk a of Kidd, Fy a of Duffy, and K of the Kell systems, were detectable for only one week at either humidity level. Of these antigens, only Fy a and K aged at 20% humidity were detected at the two-week test period. Other variants (S of MN and C, c, E, and e of Rh) were detected for various lengths of time ranging from 2 to 26 weeks. In particular, Rh factors C, c, and E were affected adversely by high moisture environments. The four enzyme systems selected were adenylate kinase (AK), adenosine deaminase (ADA), phosphoglucomutase (PGM), and erythrocyte acid phosphatase (EAP). AK and PGM isoenzymes were still identifiable at 26 weeks, and ADA and EAP at 13 weeks for the low and high humidity storage conditions. PGM isoenzymes appeared to be more stable at low humidity, and ADA and EAP at high humidity. No obvious differences in detectability resulting from phenotype or substrate were discerned, except possibly for permanent press and denim, which appeared to shorten the detectability time of PGM. Storage of the specimens at —20°C generally preserved the antigens and the enzymes better than storage at room temperature. The discrimination probability was calculated on the basis of the genetic markers still detectable at the end of each aging interval. No test error was assumed, and frequency of occurrence data for each genetic marker were taken from the literature.

The Simultaneous Identification of Seminal Acid Phosphatase and Phosphoglucomutase by Starch Gel Electrophoresis

Although elevated acid phosphatase (AP) activity in vaginal fluid is a consistent indicator for semen, differentiation between vaginal AP and seminal AP provides a more meaningful result. Detection of seminal AP in mixtures of vaginal AP, feces, and blood is accomplished by starch gel electrophoresis, employing the substrate thymolphthalein monophosphate as a selective visualization agent. Genetic phosphoglucomutase isoenzymes are simultaneously separated by this method and allow differentiation in some semen/vaginal fluid mixtures.

Characterisation of the isoenzymes of phosphoglucomutase (PGM) determined by the first (PGM1) and second (PGM2) locus observed by isoelectric focusing

The existence of four alleles of phosphoglucomutase (PGM1) in human red cell lysates has previously been demonstrated by isoelectric focusing (Bark et al., 1976; Kühnl et al., 1977; Sutton and Burgess, 1978). Experiments are now described in which the position of each of the first-locus (PGM1) and second-locus (PGM2) isoenzymes is defined, thus extending and confirming the original proposal made by Bark et al.