Three phenotypes of glucosephosphate isomerase in sheep: improved staining recipe

Contrary to results published recently, we observe three, rather than two, phenotypes for the enzyme glucosephosphate isomerase (EC 5.3.1.9) from sheep. The phenotypic electro phoretic patterns conform to the patterns observed for this dimeric enzyme in other species. Genotype frequencies in a flock of South downs do not deviate significantly from those predicted under the assumption of the Hardy-Weinberg equilibrium. A remarkable observation is that the electro phoretically distinct phenotypes of GPI are largely or entirely obliterated by the addition of 1–10 mmol/1 MgCl₂ to the electro phoretic buffers. Modification of the usual staining recipe for GPI result in greater resolution and shorter staining times.     

Polymorphism of erythrocyte glucosephosphate isomerase in sheep

An electrophoretic analysis of glucosephosphate isomerase (GPI) in seven Italian sheep populations suggests that this locus is more polymorphic than previously supposed. The observed phenotype distributions are in agreement with the hypothesis of the existence of three codominant alleles, GPI*F, GPI*S and GPI*N, GPI*S being the most frequent (0.935 ÷ 1.000).     

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.     

Unique phenotypic expression of glucosephosphate isomerase deficiency.

Studies of a Mexican kindred present evidence for a unique phenotype of erythrocyte glucosephosphate isomerase, GPI Valle Hermoso. The proband was apparently the homozygous recipient of a mutant autosomal allele governing production of an isozyme characterized by decreased activity, marked thermal instability, normal kinetics and pH optimum, and normal starch gel electrophoretic patterns. Unlike previously known cases, leukocyte and plasma GPI activities were unimpaired. This suggested that the structural alteration primarily induced enzyme instability without drastically curtailing catalytic effectiveness, thereby allowing compensation by cells capable of continued protein synthesis. Age-related losses of GPI, however, were not evident by density-gradient fractionation of affected erythrocytes.     

A new variant of glucosephosphate isomerase deficiency.

A new variant of glucose-6-phosphate isomerase deficiency is described. The enzyme kinetics and properties were studied. Genetic and electrophoretic data pointed to a double heterozygous state in the patient. These data are compared to the other variants described in the literature until now.     

Intracellular ATP in a glucosephosphate isomerase mutant ofSaccharomyces cerevisiae

The degree of ATP depletion caused by glucose in a glucosephosphate isomerase-deficient strain of Saccharomyces cerevisiae was determined. Even in the presence of a sugar normally fermentable by the mutant, the addition of glucose can decrease the intracellular ATP, depending on the competition of the sugars for transport and subsequent phosphorylation. For both parent and mutant cells, a correlation exists between the calculated velocity of ATP formation or ATP consumption during the utilization of different concentrations of sugars and the experimental intracellular ATP level. For initially resting yeast cells, a rate increase of 35 mumol per min per g ATP was calculated to increase the intracellular level of this nucleotide by 1 mumol per g cell mass.     

Genetics of glucosephosphate isomerase in Aedes togoi (Diptera: Culicidae)

The genetics of glucosephosphate isomerase (E.C. 5.3.1.9) in two strains (Malaysian and Taiwan) of Aedes togoi is reported. Three electrophoretic phenotypes were present in both sexes. The zymogram patterns were identical in both strains of A. togoi. The phenotypes were governed by a pair of codominant alleles. The allele frequency of the slow-moving band was 0.63 in the Malaysian strain and was 0.86 and 0.82 in F161 and F169 generations, respectively, of the Taiwan strain. The sample studied was in good accord with Hardy-Weinberg expectations.     

Affinity labeling of human glucosephosphate isomerase with N-bromoacetylethanolamine phosphate

N-Bromoacetylethanolamine phosphate rapidly and irreversibly inactivates glucosephosphate isomerase in a pseudo first-order fashion. Ratesaturation effects are observed with a minimum half-life of 4.5 minutes and a half-maximal rate of inactivation at 0.056 mM. Substrates, as well as competitive inhibitors, protect the isomerase from inactivation. Using ¹⁴C-labeled N-bromoacetylethanolamine phosphate, the incorporation of approximately one equivalent of inactivator per subunit of isomerase is indicated. After acid hydrolysis, the only modification appears to be the formation of carboxymethyl histidine. These studies indicate that the substrate analogue N-bromoacetylethanolamine phosphate is a specific affinity label that can be used to probe the active site of glucosephosphate isomerase.     

Characterization of Neurospora crassa mutants deficient in glucosephosphate isomerase.

Two independent mutants of Neurospora crassa lacking glucosphosphate isomerase activity (gpi) were isolated. These mutants were obtained as double mutants containing the pp or T9 mutation in addition to the gpi mutation located on linkage group IV; the pp mutation caused the inability to form protoperithecium and the loss of ascospore germination, and the T9 mutation caused the alteration in glucoamylase and several growth characteristics. The gpi mutants did not grow on fructose but grew on glucose or sucrose. Growth of these mutants on glucose was stimulated by addition of fructose. The gpi mutants showed restricted colonial growth on agar media containing glucose in contrast to the normal filamentous growth of the wild-type stain.     

A simple procedure to obtain yeast hexokinase free of glucosephosphate isomerase and mannosephosphate isomerase

A hexokinase preparation was obtained from aSaccharomyces cereviaiae mutant strain deficient in glucosephosphate isomerase (GPI) and mannosephosphate isomerase (MPI) by precipitation with ammonium sulfate. The supernatant fraction corresponding to 40 – 60 % saturation showed the lowest content in GPI and MPI activity. The fraction was used without further purification in the determination of glucose, either free or in a mixture with fructose and mannose. The results were similar to those obtained with pure commercial hexokinase.     

Immunoreactivity of the two common allozymes of murine glucosephosphate isomerase

There are two common electrophoretic variants (allozymes) of murine glucosephosphate isomerase (GPI). In order to develop immunocytochemical procedures that are allozyme-specific, the two variants (GPI-1A and GPI-1B) were purified from skeletal muscle of several mouse strains and used as antigens for the elicitation of antibodies. The allozymes were purified to a specific activity of 800 units/mg by substrate elution from cellulose phosphate. When the purified allozymes were presented as antigens to goats, rabbits, and mice (both syngeneically and allogeneically), the goats and rabbits produced high titers of anti-GPI antibody, but no humoral antibody was detected in the mice, as determined by radioimmunoassays. Antisera specific for the GPI-1B variant were enriched by absorbing selected sera with GPI-1A conjugated to Sepharose 4B. No antisera specific for GPI-1A were detected following the immunizations. The specificity of the anti-GPI-1B antisera provides a unique opportunity for the development of immunocytochemical procedures for studying the distribution of this allozyme in chimeric mouse tissues.This research was supported by grants from the Public Health Service (HD 12685) and the National Science Foundation (PCM 7923065). J.G. is the recipient of a Basil O'Connor Award from the March of Dimes-Birth Defects, and M.L.O.G. is a Fellow of the Alfred Sloan Foundation.     

Sugar synthesis in Leptospira. I. Presence of glucosephosphate isomerase

The presence of glucosephosphate isomerase, one of the key enzymes in carbohydrate metabolism, was confirmed for the first time in the cell-free extract of Leptospira biflexa. The glucosephosphate isomerase of L. biflexa was heat-labile and its optimum pH was about 8.5. The enzyme showed an optimal temperature of about 45 C but was more stable at 30 C. Km value of the enzyme was 5.6 X 10(-3)M. The activity of the enzyme was inhibited by the inhibitor, 6-phosphogluconate. From this study, the presence of a metabolic pathway, the phosphogluconate pathway, other than non-oxidative pentose phosphate pathway presented by Baseman and Cox was suggested.     

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.     

Thermostability characteristics of glucosephosphate and triosephosphate isomerase in erythrocytes from several species

Significant differences in the thermostability of both glucosephosphate and triosephosphate isomerase were noted among a series of six primate and five nonprimate species. The enzyme structural differences among species, as assessed by thermostability profiling, was greater than expected from electrophoretic mobility patterns. Microheterogeneity of GPI, i.e. differences in thermostability within a species that are not detectable by electrophoresis, was detected in two primate species. Major differences in the levels of erythrocyte enzyme activity were observed with human and cow differing by 18-fold for TPI and baboon and cow differing by seven-fold in GPI activity.     

Clinical symptoms and biochemical properties of three new glucosephosphate isomerase variants

Glucosephosphate isomerase deficiency as the cause of macrocytic congenital nonspherocytic hemolytic anemia is described in three unrelated families. The biochemical properties of the variant glucosephosphate isomerases indicate that the patients have new variants, designated as GPI Kiel, GPI Hamburg, and GPI Homburg. The severity of the clinical symptoms depended on the amount of residual GPI activity and the biochemical properties of the variant enzyme. Thus the patient with GPI Kiel (34% residual activity) whose variant GPI was slightly unstable showed a mild chronic hemolytic anemia. The patient with GPI Homburg (7% residual activity) whose variant enzyme was stable and had a reduced specific activity, suffered from severe congenital hemolytic anemia and neuromuscular symptoms. Due to the special properties of GPI Homburg, we assume that both the hematological and neuromuscular symptoms of the patient with GPI Homburg are caused by his GPI deficiency. The twins with GPI Hamburg (27% residual activity) had a distinctly unstable variant enzyme and had suffered from hemolytic crises since birth. Only GPI Homburg showed an altered electrophoretic mobility and an increased affinity for fructose-6-phosphate. The other two variants had normal values.