The Dpg gene: an intracorpuscular modifier of red cell metabolism.

The genetic locus designated Dpg has two alleles in outbred Long-Evans rats. Genotype at this locus affects quantities of red cell 2,3- diphosphoglycerate (DPG) and adenosine triphosphate, as well as activities of two important glycolytic enzymes: phosphofructokinase and pyruvate kinase. Intravascular red cell survival is shortened in low-DPG animals. In order to get closer to the specific action of this locus, we addressed the question of whether the Dpg gene acts through intracorpuscular or extracorpuscular factors. Bone marrow transplantation after total body irradiation and 51Cr red cell survival after cross transfusion were the methods used. Because the animals that were used differed in hemoglobin phenotype, donor and recipient cells could be quantified in cross-transplanted animals. Phenotypic markers of Dpg genotype were measured in animals 40 to 50 days after transplantation. Values for these markers correlated highly with the percentage of donor and recipient cells present. In vivo survival of low-DPG red cells was significantly shorter than that of high-DPG cells (P less than .05), regardless of the genotype of the recipient. From the present studies, we conclude that the action of the Dpg gene is exerted by an intracorpuscular factor.

Genetic components of variations of red cell glycolytic intermediates at two altitudes among the South American Aymara.

Red cell haemoglobin (Hb), haematocrit (Ht), 2,3-diphosphoglycerate (DPG), and adenosine triphosphate (ATP) levels were measured in 876 individuals from six villages at two altitude levels (altiplano and coast) of the Departmento de Arica of Northern Chile. Of these, data on 761 individuals are subjected to analysis to search for the evidence of genetic adaptation to a hypoxic environment at a high altitude. Total phenotypic variance for each of the variables is higher at the altiplano as compared to their counterparts at the coastal level. Data on 1127 pairs of relatives of six degrees of relationships are used to determine the genetic component of variation in each of these four traits. To a certain extent the larger familial correlations as well as higher variances at altiplano are explained by the apparent assortative mating (which may again be due to their restricted population size) at the higher elevation. Yet, at least in three variables (Hb, Ht, and DPG) no reduction in the additive genetic component of variation is noticed at the higher altitude. ATP seems to have the highest degree of genetic component of variance, particularly at the coastal level. Some implications of these results are discussed in the light of their roles in the glycolytic pathway.

Erythrocyte isozymes of phosphofructokinase in genetically high- and low-2,3-diphosphoglycerate rats.

A major locus (Dpg) with two alleles (d and D) controls erythrocyte 2,3-diphosphoglycerate (DPG) levels in Long-Evans rats and is closely linked to a locus (Hbb) determining a hemoglobin electrophoretic polymorphism. Glycolytic intermediate levels and phosphofructokinase (PFK) kinetic studies suggest that in vivo PFK activity differences underlie the differences in DPG levels. We report here chromatographic and immunologic evidence that rat erythrocyte PFK is composed of two isozymes which elute from DEAE-Sephadex at positions identical to those of the isozymes in platelets and liver, respectively. The percentage of platelet-type PFK is significantly (P less than 0.05) smaller in low-DPG (dd) hemolysates than in DD hemolysates regardless of hemoglobin phenotype. When hemolysates were prepared in a stabilizing buffer, PFK specific activity was significantly (P less than 0.005) higher in DD rats. These data suggest that the PFK kinetic differences may result from alterations in the isozyme composition of active PFK.

Erythrocyte phosphofructokinase in rat strains with genetically determined differences in 2,3-diphosphoglycerate levels.

We have studied the erythrocyte enzyme phosphofructokinase (PFK) from two strains of Long-Evans rats with genetically determined differences in erythrocyte 2,3-diphosphoglycerate (DPG) levels. The DPG difference is due to two alleles at one locus. With one probable exception, the genotype at this locus is always associated with the hemoglobin (Hb) electrophoretic phenotype, due to a polymorphism at the III beta-globin locus. The enzyme PFK has been implicated in the DPG difference because glycolytic intermediate levels suggest that this enzyme has a higher in vivo activity in High-DPG strain rats, although the total PFK activity does not differ. We report here that partially purified erythrocyte PFK from Low-DPG strain cells is inhibited significantly more at physiological levels of DPG (P less than 0.01) than PFK from High-DPG strain erythrocytes. Citrate and adenosine triphosphate also inhibit the Low-DPG enzyme more than the High-DPG enzyme. Therefore, a structurally different PFK, with a greater sensitivity to inhibitors, may explain the lower DPG and ATP levels observed in Low-DPG strain animals. These data support a two-locus (Hb and PFK) hypothesis and provide a gene marker to study the underlying genetic and physiologic relationships of these loci.

Erythrocyte enzymes in groups of Rattus norvegicus with genetic differences in 2,3-diphosphoglycerate levels.

1. A major locus with two alleles is responsible for large differences in erythrocyte 2,3-diphosphoglycerate (DPG) levels in Rattus norvegicus. Blood from homozygous High-DPG, homozygous Low-DPG and heterozygous animals was used to measure blood indices and red cell enzyme activities. 2. Significant differences between groups were found in DPG levels, white blood cell counts and hemoglobin levels. 3. The results suggest that none of the red cell enzymes assayed is structurally or quantitatively different in the three groups.

Red cell diphosphoglycerate mutase. Immunochemical studies in vertebrate red cells, including a human variant lacking 2,3-DPG.

Diphosphoglycerate mutase (DPGM) was purified to homogeneity from human erythrocytes. The enzyme and Freund adjuvant were injected into chickens and yielded a monospecific precipitating antibody. Radial immunodiffusion with this antibody was used to measure the amount of DPGM in hemolysates from human adult and cord red cells. Dog, rabbit, rat, chicken, and goat red cells all had DPGM during the neonatal period, but goat adult red cells had no detectable enzyme. Single bands with no spurs were present on Ouchterlony plates in which human hemolysate was placed adjacent to hemolysates from the other species tested. The amount of human red cell DPGM did not differ between young and old cells separated by centrifugation. Red cells from a patient with a DPGM genetic variant who had erythrocytosis and no detectable enzyme activity contained a reduced amount of DPGM as determined by radial immunodiffusion. The abnormal DPGM differed from normal by immunoelectrophoresis and in stability as measured by the amount of crossreacting material in young versus old erythrocytes.

Association between cholesterol and 2,3-diphosphoglycerate in genetically selected hooded rat lines.

We have developed two strains of hooded rats with differing erythrocyte oxygen affinities by selection on red cell 2,3-diphosphoglycerate levels. Genetic studies have shown that these strains differ at one DPG-level-determining locus. This article reports the results of a study which involved measurement of plasma cholesterol levels in rats from the strains and the F2 progeny of strain intercrosses. Low-DPG strain rats, with high oxygen affinity, had significantly higher mean cholesterol levels than High-DPG rats. Animals from the extremes of the F2 distribution of DPG levels showed similar, significantly different mean cholesterol levels, indicating that the negative association between DPG and cholesterol levels in strain rats was not due to inadvertent fixation of unrelated genes during selection on DPG. The possibility is discussed that high oxygen affinity, brought about by low DPG levels, may be causative in increasing cholesterol levels.