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-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<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.This investigation was supported in part by Grant AM 14898, National Research Service Award 5 F 32 AM 05418, and Biochemical Research Support Grant 5 S07 RR 05551 from the National Institutes of Health.     

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.     

Identification of a Major Locus Contributing to Erythrocyte 2,3-Diphosphoglycerate Variability in Hooded (Long-Evans) Rats

The erythrocyte glycolytic intermediate 2,3-diphosphoglycerate (DPG) and adenosine triphosphate (ATP) play an important role in oxygen transport and delivery by binding to hemoglobin (Hb) and reducing its affinity for oxygen. Considerable quantitative variability in the levels of DPG and ATP exists in human populations and in a population of hooded (Long-Evans) rats we have studied. This paper presents the results of studies on the genetic component of DPG-level variation in an outbred population of hooded rats. Beginning with about 100 rats, a two-way selection experiment was initiated. Pairs of rats with the highest DPG levels were mated to produce a High-DPG rat strain and animals with the lowest DPG levels were mated to produce a Low-DPG strain. Mean DPG levels responded rapidly to selection and, from generation 3 on, the differences between strain means were highly significant. Ten High-DPG strain rats were intercrossed with 10 Low-DPG strain rats of generation 10 to produce an F₁ generation in which the DPG levels were almost as high as those of High-DPG animals. This indicates partial dominance of High-DPG alleles. The F₂ DPG-level distribution showed two distinct subpopulations. The high DPG subpopulation contained three times as many animals as the low DPG subpopulation. From these results and the statistical analyses performed, it was concluded that the DPG differences between strains were due to an allelic difference at one major locus, the allele carried by the High-DPG strain showing partial dominance over the allele carried by the Low-DPG strain. It appears that this locus may also effect ATP levels to a large extent and is polymorphic in hooded rat populations. Identification of this locus gives us a useful tool for studies of the physiological effects of DPG variability, as well as providing an example of a major gene effect in a quantitatively varying trait.     

Association in Long-Evans hooded rats of red cell 2,3-diphosphoglycerate levels with hemoglobin types

Two sublines of commercially available Long-Evans hooded rats have been developed by genetic selection. These sublines have widely differing levels of erythrocyte 2,3-diphosphoglycerate (DPG) due to different alleles at a single genetic locus. In the present work, it is shown that rats from the commercial population are also polymorphic at a hemoglobin locus, probably involving two alleles of the ^III-globin chain locus. Particular hemoglobin types have been found to be strongly associated with certain DPG types, not only in the high-DPG and low-DPG lines but also in the commercial population. Two explanations for this association are considered. One is a single-locus hypothesis, with hemoglobin allelic variation causing DPG variation, and the other is a two-locus hypothesis, with marked linkage disequilibrium.This work was supported by a Michigan Heart Association Grant, by the Meyers Foundation, and by NIH Training Grant (5 T01-GM-0071).     

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.This work was supported by an NIH Training Grant (5-T01-GM-0071) and a Michigan Heart Association Grant.     

Developmental changes of 6-phosphofructo-1-kinase subunit levels in erythrocytes from normal dogs and dogs affected by glycogen storage disease type VII.

1. The subunit proportions (L:M:C) of the PFK isozymes from normal adult erythrocytes were 2:86:12. Affected adult erythrocyte 6-phosphofructo-1-kinase (PFK) isozymes contained normal L-type (31%) and C-type (61%) subunits as well as a small amount (8%) of truncated M-type subunit. 2. When measured within 24 hr of birth, both normal and affected dog erythrocytes contained high PFK activities due to elevated levels of the L-type subunit. As the dogs matured, PFK activity decreased due to a greater than 99% loss of the L-type subunit. 3. By 2 weeks of age, the M-type and C-type subunits in normal dog PFK isozymes increased several-fold and attained near adult levels. 4. During post-natal development, the L-type subunit from affected dog erythrocytes decreased more rapidly than from normal dog erythrocytes; but it was maintained at a higher level in the affected adult erythrocytes. Also, in the affected dog erythrocytes, truncated M-type subunits were detected; and the initially high levels of the C-type subunit decreased approximately 50% after 4 weeks.     

5-hydroxymethylfurfural-forming proteins in the renal glomeruli of control and streptozotocin-diabetic rats

Nonenzymatic glycosylatin of renal and erythrocyte proteins was measured by the formation of 5-hydroxymethylfurfural (5-HMF) after mild acid and heat treatments. Two weeks after the injection of streptozotocin (SZO), diabetic rats showed significantly higher levels of 5-HMF formation in hemolysates, but not in renal glomerular extracts, than the control rats. However, both the hemolysates and the renal glomerular extracts showed higher levels of nonenzymatic glycosylation in diabetic rats than in control rats 8 weeks after SZO-injection. 5-HMF-forming protiens were found in all subcellular fractions although the microsomal proteins showed the highest specific activity of 5-HMF-formation per mg.     

Ketone body utilization and its metabolic effect in resting muscles of normal and streptozotocin-diabetic rats.

By using an in situ rat hindquarter perfusion, we evaluated ketone body utilization and its metabolic effects in the resting muscle of 24 h fasted normal and streptozotocin (STZ)-diabetic rats. Under the perfusion with ketone body-supplementation (1 mM each of acetoacetic acid (AcAc) and 3-hydroxybutyric acid (3-OHB], the AcAc and 3-OHB uptake of STZ-diabetic rats was significantly (P less than 0.05) smaller than that of normal rats. This might be explained by the low enzyme activity of 3-oxoacid CoA transferase demonstrated in the hindlimb muscles of STZ-diabetic rats and this reduced ketone body uptake would be one of the causes of the development of diabetic ketoacidosis. The glucose uptake and the phosphofructokinase (PFK) activity of normal rats were significantly (P less than 0.05) higher than those of STZ-diabetic rats. In both normal and STZ-diabetic rats, the glucose utilization and PFK activity of the muscles in the ketone body-supplemented condition were significantly (P less than 0.05) lower than those in the non-supplemented condition. This inhibition of glucose utilization by ketone bodies should be due to the mechanism by which the oxidation of ketone bodies inhibits PFK in the muscle.     

Inherited erythrocyte phosphofructokinase deficiency: Molecular mechanism

Summary Erythrocyte PFK activity 50–60% that of normal controls was found in a mother and her son, without muscular or hematological symptoms.The PFK activity of the mother's muscle was normal in fresh preparations and partially unstable to storage at 4°C. Electrophoresis of muscle PFK revealed two bands, one normal and one abnormal with an anodic mobility greater than normal. Both patients were characterized as heterozygotes for an unstable muscle PFK. Unstable M'subunits disappeared in erythrocytes which are old cells devoid of protein synthesis. Consequently an increased E/M subunit ratio leads to a distribution of the five isozymes different from that of normal erythrocytes. In these patients, we observed a loss of the M₄ enzyme together with an increase in the E₄ isozyme. The kinetic and immunologic data were compatible with these modifications. Isoelectric focusing of hemolysates from the two patients revealed an acidification of the main activity band, suggesting that an increase in E₄ isozyme resulted in a change of the total electric charge.Unité de recherche d'enzymologie des cellules sanguines (INSERM U160 & ERA 573 du CNRS) Hôpital Beaujon 92118 Clichy Cedex, France     

Presence of a truncated M-type subunit and altered kinetic properties of 6-phosphofructo-1-kinase isozymes in the brain of a dog affected by glycogen storage disease type VII.

6-Phosphofructo-1-kinase (PFK) activity in the brain of a dog affected by glycogen storage disease type VII was only 31% of the PFK activity in the normal dog brain. PFK in the normal dog brain was composed of L-type, M-type and C-type subunits with apparent molecular weights of 78,000, 86,000, and 88,000, respectively, and subunit proportions (L:M:C) of 27:49:24. PFK in the affected dog brain was composed of nearly equal levels of the normal L-type and C-type subunits, but a normal M-type subunit was not detected. Using antidog muscle PFK IgG, immunoblots of gels containing partially purified PFK from the affected dog brain revealed a small amount of immunoreactive protein with an apparent molecular weight of 84,000, suggesting the presence of a truncated M-type subunit. Kinetic studies indicated that the PFK isozymes in the affected dog brain exhibited significantly different kinetic regulatory properties when compared to the PFK isozyme pool in the normal dog brain.     

Effects of potassium antimony tartrate on rat erythrocyte phosphofructokinase activity

In an earlier study, we observed a marked accumulation of antimony in erythrocytes of rats administered potassium antimony tartrate (Sb) in drinking water. This observation has raised concerns of possible adverse effects on the hematological systems. A study was therefore carried out to investigate the effects of Sb on phosphofructokinase (PFK), a rate-limiting enzyme of erythrocyte glycolysis. Preincubation of PFK with Sb caused a marked inhibition of the enzyme with 95% loss of activity at 5 mM. In comparison, 5 mM sodium arsenite, a known enzyme inhibitor, reduced PFK activity by only 38%. Increasing the concentrations of fructose-6-phosphate (F6P) or magnesium had no effects on the inhibitory potency of Sb. Varying the concentrations of ATP and Sb produced a complex effect on PFK activity. At 1 mM ATP, 0.2 mM Sb was required for 50% inhibition (IC₅₀) of PFK but only 0.05 mM Sb was required for the same inhibition when the concentration of ATP was reduced to 0.2 mM. Glutathione (2–10 mM) and hemoglobin (8–40 <μ > M) partially protected the enzyme from the Sb effect, with the protection being more effective at low antimony concentrations. When Sb was added to assay mixtures after initiation of a PFK reaction with physiological concentrations of ATP (0.2 mM) and F6P (0.1 mM), PFK activity was approximately 50% inhibited by 0.5 mM Sb and completely inhibited by 5 mM Sb. In contrast, glucose utilization in whole blood was only 16% lower over an 8 hour incubation period in the presence of 5 mM Sb. It is concluded that while PFK is markedly inhibited by Sb under in vitro assay conditions, glycolysis in erythrocytes is not significantly affected except at very high Sb concentrations. The weak effect of Sb on glycolysis in erythrocytes may be due in part to the protective effect of hemoglobin and, to a lesser extent, glutathione on PFK. © 1998 John Wiley & Sons, Inc. J Biochem Toxicol 12: 227–233, 1998     

Effects of 20-MHz radiofrequency radiation on rat hematology, splenic function, and serum chemistry

In this study, Sprague-Dawley rats were exposed in a TEM chamber to 20-MHz (HF-band) continuous-wave radiofrequency radiation (RFR) for 6 hr/day, 5 days/week up to 6 weeks. The average E-field intensity was 2686 +/- 164 V/m (mean +/- SD) and the calculated specific absorption rate was 0.3 W/kg. Randomly sampled rats killed on Days 8, 22, 39, and 42 after initiation of exposure showed no statistically significant differences from controls for body mass, spleen cell density, erythrocyte and leukocyte counts, hematocrit, hemoglobin, methemoglobin, erythrocyte fragility, bilirubin, creatinine, SGPT, alkaline phosphatase, calcium, sodium, potassium, and spleen cell chemiluminescence. Splenic mass differences were statistically significant (p less than 0.05) only on Day 22. Spleen to body mass ratios differed significantly between exposed and control groups on Days 22 and 39 (P less than 0.05 and P less than 0.025, respectively). Histologic examination of the rats revealed the successive accumulation of phagocytic cells, lymphoid proliferation, development of lesions, and tissue necrosis characteristic of respiratory mycoplasmosis. In a followup experiment, a separate set of rats was exposed for 6 weeks to identical levels of RFR. No significant differences were found in splenic parameters and spleen cell peroxidative activity. Histologic examination of these animals revealed no evidence of mycoplasma infection. The observed differences between exposed and control animals of the first experiment appear to have resulted from subclinical respiratory mycoplasmosis rather than exposure to RFR.     

Regulation of glycolysis in sea bass liver: phosphofructokinase isozymes

Sea bass (Dicentrarchus labrax L.) liver phosphofructokinase (PFK) presents biphasic kinetics with respect to fructose-6-phosphate (F-6-P) in experiments carried out with crude extract. After the enzyme had been purified, two isozymes have been detected after chromatographic treatment. The two isozymes present different kinetic behaviour PFK-L1, the first eluted phosphofructokinase activity shows positive cooperativity with respect to fructose-6-phosphate and PFK-L2, the second activity fraction, has a Hill coefficient of 0.38 (negative cooperativity). The first isozyme shows less affinity for fructose-6-phosphate than that shown by PFK-L2. The joint kinetics of both isozymes produces a biphasic kinetics with respect to fructose-6-phosphate, similar to that observed in crude extracts.     

Phosphofructokinase isozymes from human organs and blood cells.

Phosphofructokinase (PFK) isozymes of blood cells and some human tissues were studied by starch gel electrophoresis and immunoprecipitation by anti-muscle and anti-erythrocyte PFK sera. PFK from muscle, heart, brain and placenta were totally precipitated by both antisera. PFK from blood cells (erythrocytes, lymphocytes, granulocytes, platelets) were precipitated more strongly by anti-erythrocyte PFK serum than by anti-muscle PFK serum. Liver, kidney and monoblast PFK were slightly precipitated by both antisera. From the electrophoretic patterns and the immunoprecipitation curves we may conclude that muscle contains the homotetrameric M4 forms; platelet, liver and kidney the homotetrameric E4 form, and blood cells the M-E hybrids. Monoblasts probably contain a E4 type PFK precursor, and heart, placenta and brain, a modified M4 type PFK. Other isozymes, unrelated with muscle and erythrocyte, were revealed in liver and kidney.     

An immunoelectrophoretic study of protein polymorphism in hemolysates of pigs and cattle

By means of immunoelectrophoretic analysis, the antigenic spectrum of proteins in pig and cattle hemolysate was studied. Based on the immunological similarity of the electrophoretic variants of one and the same polymorphic system, two polymorphic systems were established in pig hemolysates, designated preliminarily as Ph-A and Ph-B systems. In cattle hemolysates, two polymorphic systems were found: Ch-B and Ch-C systems. The proteins of the Ph-B and Ch-B systems seem to be isozymes of an erythrocyte catalase and give an immunological cross-reaction. The hereditary nature of the established polymorphism was confirmed by genetic analysis of the Ph-B system. Based on this, the suggestion is advanced that erythrocyte catalase in pigs is controlled by at least two codominant alleles at the Ph-B locus.     

Adenine phosphoribosyl transferase polymorphism in baboons

Two allelic isozymes of adenine phosphoribosyl transferase (APRT) were detected by starch gel electrophoresis of baboon hemolysates. Extensive family data verified autosomal codominant inheritance. The gene frequencies of five subspecies of baboons differed significantly. The activity of erythrocyte APRT is sufficiently high to enable the use of this enzyme as a sensitive marker for assessing chimerism in research involving bone marrow transplantation.This research was supported in part by NIH Grants HL28972, HG00336, and HV53030.     

Genetic variation in erythrocyte NAD levels in the mouse and its effect on glyceraldehyde phosphate dehydrogenase activity and stability

During a screening of inbred strains for enzyme variation in glycolysis, differences were found in glyceraldehyde phosphate dehydrogenase (GAPDH) activity between C57BL/6J and SM/J mice. Segregation analysis did not reveal unequivocal monogenic inheritance. Dialysis of hemolysates caused decay of enzyme activity, especially in C57BL animals, which could be prevented by the presence of NAD. This led to the finding that erythrocyte NAD levels were threefold higher in SM than C57BL animals and, although additively inherited, did not appear to be monogenic. This is comparable with but independent of the differences and effect of erythrocyte NADP levels on glucose-6-phosphate dehydrogenase activity reported by R. P. Erickson [(1974) Biochem. Genet. 11:33] and emphasizes the range of mechanisms that can be involved in the genetic control of enzyme activity in mammalian systems.We thank the MRC for financial support.     

Pressure increases oxygen affinity of whole blood and erythrocyte suspensions

Effect of hydrostatic pressure (HP) on whole blood (WB) or erythrocyte suspension hemoglobin (Hb) O2 affinity has been studied using newly developed techniques. O2 partial pressure at which hemoglobin is half-saturated with O2 (P50) measurements were made at 5 HP (1, 26, 51, 76, and 126 ATA) on thin films of human WB or erythrocytes at 37 degrees C. CO2 partial pressure of WB was either 28 or 57 Torr (film pH 7.51 or 7.31). HP increased affinity of erythrocytes and WB. For erythrocytes in tris(hydroxymethyl)aminomethane buffer, the ratio (r) of P50 (1 ATA)/P50 (51 ATA) was 1.089 (P less than 0.01) at pH 7.0. WB P50 decreased with HP at a rate of -3.3 X 10(-2) Torr X atm-1; change in P50 at higher HP vs. 1 ATA was highly significant (P less than 0.01). No effect of HP was seen on the CO2 Bohr coefficient. Inert gas choice, N2 vs. helium (He), had no effect. Measurement of decrease of P50 with HP at 76 ATA in hemolyzed WB gave an r of 1.15, as great or greater than that found in WB, indicates that Donnan equilibrium alteration is not involved. No effect of HP was found in WB on the ratio of P50 of erythrocytes with normal (5 mmol/l erythrocytes) 2,3-diphosphoglycerate (DPG) to P50 of erythrocytes with less than 5% of normal DPG; i.e., no effect of pressure was seen on the independent influence of DPG on P50. WB measurements of Hb O2 uptake under simulated physiological conditions are characterized by a net decrease in partial molal volume on oxygenation of 30-35 ml/mol Hb4.     

Reversible phosphorylation control of skeletal muscle pyruvate kinase and phosphofructokinase during estivation in the spadefoot toad,Scaphiopus couchii

Both pyruvate kinase (PK) and phosphofructokinase (PFK) occur in two different forms, separable by isoelectric focusing (IEF), in skeletal muscle of the spadefoot toad Scaphiopus couchii. During estivation (aerobic dormancy) the proportions of the two forms changed compared with controls; in both cases the amount of enzyme in Peak I (pI = 5.3-5.4) decreased whereas activity in Peak II (isoelectric point = 6.2-6.4) increased. In vitro incubation of crude muscle extracts with 32P-ATP under conditions that promoted the activity of cAMP-dependent protein kinase led to strong radiolabeling associated with Peak I, but not Peak II, and reverse phase HPLC confirmed that 32P was associated with the subunits of both PK and PFK found in Peak I. Specific radiolabeling of Peak I PK and PFK by protein kinase A was further confirmed using immunoprecipitation. In total, this information allowed identification of the Peaks I and II enzymes as the phosphorylated and dephosphorylated forms, respectively, and the effect of estivation was to increase the proportion of dephosphorylated PK and PFK in muscle. Analysis of the kinetic properties of partially purified PK and PFK revealed significant kinetic differences between the two forms of each enzyme. For PK, the Peak II (low phosphate) enzyme showed a 1.6-fold higher Km for phosphoenolpyruvate and a 2.4-fold higher Ka for fructose-1,6-bisphosphate than did the Peak I (high phosphate) form. These kinetic properties suggest that Peak II PK is the less active form, and coupled with the shift to predominantly the Peak II form during estivation (87% Peak II vs. 13% Peak I), are consistent with a suppression of PK activity in estivating muscle, as part of the overall metabolic rate depression of the estivating state. A similar shift to predominantly the Peak II, low phosphate, form of PFK (75% Peak II, 25% Peak I) in muscle of estivating animals is also consistent with metabolic suppression since phosphorylation of vertebrate skeletal muscle PFK is typically stimulated during exercise to enhance enzyme binding to myofibrils in active muscle. Peak II PFK also showed reduced sensitivity to inhibition by Mg:ATP (I50 50% higher) compared with the Peak I form suggesting that the enzyme in estivating muscle is less tightly regulated by cellular adenylate status than in awake toads. The data indicate that reversible phosphorylation control over the activity states of enzymes of intermediary metabolism is an important mechanism for regulating transitions between dormant and active states in estivating species.     

Temperature and phosphate effects on allosteric phenomena of phosphofructokinase from a hibernating ground squirrel (Spermophilus lateralis)

Temperature effects on the kinetic properties of phosphofructokinase (PFK) purified from skeletal muscle of the golden-mantled ground squirrel, Spermophilus lateralis, were examined at 37 degrees C and 5 degrees C, values characteristic of body temperatures in euthermia vs. hibernation. The enzyme showed reduced sensitivity to all activators at 5 degrees C, the K(a) values for AMP, ADP, NH(4) (+) and F2,6P(2) were 3-11-fold higher at 5 degrees C than at 37 degrees C. Inhibition by citrate was not affected whereas phosphoenolpyruvate, ATP and urea became more potent inhibitors at low temperature. While typically considered an activator of PFK activity, inorganic phosphate performed as an inhibitor at 5 degrees C. Decreasing temperature alone causes the actions of inorganic phosphate to change from activation to inhibition. We found that K(m) values for ATP remained constant while V(max) dropped significantly upon the addition of phosphate. Phosphate inhibition at 5 degrees C was noncompetitive with respect to ATP and the K(i) was 0.15 +/- 0.01 mm (n = 4). The results indicate that PFK is less likely to be activated in cold torpid muscle; PFK is less sensitive to changing adenylate levels at the low temperatures characteristic of torpor, and PFK is clearly much less sensitive to biosynthetic signals. All of these characteristics of hibernator PFK would serve to reduce glycolytic rate and help to preserve carbohydrate reserves during torpor.