In vitro manipulation of 2,3-diphosphoglycerate levels in acid-citrate-dextrose blood with steroids

Erythrocyte 2, 3-DPG content has a marked effect on the oxyhemoglobin dissociation curve and is known to decrease in ACD stored blood. This study revealed the effect of various steroids on the level of 2, 3-DPG. Hydrocortisone sodium succinate and methylprednisolone sodium succinate were found to increase the levels of 2, 3-DPG in 3, 10, and 13 day old ACD stored blood during incubation. Hydrocortisone sodium succinate was more effective than methylprednisolone sodium succinate in increasing the level of 2, 3-DPG. In three day old blood the increased 2, 3-DPG approached the lower limits of normal values.     

Biogenesis of erythrocyte membrane proteins. In vitro studies with rabbit reticulocytes.

The capability of rabbit reticulocytes to synthesize red cell membrane proteins has been tested in vitro. Reticulocyte-rich blood from phenylhydrazine-treated rabbits was incubated in vitro in a complete amino acid medium containing ferrous salts, glucose, rabbit plasma and [3-H]leucine. Red cell ghost membranes were prepared by hypotonic lysis and leucine incorporation into hemoglobin and total membrane proteins determined. The pattern of incorporation into individual peptides was determined by polyacrylamide gel electrophoresis of labeled membranes on large (19 mm) gels which were then sliced into 1 mm sections; radioactivity was compared with densitometric tracings of Coomassie blue stained analytical (6 mm) gels. Incorporation of [3-H]leucine into both hemoglobin and membrane protein was linear over 1 h. Gel analysis of labeled membranes revealed that the amino acid was primarily incorporated into peptides with molecular weights of 90 000 or less; three peptides of molecular weights 90 000, 60 000 and 33 000 showed the highest specific activity. Synthesis of the four largest peptide species was negligible. Removable of ferrous salts inhibited synthesis of both globin and membrane protein equally (approx. 50%). However, puromycin and cycloheximide preferentially inhibited the synthesis of globin as compared to membrane proteins. Reticulocytes remain capable of synthesizing a number of membrane proteins; these results are consistent with studies of red cell membrane synthesis in anemic rabbits in vivo.     

In vitro studies of cerebral cortical RNA and nucleotide metabolism in hypothyroidism.

—Thyroid hormone deficiency induced during the neonatal period in the rat, resulted in an enhanced incorporation of [2-¹⁴C]uridine and [8-¹⁴C]adenosine in vitro into cerebral cortical RNA at 25 days of age. An examination of the acid-soluble pool constituents separated by polyethyleneiminecellulose TLC, revealed that all phosphorylated derivatives were more highly labelled compared to controls. These differences were not apparent at a lower incubation temperature (4°C). When the average specific activity of precursor pool ATP labelled from adenosine was utilized for the calculation of the rate of RNA synthesis, no change was observed in hypothyroidism. The results are compatible with a maturational-dependent increase in nucleoside transport and rate of phosphorylation in hypothyroidism which is reflected in the stimulated incorporation into cerebral RNA. The apparent normal rate of RNA synthesis coupled with a diminished cellular RNA concentration in thyroid hormone deficiency, suggests an increased RNA turnover. Experiments with actinomycin D revealed no apparent difference in the rate of decay of rapidly-labelled (nuclear) RNA. The possibility is discussed that the processing of nuclear RNA, the formation of stable ribosomal complexes and events at the translational level are subject to modification in developing hypothyroid rats.     

Autologous transfusion of erythrocytes with high or low concentration of 2,3-diphosphoglycerate. Survival rate and time and hemoglobin-oxygen affinity]

1. In human erythrocytes the 2.3 DPG concentration was increased three to fourfold of the norm as IPP re-suspension by an incubation time of four hours at 37 degrees C or as ACD-AG blood was lowered below 20% of the norm respectively. After an autologous transfusion the 24 hours' surviving rate and the apparent half survival time of cells as well as the affinity of haemoglobin to oxygen in the total blood were measured. 2. The 24 hours' surviving rate for fresh erythrocytes with increased 2.3 DPG and ATP concentration amounts to 73% and the apparent half survival time amounts to 6 days. If erythrocytes are stored for four weeks as IPP resuspension at 4 degrees C, the 24 hours' surviving rate is 59%. Erythrocytes from fresh ACD-AG blood with lowered 2.3 DPG and a normal ATP concentration have a 24 hours' surviving time of 85% and an apparent half survival time of 24 days. 3. After autologous transfusion of 400 ml of erythrocytes with increased 2.3 DPG concentration the P50 value of the total blood will increase by 3 mm of Hg, after administering 400 ml of erythrocytes with lowered 2.3 DPG concentration it will fall by 1.8 mm of Hg. 4. The findings are discussed in connection with the significance of the changes of affinity of haemoglobin to oxygen produced by 2.3 DPG for the oxygen supply of tissues and under the aspect of using stored blood with increased 2.3 DPG concentration for practical purposes.     

Affinity of hemoglobin for the cytoplasmic fragment of human erythrocyte membrane band 3. Equilibrium measurements at physiological pH using matrix-bound proteins: the effects of ionic strength, deoxygenation and of 2,3-diphosphoglycerate

The cytoplasmic fragment of band 3 protein isolated from the human erythrocyte membrane was linked to a CNBr-activated Sepharose matrix in an attempt to measure, in batch experiments, its equilibrium binding constant with oxy- and deoxyhemoglobin at physiological pH and ionic strength values and in the presence or the absence of 2,3-diphosphoglycerate. All the experiments were done at pH 7.2, and equilibrium constants were computed on the basis of one hemoglobin tetramer bound per monomer of fragment. In 10 mM-phosphate buffer, a dissociation constant KD = 2 X 10(-4)M was measured for oxyhemoglobin and was shown to increase to 8 X 10(-4)M in the presence of 50 mM-NaCl. Association could not be demonstrated at higher salt concentrations. Diphosphoglycerate-stripped deoxyhemoglobin was shown to associate more strongly with the cytoplasmic fragment of band 3. In 10 mM-bis-Tris (pH 7.2) and in the presence of 120 mM-NaCl, a dissociation constant KD = 4 X 10(-4)M was measured. Upon addition of increasing amounts of 2,3-diphosphoglycerate, the complex formed between deoxyhemoglobin and the cytoplasmic fragment of band 3 was dissociated. On the reasonable assumption that the hemoglobin binding site present on band 3 fragment was not modified upon linking the protein to the Sepharose matrix, the results indicated that diphosphoglycerate-stripped deoxyhemoglobin or partially liganded hemoglobin tetramers in the T state could bind band 3 inside the intact human red blood cell.     

Decreased level of 2,3-diphosphoglycerate and alteration of structural integrity in erythrocytes infected with Plasmodium falciparum in vitro

2,3-Diphosphoglycerate (2,3-DPG), an intracellular metabolite of glycolytic pathway is known to affect the oxygen binding capacity of haemoglobin and mechanical properties of the red blood cells. 2,3-DPG levels have been reported to be elevated during anaemic conditions including visceral leishmaniasis. 2,3-DPG activity in P. falciparum infected red blood cells, particularly in cells infected with different stages of the parasite and its relationship with structural integrity of the cells is not known. Chloroquine sensitive and resistant strains of P. falciparum were cultured in vitro and synchronized cultures of ring, trophozoite and schizont stage rich cells along with the uninfected control erythrocytes were assayed for 2,3-DPG activity and osmotic fragility. It was observed that in both the strains, in infected erythrocytes the 2,3-DPG activity gradually decreased and osmotic fragility gradually increased as the parasite matured from ring to schizont stage. The decrease in 2,3-DPG may probably be due to increased pyruvate kinase activity of parasite origin, which has been shown in erythrocytes infected with several species of Plasmodium. The absence of compensatory increase in 2,3-DPG in P. falciparum infected erythrocytes may aggravate hypoxia due to anaemia in malaria and probably may contribute to hypoxia in cerebral malaria. As 2,3-DPG was not found to be increased in erythrocytes parasitized with P. falciparum, the increased osmotic fragility observed in these cells is not due to increased 2,3-DPG as has been suggested in visceral leishmaniasis.     

Influence of mouse age and erythrocyte age on glutathione metabolism.

In order to determine whether the biological age of a mouse influences erythrocyte metabolism and erythrocyte aging in vivo, blood samples were collected from male C57/BL6J mice of different biological ages ranging from mature (10 months) to "very old" (37 months). In the very old mouse, compared with the mature mouse, the erythrocyte survival time was decreased, erythrocyte densities were increased, the concentrations of total free thiol and reduced glutathione, and glutathione reductase activity were decreased. Erythrocytes were separated into different density (age) groups by phthalate ester two-phase centrifugation or by albumin density-gradient centrifugation. The density-age relationship of erythrocytes was established by pulse-labelling with 59Fe in vivo and by subsequent determinations of specific radioactivity of erythrocyte fractions of different densities prepared during a chase period of 60 days. The age of erythrocytes in mice of all ages was directly related to density. Also, in older erythrocytes compared with younger erythrocytes, decreased concentrations of total free thiol and reduced glutathione, and decreased glutathione reductase activity were observed. These were the lowest in the old erythrocytes of very old mice. These results in aging erythrocytes from aging mice suggest that the glutathione status the erythrocyte may be an index of aging, not only of the cell but also of the organism.     

Spectroscopic studies of myoglobin at low pH: heme structure and ligation

We explore heme structure and ligation subsequent to a low-pH conformational transition in sperm whale myoglobin. Below pH 4.0, the iron-histidine bond breaks in metMb and deoxyMb. In MbCO, the majority of the iron-histidine bonds remain intact down to pH 2.6; however, the observation of a weak Fe-CO mode at 526 cm-1 indicates that a small fraction of the sample has the histidine replaced by a weak ligand, possibly water. The existence of a sterically hindered CO subpopulation in MbCO and the continued association of the four-coordinate heme with the protein in deoxyMb suggest that the heme pocket remains at least partially intact in the acid-induced conformation. The global pH-dependent conformational change described here is clearly distinguished from the local "closed" to "open" transition described previously in MbCO [Morikis et al. (1989) Biochemistry 28, 4791-4800]. Further observations of the four-coordinate heme state yield insights on the mechanism of heme photoreduction and the assignment of the 760-nm band in deoxyMb.     

Mechanistic studies of carboxypeptidase Y from Saccharomyces cerevisiae. pH and pD profiles and inactivation at low pH (pD) values.

Steady-state kinetics of carboxypeptidase Y, a proteinase from yeast, were studied by using the reaction of 4-nitrophenyl trimethylacetate as a probe. The pH profile of kcat. is sigmoidal in H2O-based buffers for the carboxypeptidase Y-catalysed hydrolysis of this ester (kcat. referring to the rate of deacylation of trimethylacetyl-carboxypeptidase Y). The corresponding pD profile in 2H2O is doubly sigmoidal, with inflexions at pD approximately 3.8 and approximately 6.8. The ionization of pKDapp. approximately 3.8 is caused by a rapid inactivation in 2H2O media by a process that is only slowly reversed on transfer to pH 7.00 phosphate buffer in H2O. The corresponding inactivation in H2O-based buffers of low pH is considerably slower (approximately 30-fold), follows a first-order rate-dependence and is very strongly pH-dependent, indicating some form of co-operative change in enzyme tertiary structure.     

Studies on avian erythrocyte metabolism. XVI. Accumulation of 2,3-bisphosphoglycerate with shifts in oxygen affinity of chicken erythrocytes

The ability of the chicken erythrocyte to accumulate 2,3-bisphosphoglycerate (2,3-P2-glycerate) and its effect upon the oxygen affinity (P50) of the cell suspensions have been determined. Erythrocytes from chick embryos, which contain 4-6 mM 2,3-P2-glycerate, and from chickens at various ages, which contain 3-4 mM inositol pentakisphosphate but no 2,3-P2-glycerate, were incubated with inosine, pyruvate, and inorganic phosphate. Red blood cells from 20-day chick embryos incubated in Krebs-Ringer, pH 7.45, containing 20 mM inosine and 20 mM pyruvate had an increase in 2,3-P2-glycerate from 4.3 to 11.9 mM after 4 h. Importantly, as 2,3-P2-glycerate concentration increased there was a corresponding increase in P50 of the cell suspension. Further, erythrocytes from 9- and 11-week, and 7-, 14-, 24-, and 28-month-old chickens when incubated similarly with inosine and pyruvate accumulated 2,3-P2-glycerate with corresponding increases in P50 of the cell suspensions. The ability of the red cell to accumulate this compound under the incubation conditions used apparently decreases with age of the bird (e.g., 11.9 mM in the 20-day embryo to 1.1 mM in the 28-month-old chicken after 4 h incubation). Despite the presence of significant amounts of inositol-P5, the accumulation of 2,3-P2-glycerate markedly decreases oxygen affinity of the cell suspensions. The delta P50/mumol increase in 2,3-P2-glycerate in the red cells of the 20-day chick embryo after 4 h incubation is 1.5 Torr; conversely, the delta P50/mumol decrease in 2,3-P2-glycerate in the red cells of the 17-day embryo after 6 h incubation in the presence of sodium bisulfite is 2.8 Torr. The demonstrated ability of the chicken erythrocyte to accumulate 2,3-P2-glycerate in response to certain substrates suggests that regulation of concentration of this compound could contribute significantly to regulation of blood oxygen affinity in birds.     

Molecular dynamics simulations of human alpha-lactalbumin: changes to the structural and dynamical properties of the protein at low pH.

Two 700-ps molecular dynamics simulations of human alpha-lactalbumin have been compared. Both were initiated from an X-ray structure determined at pH 6.5. One simulation was designed to represent native conditions and the other the protein in solution at pH 2.0 without a bound calcium ion. The low pH conditions were modelled by protonating the aspartate, glutamate, and histidine side chains and the protein C-terminus. Significant changes were observed for the C-terminal region of the sequence in the simulation at low pH. Most notably an alpha-helix, helix D, and the C-terminal 3(10) helix were substantially disrupted relative to the simulation at high pH. These perturbations to the native fold are similar to those observed in an X-ray structure of alpha-lactalbumin at pH 4.2. In addition, larger fluctuations about side chain torsion angles were observed in the low pH simulation than in that corresponding to the higher pH. These structural and dynamical changes might be representative of the early stages of the transition to the molten-globule state of the protein known to be formed under low pH conditions in solution.