PubMed · 6757143
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H L Zauder. 1982. Massive transfusion.. https://pubmed.ncbi.nlm.nih.gov/6757143/
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3-Phosphoglycerate kinase is a typical two-domain "hinge-bending" enzyme, which is known to be regulated by multivalent anions. Here a relationship between this regulation and the hinge-bending domain closure is proposed on the basis of enzyme kinetic analysis and molecular modeling. Activation of the pig muscle enzyme at low concentrations and inhibition at high concentrations of various anionic analogues of the substrate 3-phosphoglycerate or of the nonsubstrate metal-free ATP are described by a two-site model assuming separate sites for activation and inhibition, respectively. Kinetic experiments with various pairs of analogues suggest the presence of a common site for activation by all effectors, separate from the catalytic site for 3-phosphoglycerate; and a common site for inhibition, except for metal-free ATP, identical with the catalytic site of 3-phosphoglycerate. An additional inhibiting site for all of the anions investigated, including metal-free ATP, is also proposed. A similar two-site model can describe activation of the enzyme by a large excess of each substrate; here the ligand binds to the catalytic site as a substrate and to the regulatory site as an activator. Activation is exerted not only by the physiological substrate, 3-phophoglycerate, but also by a synthetic weak substrate. The activity in the reaction with 3-phosphoglycerate and MgATP is greatly enhanced by the simultaneous presence of the weak substrate. This finding clearly proves the existence of a regulatory site, separate from the catalytic site. This regulatory site, however, may only exist in the catalytically competent closed conformation of the enzyme, as indicated by molecular modeling. Docking of the regulator anions into the known X-ray structures of the enzyme revealed the appearance of an anion binding site between the two domains, including the invariant residues of Lys-215 (C-domain) and of Arg-65 among other residues of the basic cluster (N-domain), as a consequence of the large-scale substrate-induced conformational change that leads to domain closure.
Recent work has enabled us to quantitate the four variables (2,3-DPG concentration, pHi, non-S hemoglobin composition, and O2 saturation) that modulate the equilibrium solubility (csat) of Hb S inside sickle erythrocytes (SS RBCs). Using measured values of mean corpuscular hemoglobin concentration (MCHC), 2,3-DPG concentration, and %Hb (F+A2), along with estimates of pHi and the Deltacsat due to partial oxygenation of SS RBCs in the microcirculation, we calculated the mean polymer fraction (fp) in erythrocytes from 46 SS homozygotes. Values of fp derived from the conservation of mass equation ranged from 0.30 to 0.59. MCHC and %Hb F were major determinants of the magnitude of fp; 2,3-DPG concentration and pHi also contributed, but to a lesser extent. A clinical severity score (CSS) was assigned to each patient based on mean hospitalization rate. There was a weak, but statistically significant, negative correlation between fp and steady state hematocrit (P = .017), but none between fp and whole blood hemoglobin concentration (P = .218). Although there was no correlation between fp and mean number of hospitalization days per year, patients with the greatest number of admissions and hospitalization days were found only among those who had an fp > 0.45. All five patients who died during the follow-up period (median, 7 years; range, 3 to 10 years) had fp values >/=0.48. However, patients with few admissions, low hospitalization days, and long survivals occurred at all fp levels. These results suggest that the clinical course of homozygous SS disease cannot be predicted by mean fp calculations, which assume a homogeneous distribution of the five variables that modulate intraerythrocytic polymerization. A heterogeneous distribution is more likely; so the amount of polymerized Hb S could vary considerably among cell populations. Factors such as membrane abnormalities and endothelial cell interactions may also contribute to clinical severity.
OBJECTIVE: To examine metabolic responses of chronically starved horses to refeeding with 3 isoenergetic diets. DESIGN: Uncontrolled clinical trial. ANIMALS: 22 mature mixed-breed horses that were emaciated but otherwise clinically normal. PROCEDURE: Horses were fed 1 of 3 diets: alfalfa hay, oat hay, or a combination diet of half oat hay and half commercially prepared ration. Digestible energy of diets was gradually increased throughout the refeeding period. One pre- and 4 postprandial blood samples were obtained daily, and analyses included RBC count, Hct, and determination of hemoglobin, glucose, insulin, free fatty acid, total bilirubin, 2,3-diphosphoglyceric acid, phosphorus, magnesium, calcium, sodium, and potassium concentrations. Body weight, fecal output, and feed and water consumption were measured and recorded daily. Repeated-measures ANOVA was used to examine dietary and temporal (day) effects of the 3 dietary regimens during 10-day trials. RESULTS: 19 Horses survived. Three horses (2 fed alfalfa diet, 1 fed combination diet) died of metabolic or gastrointestinal problems. Increasing temporal effects in serum concentrations of glucose, insulin, magnesium, calcium, and sodium; decreasing temporal effects in serum concentrations of free fatty acid, 2,3-diphosphoglyceric acid, and phosphorus; and dietary effects in serum concentrations of glucose, insulin, magnesium, and potassium were detected in the 19 surviving horses. Serum phosphorus and free fatty acid concentrations decreased dramatically during the first 5 days of refeeding with all 3 diets. Serum magnesium concentrations increased in horses fed the alfalfa hay diet, whereas improvement was not evident in horses fed oat hay or combination diets. Horses receiving the alfalfa and oat hay diets had lower postprandial glucose and insulin concentrations than horses receiving the combination diet. Horses fed oat hay alone ate 92% of feed offered, compared with 98% feed consumption for horses fed alfalfa hay or combination diets. CLINICAL IMPLICATIONS: Clinically normal emaciated horses can be successfully rehabilitated by gradual refeeding with a high forage diet.