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Trypanosoma cruzi, the protozoan that causes Chagas disease, possesses a complex life cycle involving different developmental stages. Experimental conditions for two-dimensional electrophoresis (2-DE) analysis of T. cruzi trypomastigote, amastigote and epimastigote proteomes were optimized. Comparative proteome analysis of the cell-cycle stages were carried out, revealing that few proteins included in the 2-DE maps displayed significant differential expression among the three developmental forms of the parasite. In order to identify landmark proteins, spots from the trypomastigote 2-DE map were subjected to matrix-assisted laser desorption/ionization-time of flight mass spectrometry peptide mass fingerprinting, resulting in 26 identifications that corresponded to 19 different proteins. Among the identified polypeptides, there were heat shock proteins (HSP; chaperones, HSP 60, HSP 70 and HSP 90), elongation factors, glycolytic pathway enzymes (enolase, pyruvate kinase and 2,3 bisphosphoglycerate mutase) and structural proteins (KMP 11, tubulin and paraflagellar rod components). The relative expression of the identified proteins in the 2-DE maps of the T. cruzi developmental stages is also presented.

A 28-year-old asymptomatic male of Iranian Jewish (Meshadi) heritage was found on routine exam to have an erythrocytosis (RBC = 6.22 × 1012/l, Hgb = 19.2 g/dl, Hct = 58.9%). Splenomegaly was absent on physical exam. There was no family history of erythrocytosis. His oxygen dissociation curve was left-shifted with a p50 of 19 mmHg (normal = 25–32 mmHg). Hemoglobin electrophoresis showed no abnormalities. DNA sequencing of the hemoglobin β globin gene and both α globin genes did not reveal a mutation. A 2,3-bisphosphoglycerate (BPG) level was markedly decreased at 0.3 μmol/g Hb (normal = 11.4–19.4 μmol/g Hb). The patient's bisphosphoglycerate mutase (BPGM) enzyme activity was also markedly decreased at 0.16 IU/g Hb (normal = 4.13–5.43 IU/g Hb). A red cell enzyme panel revealed a markedly decreased G-6-PD level (0.3 U/g Hb, normal = 8.6–18.6 U/g Hb). His parents and a brother were also available for evaluation. Both parents showed normal 2,3-BPG levels but BPGM activity approximately 50% of normal. Paradoxically, the brother showed normal BPGM activity but a slightly decreased 2,3-BPG level. All family members had markedly decreased G-6-PD activity. DNA sequencing of the BPGM gene showed the propositus to be homozygous for 185 G→A, Arg 62 Gln in exon 2. Thus, the erythrocytosis in this patient is secondary to low 2,3-BPG levels, due to a deficiency in BPG mutase. This appears due to consanguinity within this family. Am. J. Hematol. 75:205–208, 2004. © 2004 Wiley-Liss, Inc.

A system has been developed to allow the convenient production, expression and purification of site-directed mutants of the enzyme phosphoglycerate mutase from Saccharomyces cerevisiae. This enzyme is well characterised; both the amino acid sequence and crystal structure have been determined and a reaction mechanism has been proposed. However, the molecular basis for catalysis remains poorly understood, with only circumstantial evidence for the roles of most of the active site residues other than His8, which is phosphorylated during the reaction cycle. A vector/host expression system has been designed which allows recombinant forms of phosphoglycerate mutase to be efficiently expressed in yeast with no background wild-type activity. A simple one-column purification protocol typically yields 30 mg pure enzyme/1 l of culture. The active-site residue, His181, which is thought to be involved in proton transfer during the catalytic cycle, has been mutated to an alanine. The resultant mutant has been purified and characterised. Kinetic analysis shows a large decrease (1.6 x 10(4)) in the catalytic efficiency, and an 11-fold increase in the Km for the cofactor 2,3-bisphosphoglycerate. These observations are consistent with an integral role for His181 in the reaction mechanism of phosphoglycerate mutase, probably as a general acid or base.

We examined developmental changes in the relative activities of three different isozyme systems: aldolase, enolase and phosphoglycerate mutase, in tissues of fetal mice with trisomy 16 and of fetal euploid littermates. We wanted to determine whether morphological abnormalities such as reduced weight and size, which are generally observed in murine trisomy, are reflected at the molecular level. Following electrophoretic separation and subsequent measurement of relative activities of enolase isozymes in brain and phosphoglycerate mutase isozymes in heart, we found no significant differences between trisomy 16 fetuses and their euploid littermates. Synthesis of liver-specific aldolase was, however, delayed in trisomy 16 fetuses.

1 Large quantities of 2,3-bisphosphoglycerate accumulate in the red blood cells of the chick embryo during the week prior to hatching; the 2,3-bisphosphoglycerate then abruptly decreases to very small amounts within a few days after hatching. 2 The enzyme 2,3-bisphosphoglycerate phosphatase was purified from the red blood cells of the day old chick. The elution profiles for bisphosphoglycerate phosphatase and 2,3-bisphosphoglycerate mutase were identical upon gradient elution from columns of hydroxylapatite and diethylaminoethylcellulose and by gel filtration on Sephadex G-100. At each stage of purification the ratio of phosphatase to mutase activity was the same. It was concluded that, just as in the human erythrocyte, both bisphosphoglycerate phosphatase and 2,3-bisphosphoglycerate mutase activities in chick erythrocytes reside on one protein. 3 Bisphosphoglycerate phosphatase has a pH of optimal activity of 7.0. Its activity is stimulated by 2-phosphoglycolate, phosphoenolpyruvate, bisulfite and dithionite and is inhibited by 2-phosphoglycerate, 3-phosphoglycerate, inorganic pyrophosphate and phytic acid. These properties are very similar to those described for bisphosphoglycerate phosphatase purified from human erythrocytes. 4 The purified bisphosphoglycerate phosphatase-mutase contained 3-phosphoglycerate mutase activity. Although the 2,3-bisphosphoglycerate mutase and 3-phosphoglycerate mutase activities were nearly equal in the purified enzyme, this protein accounts for a maximum of only 6% of the 3-phosphoglycerate mutase activity in these cells. 5 The total activity of the bisphosphoglycerate phosphatase in the erythrocytes was measured at intervals during development of the embryo, in the young chick, and in the mature chicken. Its activity increases during the time of 2,3-bisphosphoglycerate accumulation. The enzyme activity decreases gradually from its maximum in the two-day-old chick (1.67 μmol h−1 g hemoglobin−1) and by 27 days it has fallen nearly to the levels seen in the adult chicken (0.11 μmol h−1 g hemoglobin−1). 6 The factors which might be involved in inducing the 2,3-bisphosphoglycerate accumulation and degradation in the late chick embryo and young chick are discussed. It is concluded that this system is very similar to that found in the erythrocytes of man.

The intracellular distribution of ATP, 2,3-bisphosphoglycerate(P2-glycerate) and Mg2+ was calculated for the oxygenated and deoxygenated human erythrocyte for the normal range and that of pathophysiological variations based on the association constants of the relevant complexes. The data indicate that about 20% of ATP is bound both in the oxygenated and deoxygenated cells, while 39 and 73% of P2-glycerate is bound under these conditions. An increase of the free Mg2+ concentration from 0.7 to 1.1 mM is produced by complete deoxygenation of haemoglobin. The calculations would indicate that during deoxygenation of haemoglobin the hexokinase reacts to the increased concentration of the activator Mg2+ and the decline of the inhibitor P2-glycerate with an elevation of its activity which corresponds to experimental data on intact erythrocytes. The P2-glycerate formation rate in deoxygenated cells is stimulated about 2.5 times in comparison to oxygenated cells as estimated from the free concentration of P2-glycerate and the kinetic constants of bisphosphoglycerate mutase. An assessment of the possible influence of other anions including bicarbonate shows that the distribution of the species of ATP, P2-glycerate and Mg2+ are changed by less than 20%. Together with the data presented in the accompanying paper, the results given here indicate that the constants and estimates are approximately valid for intracellular conditions.