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Dominant megacolon (Dom) is one of four mutations in the mouse that can produce a phenotype similar to Hirschsprung disease in human. The Dom gene product is not known, and no candidate region has been defined for a possible human homolog. In this publication we report mapping the Dom locus with high definition, using several intra-and interspecific crosses and a set of 16 Chr 15-specific microsatellites flanking this locus.

Two consecutive cases of severe neonatal anaemia due to severe deficiency in pyruvate kinase were observed in the same sibhood. The first child died one hour after birth and the second required major transfusion support. Pyruvate kinase deficiency is a rare cause of congenital anaemia with recessive autosomic inheritance. Clinically, this deficiency has a very variable expression, and neonatal forms are not always very severe. Several variant molecules in pyruvate kinase deficiency have been described. Recent progress in our understanding of the gene would suggest the possibility of new diagnostic and prognostic approaches.

Human bisphosphoglycerate mutase (GriP2 mutase) is a trifunctional enzyme which synthesizes and degrades GriP2 in red cells. Among the amino acid residues involved in its active site there are two conserved histidine residues, His10 which is phosphorylated during the catalytic process and His187 for which only speculative data have been made about the potential role during the reactions. Another amino acid residue, Arg89, had not been described as part of this active site but we have recently shown that a natural mutant Arg89-->Cys was highly thermolabile and showed severe perturbations of its enzymatic properties. To understand better the exact role of these residues, replacements of His10 by Gly (H10G) or Asp (H10D), His187 by Asn (H187N), Tyr (H187Y) or Asp (H187D) and Arg89 by Cys (R89C), Ser (R89S), Gly (R89G) or Lys (R89K) were performed by site-directed mutagenesis. The results obtained in this report show that replacement of the His10 residue completely abolished the enzymatic activities. Concerning the His187 residue, our results afford arguments that it plays an essential role in the three catalytic activities. Indeed all these activities are abolished in the two H187Y and H187D variants, whereas they are detectable though strongly diminished, for the H187N variant. In addition mutations at His187 could be distinguishable from those at His10 since the former resulted in a thermolabile enzyme, whereas no significant change in heat stability was observed for the latter. It is noteworthy that the H187N variant is protected against thermal instability by glycerate 2,3-bisphosphate (GriP2). Concerning the Arg89 mutants, R89C, R89S and R89G, the three variants showed characteristics identical to those found in the natural R89C mutant, i.e. loss of 99% of synthase activity, consistent decrease of mutase and 2-phosphoglycolate-stimulated phosphatase activities whereas the unstimulated phosphatase activity was normal. Moreover these mutants were unstable at 55 degrees C but GriP2 was able to protect them against thermal instability. In contrast, the R89K mutant was stable at 55 degrees C. Its synthase and unstimulated phosphatase activities were normal but its mutase and 2-phosphoglycolate-stimulated phosphatase activities were decreased. In addition, Km values for monophosphoglycerates were increased (3.2-fold) in the synthase but normal in mutase activities, whereas Km values for GriP2 were normal in mutase and phosphatase activities.(ABSTRACT TRUNCATED AT 400 WORDS)

Bisphosphoglycerate mutase (EC 5.4.2.4.) is an erythrocyte-specific enzyme whose main function is to synthesize 2,3-diphosphoglycerate (glycerate-2,3-P2) an effector of the delivery of O2 in the tissues. In addition to its main synthase activity the enzyme displays phosphatase and mutase activities both involving 2,3-diphosphoglycerate in their reaction. Using a prokaryotic expression system, we have developed a recombinant system producing human bisphosphoglycerate mutase in E coli. The expressed enzyme has been extracted and purified to homogeneity by 2 chromatographic steps. Purity of this enzyme was checked with sodium dodecyl sulfate polyacrylamide gel and Cellogel electrophoresis and structural studies. The bisphosphoglycerate mutase expressed in E coli was found to be very similar to that of human erythrocytes and showed identical trifunctionality, thermostability, immunological and kinetics' properties. However, the absence of a blocking agent on the N-terminus results in a slight difference of the electrophoretic mobility of the enzyme expressed in E coli compared to that of the erythrocyte.

2,3-bisphosphoglycerate mutase is a multifunctional enzyme which catalyses in red blood cells the synthesis and the degradation of 2,3-bisphosphoglycerate, the allosteric effector of hemoglobin. In order to study the structure-function relationships in BPGM, an expression vector was constructed which yielded an active protein, but with a modified electrophoretic mobility, due to a non-blocked N-terminal residue. Using site directed mutagenesis, mutants were produced with shortened chains. Results indicated the importance of residues 252-256 for the function. A natural deficient mutant with the substitution 89 Arg----Cys was described. Artificial mutant with the same substitution reproduced the same defect, as well as mutants Arg----Gly and Arg----Ser, indicating the key role of Arg 89 in the enzymatic mechanism.

1. Treatment of purified rabbit phosphoglyceromutase (M type) with N-ethylmaleimide or with iodoacetamide produces the concurrent loss of phosphoglyceromutase activity with its collateral glycerate-2,3-P2 phosphatase activity. 2. Differences are observed in the protective effect of glycerate-2,3-P2 and of glycolate-2-P against N-ethylmaleimide and iodoacetamide treatments. 3. Specific chicken antibodies obtained by injection of the purified rabbit M type phosphoglyceromutase do not cross-react with the B type but neutralize both rabbit and human M type phosphoglyceromutase. 4. Purified rabbit M type phosphoglyceromutase can hybridize in vitro with the purified human B type or with purified human glycerate-2,3-P2 synthase. 5. Its ability to hybridize with glycerate-2,3-P2 synthase is unchanged after iodoacetamide treatment.

New cases of diphosphoglyceromutase (DPGM) have been detected, associated with erythrocytosis in two unrelated families. The deficiency appears to be inherited as an autosomal dominant trait. Diphosphoglycerate phosphatase activity paralleled DPGM activity in all the subjects. Three of the latter displayed complete DPGM deficiency with about 0.4% of the normal 2,3-diphosphoglycerate (2,3 DPG) level. The other four showed partial deficiency (about 50% normal mean) with a similar decrease in 2,3-DPG level. The P50 values are in agreement with the red cell 2,3-DPG concentrations. Il all the deficient subjects the ATP level was elevated and the pattern of glycolytic intermediates was disturbed, with an increase in fructose 1,6-diphosphate, triose-phosphates, 3-phosphoglycerate, glucose 1,6-diphosphate, and reduced or normal levels of glucose-6-phosphate and fructose-6-phosphate.

Phosphoglyceromutase (2,3-bisphospho-D-glycerate:2- phospho-D-glycerate phosphotransferase, EC 2.7.5.3) from the red cell is compared with the muscle and the liver forms of the enzyme obtained from rabbits. Partially purified samples were used for this study. The electrophoretic mobility of red cell phosphoglyceromutase is completely different from that of muscle and similar to that of liver. The muscle isozyme can hybridize with liver or red blood cell phosphoglyceromutase. N-Ethylmaleimide, an SH group reagent, completely inhibits muscle phosphoglyceromutase activity and slightly inhibits the activity of the enzyme from liver and from red cells. Both liver and red cell isozymes are completely inactivated by heating at 60 degrees C for 15 min, whereas a 25% decrease in inactivity is noted for the muscle enzyme. Finally, chicken antibody directed against human red cell phosphoglyceromutase reacts with the enzyme isolated from rabbit red cells and liver, but not with that obtained from muscle.

An inherited and complete deficiency of diphosphoglycerate mutase was discovered in the erythrocytes of a 42-yr-old man of French origin whose blood hemoglobin concentration was 19.0 g/dl. Upon physical examination he was normal with the exception of a ruddy cyanosis. The morphology of his erythrocytes was also normal and there was no evidence of hemolysis. The erythrocyte 2,3-diphosphoglycerate level was below 3% of normal values and, as a consequence, the affinity of the cells for oxygen was increased. Diphosphoglycerate mutase activity was undetectable in erythrocytes as was that of diphosphoglycerate phosphatase. The activities of all the other erythrocyte enzymes that were tested were normal except for nomophosphoglycerate mutase which was diminished to 50% of the normal value. The levels of reduced glutathione, ATP, fructose 1,6-diphosphate, and of triose phosphates were elevated, whereas those of glucose 6-phosphate and fructose 6-phosphate were decreased. This report sheds new light on the role of diphosphoglycerate mutase in the metabolism of erythrocytes.

A new method for the purification of M-type phosphoglyceromutase from rabbit muscle involving affinity chromatography on dye ligand media in the presence of 2,3-diphosphoglycerate is described. The method is rapid and technically simple. The purity of the enzyme was verified by electrophoresis in polyacrylamide gel in the presence of sodium dodecyl sulphate and by Cellogel electrophoresis. Immunological techniques showed that the anti-M antibodies are specific and do not cross-react with the B isozyme.

Seven new homozygous cases of hereditary triosephosphate isomerase (TPI) deficiency have been detected in five unrelated families. Two of the families originate in France, the others from Algeria, Yugoslavia, and Morocco. Only the parents coming from Algeria and Morocco were first cousins. In the other parents no evidence of consanguinity was found. All seven patients exhibited the same symptoms, i.e. hemolytic anemia appearing very early after birth associated with progressive neuromuscular symptoms. Expression of the deficiency is heterogeneous; this had previously been pointed out in the previously reported cases of TPI deficiency. Red cell TPI activity was 3 to 4% of the normal mean in the patients and 50 to 60% in the parents. The latter did not exhibit any clinical symptoms. The levels of red cell glycolytic intermediates and the characteristics of the mutated TPI could be studied in four of the patients only. Substantial increases of red cell dihydroxyacetone phosphate and of fructose 1,6-diphosphate, normal Km of TPI for glyceraldehyde phosphate, and thermoinstability of the enzyme were found. In addition the electrophoretic pattern showed no significant modification of the mobility of the TPI bands, but abnormal decreased staining of the two more anodal bands.

Screening for abnormal hemoglobins and G6PD deficiency was conducted in adult and new born Maroccans from Casablanca. The results presented deal with HbS and HbC traits, alpha, gamma, and delta mutations, the presence of detectable amount of Hb Bart's and the G6PD deficiency frequency.

Prenatal diagnosis has been performed on umbilical cord blood of an 18 weeks fetus of heterozygous triosephosphate isomerase (TPI) deficient parents. After excluding maternal blood contamination, TPI activity was measured and found to be 60 per cent of the normal mean whereas the value of glucose-6-phosphate dehydrogenase activity was in the normal range of fetal blood. In addition, the analysis of the characteristics of fetal TPI, i.e. Km measurements for glyceraldehyde-3-phosphate, heat stability tests and electrophoretic studies, did not show any evidence of a special form of TPI in fetal blood. These results were consistent with the heterozygous state and were confirmed at birth.