We have studied the effect of the P6-inositol (IHP)-induced change from the quaternary oxy (R) to the deoxy (T) structure in derivatives of human, trout IV, and carp methemoglobins. Addition of IHP to human fluoroand aquomethemoglobin leads to the appearance of the slowly exchanging proton resonance at about -10 ppm from HDO diagnostic of the T structure. This experiment, and the crystallization of aquomethemoglobin + IHP by G. Fermi & M. F. Perutz ((1977) J. Mol. Biol. 114, 421) confirmed that the spectral change in the UV which IHP induces in these compounds can be used as a reliable indicator of the R-*-T transition. Judged by this spectral change, IHP converts all derivatives of carp hemoglobin from the R to the T structure. The pH at which the midpoint of the IHP-induced transition occurs increases with rising spin, being lowest in cyano, intermediate in azido, and highest in thiocyanate and aquomethemoglobin of carp. Conversely the replacement of water by fluoride or thiocyanate as the sixth ligand is unaffected by IHP because all three derivatives are predominantly high spin, but the affinity of azide for carp aquomethemoglobin is reduced 2.7-fold and that of cyanide 3.3-fold by IHP, corresponding to changes in the free energy of binding of 600 and 700 cal/mol heme. Conversion to the T structure of all carp methemoglobin derivatives except the cyanide one is accompanied by large changes in the visible absorption spectra, the most spectacular being that of the nitrite derivative whose color is changed from red to brown. IHP converts all human methemoglobin derivtleme-heme interaction arises from an equilibrium between states which differ in the tertiary structure of the a and 0 subunits and in their quaternary structure in the tetramer. This equilibrium is linked to the stereochemistry at the heme. In deoxyhemoglobin where the tense (T) quaternary structure is dominant, the heme irons are five coordinated and high spin.

Glucose-6-phosphate isomerase (GPI) deficiency, an autosomal recessive genetic disorder with the typical manifestation of nonspherocytic haemolytic anaemia, can be associated in some cases with neurological impairment. GPI has been found to be identical to neuroleukin (NLK), which has neurotrophic and lymphokine properties. To focus on the possible effects of GPI mutations on the central nervous system through an effect on neuroleukin activity, we analysed DNA isolated from two patients with severe GPI deficiency, one of them with additional neurological deficits, and their families. The neurologically affected patient (GPI Homburg) is compound heterozygous for a 59 A-->C (H20P) and a 1016 T-->C (L339P) exchange. Owing to the insertion of proline, the H20P and L339P mutations are likely to affect the folding and activity of the enzyme. In the second family studied, the two affected siblings showed no neurological symptoms. The identified mutations are 1166 A-->G (H389R) and 1549 C-->G (L517V), which are located at the subunit interface. We propose that mutations that lead to incorrect folding destroy both catalytic (GPI) and neurotrophic (NLK) activities, thereby leading to the observed clinical symptoms (GPI Homburg). Those alterations at the active site, however, that allow correct folding retain the neurotrophic properties of the molecule (GPI Calden).

The separation of red blood cells into reticulocytes and young and old erythrocytes enables investigations of fractions with different contents of reticulocytes. Activities of hexokinase, glucose phosphate isomerase, phosphofructokinase, pyruvate kinase and glucose-6-phosphate dehydrogenase showed a linear relationship to reticulocyte counts. The dependence of these enzyme activities on the age of the red blood cells exhibited a strong decline from the reticulocyte to the young erythrocyte stage followed by only little further loss of activity, thus leading to a biphasic decay of enzyme activities. By linear regression analysis enzyme activities in erythrocytes (AE) and reticulocytes (AR) could be evaluated. The activity of a given enzyme in the reticulocyte exceeded that of the erythrocyte; the quotient AR/AE represents the decline of enzyme activity from the reticulocyte to the erythrocyte stage. This value AR/AE is 16.7 for pyruvate kinase and 9.4 for hexokinase and thus considerably higher than that for the other enzymes investigated (glucose phosphate isomerase: 2.9, phosphofructokinase: 4.3, glucose-6-phosphate dehydrogenase: 4.5). In patients suffering from erythrocyte enzymopathies, the AR/AE for pyruvate kinase was 16.2 and thus almost identical to the normal enzyme. Calibration curves where the enzyme activity is plotted versus the fraction of reticulocytes enable the determination of normal activity of a given erythrocyte enzyme depending on the content of reticulocytes in red blood cell suspensions. Thus an unambiguous diagnosis of enzyme defects irrespective of reticulocyte counts becomes possible.

The molecular alterations responsible for the characteristic enzyme abnormalities in pyruvate kinase (PK) deficiency were investigated in two unrelated children homozygous for PK deficiency. Both variant enzymes were characterized according to the recommendations of the International Committee for Standardization in Haematology. Genomic DNA was specifically amplified by the polymerase chain reaction. Normal and mutant alleles of the L-type PK gene were analyzed by nucleotide sequencing. Heterozygosity of the parents was confirmed by allele- specific oligonucleotide hybridization. In PK Linz a C to T base exchange at position 394 of the L-type PK gene was found. As a result, the 132nd amino acid of the mutant enzyme, arginine (CGC), is replaced by cysteine (TGC). The affected amino acid residue is located within the deduced active site of the protein and the enzyme variant shows strongly altered allosteric properties. PK Beirut shows a C for T substitution at position 1058, changing the 353 amino acid from threonine (ACG) to methionine (ATG). In contrast to PK Linz, this amino acid lies outside the deduced substrate binding site and kinetic parameters of PK Beirut are close to normal. Both enzyme variants show a markedly reduced specific activity and thermolability.

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