Relative binding affinities of OmpR and OmpR-phosphate at the ompF and ompC regulatory sites.

In Escherichia coli, porin gene expression is regulated, in part, by the two-component regulatory system consisting of the two proteins EnvZ and OmpR. EnvZ is an integral inner membrane protein that is phosphorylated by cytoplasmic ATP on a histidine residue. EnvZ modulates the activity of OmpR by phosphorylation and dephosphorylation. Phospho-OmpR (OmpR-P) binds to the porin genes ompF and ompC to regulate their expression. The simple affinity model predicts that as the concentration of OmpR-P increases, initially high-affinity binding sites on ompF are filled. Then binding sites of lower affinity on ompF and ompC are occupied and this ordered binding accounts for the differential expression of the porin genes. We demonstrate that acetyl phosphate phosphorylates OmpR at aspartate 55, the same residue phosphorylated by the kinase EnvZ. Quantification of the level of OmpR-P by HPLC and direct measurement of the binding affinities enabled us to test the affinity model. Our results indicate that phosphorylation dramatically increases the affinity of OmpR for its binding sites (greater than tenfold). We also show that the affinities of OmpR-P for F1 and C1 binding sites are not sufficiently different to provide a strong basis for discrimination. The consequences of these observations for the simple affinity model are considered.

Hemoglobin Old Dominion/Burton-upon-Trent, beta 143 (H21) His-->Tyr, codon 143 CAC-->TAC--a variant with altered oxygen affinity that compromises measurement of glycated hemoglobin in diabetes mellitus: structure, function, and DNA sequence.

OBJECTIVE: To determine the nature and characteristics of a unique hemoglobin variant that causes a spurious increase in glycated hemoglobin (HbA1c). MATERIAL AND METHODS: Blood specimens from four unrelated persons with this hemoglobin variant were examined by conventional laboratory methods, including electrophoresis, high-performance ion-exchange chromatography, and isoelectric focusing; by amino acid sequence analysis, polymerase chain reaction-based DNA sequence analysis, and electrospray ionization mass spectrometry, to establish the molecular structure; and by studies of oxygen affinity under varied conditions, to define the functional characteristics of the hemoglobin variant. RESULTS: The unique hemoglobin variant observed in these four cases is due to the mutation CAC-->TAC, at beta-globin gene codon 143, corresponding to beta 143 (H21) His-->Tyr. This amino acid substitution affects an important 2,3-diphosphoglycerate binding site and slightly increases the oxygen affinity of the hemoglobin variant. CONCLUSION: A hitherto unrecognized hemoglobin variant, encountered in four unrelated persons of Irish or Scots-Irish ancestry, hemoglobin Old Dominion/Burton-upon-Trent, beta 143 (H21) His-->Tyr, has now been characterized at the molecular, structural, and functional levels. Although it is associated with a slight increase in oxygen affinity, it is without hematologic effect, and its only clinical significance is that it coelutes with HbA1c on ion-exchange chromatography and thereby causes a spurious increase in HbA1c and compromises the use of this analyte to monitor the treatment of diabetes mellitus.

Hb Long Island: a hemoglobin variant with a methionyl extension at the NH2 terminus and a prolyl substitution for the normal histidyl residue 2 of the beta chain.

Hb Long Island was found in a diabetic man and his nondiabetic mother as the result of a routine clinical measurement of Hb AIc. It is present in amounts approximately equal to Hb A. Its alpha chains are normal but its beta chains have two alterations compared to the normal. A methionyl residue is attached to the usual NH2-terminal valyl residue. This valyl residue is followed by prolyl residue in place of the usual histidyl residue 2. The remaining sequence of the beta chain is normal. No hemoglobin or abnormal beta chain containing only the prolyl substitution could be detected by several different electrophoretic and HPLC procedures. We postulate that Hb Long Island is the result of a mutation in which a single nucleotide change causes the substitution of a prolyl residue for the normal histidyl residue at position 2 of the beta chain. We further postulate that this abnormal prolyl residue inhibits enzymatic cleavage of the initiator methionyl residue from the abnormal beta chain during posttranslational processing. Although the oxygen affinities of the whole blood, suspended cells, and hemolysate are normal, the affinity of the isolated Hb Long Island is slightly decreased and the effects of organic phosphates are reduced compared to normal. These changes are consistent with the loss of the normal histidyl residue 2 and the extension of the NH2-terminal end of the beta-chain molecule.

Modification of human hemoglobin with methyl acyl phosphates derived from dicarboxylic acids. Systematic relationships between cross-linked structure and oxygen-binding properties.

Human hemoglobin was reacted with five dicarboxylic acid bis(methyl phosphate) reagents under different ligand conditions. The bis(methyl phosphate) reagents tested were derived from fumaric, isophthalic, terephthalic, trans-stilbene-3,3'-dicarboxylic, and trans-stilbene-4,4'-dicarboxylic acids. These acyl phosphate mixed anhydrides are anionic electrophiles and will react with N-terminal amino and lysyl epsilon-amino groups to form amides. The major and many of the minor reaction products that result have been isolated and structurally characterized by globin chain and peptide analysis. Products which are not cross-linked, intrachain linked, and interchain singly and doubly cross-linked occur in proportions which depend upon the reaction conditions and reagent. Modifications of the beta chains were limited to the amino groups of beta 1Val, beta 82Lys, and, to a minor extent, beta 144Lys. In the case of the smaller reagents, the amino groups of alpha 1Val, alpha 99Lys, and, to a minor extent, alpha 139Lys were modified. The oxygen binding affinities of most of the major modified hemoglobins have been measured and are characterized by P50 values from about 1/2 to over 5 times that of unmodified human hemoglobin. Most show strong cooperativity with Hill coefficients (n) of 2.0 or greater. Several of the products that are cross-linked between the beta 1Val of one chain and the beta 82Lys of the other chain have oxygen affinities in a physiologically useful range for oxygen transport and delivery. An inverse linear correlation has been found between the log of P50 and bridging distances for the hemoglobins cross-linked between beta 1Val of one chain and the beta 82Lys of the other chain.(ABSTRACT TRUNCATED AT 250 WORDS)