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PubMed1992

Alloalbuminemia in Sweden: structural study and phenotypic distribution of nine albumin variants.

Plasma samples exhibiting alloalbuminemia on electrophoresis at pH 8.6 were requested from clinical laboratories throughout Sweden. Nine variants, each representing a different single point mutation, were found in 100 apparently unrelated Swedes. The overall prevalence of alloalbuminemia was estimated at 1:1700. Mutations were identified by protein-structural analysis followed by allele-specific DNA hybridization to verify the most common types. Slightly retarded (+1) mobility was seen in 80 cases. Of these, 71 had the Arg(-2)----Cys proalbumin variant previously called Malmö I proalbumin. Thirteen examples of the second most frequent type, the substitution Lys313----Asn and a mobility change of -1 charge unit, were found, as well as six cases of Glu570----Lys (albumin B) and a single case of Arg-1----Gln (proalbumin Christchurch). Five previously unreported types of alloalbuminemia were identified: four instances of Glu376----Gln, which is the second known mutation at this site; two examples of Asp550----Ala, the second mutation reported at this site; and one example each of Asp63----Asn, Gln268----Arg, and Asn318----Lys. Other mutations were identified among eight subjects of foreign descent. The high frequency and relatively uniform geographic distribution of the Arg-2----Cys mutation suggest that it may have occurred in a founder individual many generation ago in Sweden.

Amino Acid Sequence↗
PubMed1982

Structures of branched blood group A-active glycosphingolipids in human erythrocytes and polymorphism of A- and H-glycolipids in A1 and A2 subgroups.

The structures of two branched variants of blood group A-active glycolipid, Ac and Ad (Hakomori, S., Stellner, K., and Watanabe, K. (1972) Biochem. Biophys. Res. Commun. 49, 1061-1068), isolated from human type A-erythrocytes, were elucidated. The intact glycolipids and fragments produced by treatment with endo-beta-galactosidase from Escherichia freundii (Fukuda, M. N., and Matsumura, G. (1976) J. Biol. Chem. 251, 6218-6225) were analyzed by methylation. In addition, a specific limited enzyme digestion of Ad-glycolipid was made by incorporating the glycolipid into liposomes before endo-beta-galactosidase digestion. The method preferentially hydrolyzed one of the side chains carrying an A-determinant leaving a core which was recovered from the liposomes. The Ac- and Ad- glycolipids have two branched carbohydrate chains with 12 and 14 sugar residues, respectively, and both have two A-determinants as shown below. (formula see text). The structures of A- and H-active variants isolated from subgroups A1 and A2 have been compared. The results indicate that A variants from A1 or A2 erythrocytes have identical structures, and that H-variants isolated from A2 erythrocytes are identical to those isolated from O erythrocytes. Thus, there is no qualitative structural difference in A-active glycolipids between A1 and A2 erythrocytes.

ABO Blood-Group System↗
PubMed2001

Structural and functional aspects of filamins.

Filamins are a family of high molecular mass cytoskeletal proteins that organize filamentous actin in networks and stress fibers. Over the past few years it has become clear that filamins anchor various transmembrane proteins to the actin cytoskeleton and provide a scaffold for a wide range of cytoplasmic signaling proteins. The recent cloning of three human filamins and studies on filamin orthologues from chicken and Drosophila revealed unexpected complexity of the filamin family, the biological implications of which have just started to be addressed. Expression of dysfunctional filamin-A leads to the genetic disorder of ventricular heterotopia and gives reason to expect that abnormalities in the other isogenes may also be connected with human disease. In this review aspects of filamin structure, its splice variants, binding partners and biological function will be discussed.

Actins↗
PubMed1999

Refined crystal structures of native human angiogenin and two active site variants: implications for the unique functional properties of an enzyme involved in neovascularisation during tumour growth.

Human angiogenin (Ang), an unusual member of the pancreatic RNase superfamily, is a potent inducer of angiogenesis in vivo. Its ribonucleolytic activity is weak (10(4) to 10(6)-fold lower than that of bovine RNase A), but nonetheless seems to be essential for biological function. Ang has been implicated in the establishment of a wide range of human tumours and has therefore emerged as an important target for the design of new anti-cancer compounds. We report high-resolution crystal structures for native Ang in two different forms (Pyr1 at 1.8 A and Met-1 at 2.0 A resolution) and for two active-site variants, K40Q and H13A, at 2.0 A resolution. The native structures, together with earlier mutational and biochemical data, provide a basis for understanding the unique functional properties of this molecule. The major structural features that underlie the weakness of angiogenin's RNase activity include: (i) the obstruction of the pyrimidine-binding site by Gln117; (ii) the existence of a hydrogen bond between Thr44 and Thr80 that further suppresses the effectiveness of the pyrimidine site; (iii) the absence of a counterpart for the His119-Asp121 hydrogen bond that potentiates catalysis in RNase A (the corresponding aspartate in Ang, Asp116, has been recruited to stabilise the blockage of the pyrimidine site); and (iv) the absence of any precise structural counterparts for two important purine-binding residues of RNase A. Analysis of the native structures has revealed details of the cell-binding region and nuclear localisation signal of Ang that are critical for angiogenicity. The cell-binding site differs dramatically from the corresponding regions of RNase A and two other homologues, eosinophil-derived neurotoxin and onconase, all of which lack angiogenic activity. Determination of the structures of the catalytically inactive variants K40Q and H13A has now allowed a rigorous assessment of the relationship between the ribonucleolytic and biological activities of Ang. No significant change outside the enzymatic active site was observed in K40Q, establishing that the loss of angiogenic activity for this derivative is directly attributable to disruption of the catalytic apparatus. The H13A structure shows some changes beyond the ribonucleolytic site, but sites involved in cell-binding and nuclear translocation are essentially unaffected by the amino acid replacement.

Amino Acid Sequence↗
PubMed2002

Bi-directional allelic recognition of the human minor histocompatibility antigen HB-1 by cytotoxic T lymphocytes.

Human minor histocompatibility antigens (mHag) are target antigens of the graft-versus-leukemia response observed after allogeneic HLA-identical stem cell transplantation. We previously defined the molecular nature of the B cell lineage-specific mHag HB-1. The CTL epitope was identified as the decamer peptide EEKRGSLHVW presented in the context of HLA-B44. The HB-1 antigen is encoded by a locus of yet unknown function on chromosome 5q32. A single nucleotide polymorphism within this locus results in an amino acid change from histidine (H) to tyrosine (Y) at position P8 within the CTL epitope. Based on genomic information, we have developed a PCR-RFLP assay to perform HB-1 typing at the DNA level. We determined that the allelic frequency for the H and Y variant is 0.79 and 0.21, respectively. From these data, we calculated that the expected recipient disparity between HLA-B44-matched sibling pairs for HB-1H is 2.8%, whereas recipient disparity for HB-1Y is expected to be 12.4%. Therefore, we addressed whether the HB-1Y peptide is reciprocally immunogenic. We revealed that both peptide variants bind equally efficient to HLA-B44 molecules and that the H/Y substitution has no influence on formation of epitope precursor peptides by 20 S proteasome-mediated degradation. More directly, CTL recognizing the naturally presented HB-1Y peptide could be generated from a HB-1H homozygous donor using peptide-pulsed dendritic cells. Using a set of synthetic structurally related peptide variants, we found that the H/Y substitution has a major impact on TCR recognition by CTL specific for either of the HB-1 allelic homologues. HB-1 is the first human mHag described that induces bi-directional allogeneic CTL responses that may contribute to a specific graft-versus-leukemia response following allogeneic stem cell transplantation.

Alleles↗
PubMed1998

Crystal structure of chemically synthesized [N33A] stromal cell-derived factor 1alpha, a potent ligand for the HIV-1 "fusin" coreceptor.

Stromal cell-derived factor-1alpha (SDF-1alpha ) is a member of the chemokine superfamily and functions as a growth factor and chemoattractant through activation of CXCR4/LESTR/Fusin, a G protein-coupled receptor. This receptor also functions as a coreceptor for T-tropic syncytium-inducing strains of HIV-1. SDF-1alpha antagonizes infectivity of these strains by competing with gp120 for binding to the receptor. The crystal structure of a variant SDF-1alpha ([N33A]SDF-1alpha ) prepared by total chemical synthesis has been refined to 2.2-A resolution. Although SDF-1alpha adopts a typical chemokine beta-beta-beta-alpha topology, the packing of the alpha-helix against the beta-sheet is strikingly different. Comparison of SDF-1alpha with other chemokine structures confirms the hypothesis that SDF-1alpha may be either an ancestral protein from which all other chemokines evolved or the chemokine that is the least divergent from a primordial chemokine. The structure of SDF-1alpha reveals a positively charged surface ideal for binding to the negatively charged extracellular loops of the CXCR4 HIV-1 coreceptor. This ionic complementarity is likely to promote the interaction of the mobile N-terminal segment of SDF-1alpha with interhelical sites of the receptor, resulting in a biological response.

Binding Sites↗
PubMed2004

Structural and functional diversity of lectin repertoires in invertebrates, protochordates and ectothermic vertebrates.

During the past few years, substantial progress has been accomplished in the elucidation of the structural diversity of the lectin repertoires of invertebrates, protochordates and ectothermic vertebrates, providing particularly valuable information on those groups that constitute the invertebrate/vertebrate 'boundary'. Although representatives of lectin families typical of mammals, such as C-type lectins, galectins and pentraxins, have been described in these taxa, the detailed study of selected model species has yielded either novel variants of the structures described for the mammalian lectin representatives or novel lectin families with unique sequence motifs, multidomain arrangements and a new structural fold. Along with the high structural diversity of the lectin repertoires in these taxa, a wide spectrum of biological roles is starting to emerge, underscoring the value of invertebrate and lower vertebrate models for gaining insight into structural, functional and evolutionary aspects of lectins.

Amino Acid Sequence↗
PubMed2003

Biophysical and structural analysis of a novel heme B iron ligation in the flavocytochrome cellobiose dehydrogenase.

The fungal extracellular flavocytochrome cellobiose dehydrogenase (CDH) participates in lignocellulose degradation. The enzyme has a cytochrome domain connected to a flavin-binding domain by a peptide linker. The cytochrome domain contains a 6-coordinate low spin b-type heme with unusual iron ligands and coordination geometry. Wild type CDH is only the second example of a b-type heme with Met-His ligation, and it is the first example of a Met-His ligation of heme b where the ligands are arranged in a nearly perpendicular orientation. To investigate the ligation further, Met65 was replaced with a histidine to create a bis-histidyl ligated iron typical of b-type cytochromes. The variant is expressed as a stable 90-kDa protein that retains the flavin domain catalytic reactivity. However, the ability of the mutant to reduce external one-electron acceptors such as cytochrome c is impaired. Electrochemical measurements demonstrate a decrease in the redox midpoint potential of the heme by 210 mV. In contrast to the wild type enzyme, the ferric state of the protoheme displays a mixed low spin/high spin state at room temperature and low spin character at 90 K, as determined by resonance Raman spectroscopy. The wild type cytochrome does not bind CO, but the ferrous state of the variant forms a CO complex, although the association rate is very low. The crystal structure of the M65H cytochrome domain has been determined at 1.9 A resolution. The variant structure confirms a bis-histidyl ligation but reveals unusual features. As for the wild type enzyme, the ligands have a nearly perpendicular arrangement. Furthermore, the iron is bound by imidazole N delta 1 and N epsilon 2 nitrogen atoms, rather than the typical N epsilon 2/N epsilon 2 coordination encountered in bis-histidyl ligated heme proteins. To our knowledge, this is the first example of a bis-histidyl N delta 1/N epsilon 2-coordinated protoporphyrin IX iron.

Blotting, Western↗
PubMed1995

Structural basis for the biological specificity of cystatin C. Identification of leucine 9 in the N-terminal binding region as a selectivity-conferring residue in the inhibition of mammalian cysteine peptidases.

The structural basis for the biological specificity of human cystatin C has been investigated. Cystatin C and other inhibitors belonging to family 2 of the cystatin superfamily interact reversibly with target peptidases, seemingly by independent affinity contributions from a wedge-shaped binding region built from two loop-forming inhibitor segments and a binding region corresponding to the N-terminal segment of the inhibitor. Human cystatin C variants with Gly substitutions for residues Arg-8, Leu-9, and/or Val-10 of the N-terminal binding region, and/or the evolutionarily conserved Trp-106 in the wedge-shaped binding region, were produced by site-directed mutagenesis and Escherichia coli expression. A total of 10 variants were isolated, structurally verified, and compared to wild-type cystatin C with respect to inhibition of the mammalian cysteine peptidases, cathepsins B, H, L, and S. Varying contributions from the N-terminal binding region and the wedge-shaped binding region to cystatin C affinity for the four target peptidases were observed. Interactions from the side chains of residues in the N-terminal binding region and Trp-106 are jointly responsible for the major part of cystatin C affinity for cathepsin L and are also of considerable importance for cathepsin B and H affinity. In contrast, for cathepsin S inhibition these interactions are of lesser significance, as reflected by a Ki value of 10(-8) M for the cystatin C variant devoid of Arg-8, Leu-9, Val-10, and Trp-106 side chains. The side chain of Val-10 is responsible for most of the affinity contribution from the N-terminal binding region, for all four enzymes. The contribution of the Arg-8 side chain is minor, but significant for cystatin C interaction with cathepsin B. The Leu-9 side chain confers selectivity to the inhibition of the target peptidases; it contributes to cathepsin B and L affinity by factors of 200 and 50, respectively, to cathepsin S binding by a factor of 5 only, and results in a 10-fold decreased affinity between cystatin C and cathepsin H.

Amino Acid Sequence↗
PubMed1997

ACTBP2 gene frequency distribution and sequencing of the allelic ladder and variants in the Japanese and Chinese populations.

The human beta-actin related pseudogene H-beta-Ac-psi-2 (ACTBP2) gene frequency distributions in the Japanese and Chinese Han populations were investigated and compared. Analysis was carried out by applying fluorescently labeled samples and a differently labeled sequenced allelic ladder within the same lanes in denaturing gels, followed by laser detection and automated analysis using Genescan software 672. The discrimination index and the heterozygosity index were calculated to be 0.993 and 0.916 in the Japanese population, and 0.993 and 0.944 in the Chinese Han population, respectively. No deviation from Hardy-Weinberg equilibrium was observed in these two populations. The allelic ladder, which ranged from 233 bp to 319 bp, was constructed from a combination of 23 regularly occurring alleles. The allelic ladder and 12 variants observed in 24 individuals in these two populations were sequenced. The variants could be divided into three types according to their structural variation characteristics. These variants differed from the alleles of the same repeats in the allelic ladder by the presence or absence of hexanucleotides in the central repeat regions, base deletions in the flanking regions, and base insertions in the repeat units.

Actins↗
PubMed2005

An analysis of genetic variation across the MBL2 locus in Dutch Caucasians indicates that 3' haplotypes could modify circulating levels of mannose-binding lectin.

Mannose-binding protein (MBL) is a critical component of innate immunity and provides first-line protection against pathogens. Both circulating MBL serum levels and functional activity have been correlated with common genetic variants in the MBL2 gene. Associations between MBL deficiency and severe infections have been reported in immuno-incompetent patients and for autoimmune disorders; however, measured MBL serum levels do not fully correlate with the 'secretor haplotypes'. Previously, the MBL2 locus was resequenced and determined that a recombination hotspot divides MBL2 into two haplotype blocks. It was sought to investigate whether additional variants, in either block structure could associate with MBL serum levels. Therefore, 31 common variants were analysed across the locus in 212 DNA samples of healthy Caucasian individuals with known MBL serum concentrations. The additional 5' variants were in strong linkage to the elements of the 'secretor haplotypes'; functional alleles B, C and D also lie on restricted haplotypes. Four variants in the 3' block (Ex4-1483T>C, Ex4-1067G>A, Ex4-901G>A and Ex4-710G>A) are components of a distinct haplotype block. The results of this study suggest that additional 5' variants as well as markers of distinct 3' haplotype blocks in MBL2 may contribute to circulating protein levels, but further studies are required to confirm these observations. Last, there could be a selective advantage for diversification of the 3' region of the gene.

Adolescent↗
PubMed1999

Rupestris stem pitting associated virus-1 consists of a family of sequence variants.

Rupestris stem pitting (RSP) seems to be one of the most widespread virus diseases of grapevines. A virus, designated as rupestris stem pitting associated virus-1 (RSPaV-1), is consistently associated with, and likely to be the causative agent of RSP. Sequence analyses of cDNA clones derived from several RSP-affected grapevines suggested that a family of sequence variants of RSPaV-1 was associated with RSP. The genome structure of the sequence variants is identical to that of RSPaV-1 in that they had five open reading frames (ORF) and sequence identities ranging from 75 to 93% in nucleotide sequence and from 80 to 99% in amino acid sequence. ORF5 (coat protein) and the carboxyl-terminal portion of ORF1 (replicase) appeared to be the most conserved regions. The coat proteins of the sequence variants exhibited highly similar antigenic indices, suggesting serological relatedness among them. The cDNA clones obtained through reverse transcription-polymerase chain reaction from RSP-infected grapevines were heterogeneous in nt sequence with identities of 77-99% relative to RSPaV-1. Furthermore, a number of sequence variants were identified in several grapevines infected with RSP. Baselines for defining RSPaV-1 and possible mechanisms accounting for infection of grapevines with multiple sequence variants of RSPaV-1 are proposed. Findings from this study should have practical applications toward understanding the etiology of RSP and developing reliable assays to rapidly detect the disease.

Amino Acid Sequence↗
PubMed1999

Structure and chromosomal locations of mouse steroid receptor coactivator gene family.

The newly recognized steroid receptor coactivators (SRC-1, SRC-2, and SRC-3) belong to a homologous gene family and are important transcriptional mediators for nuclear receptors. Through fluorescence in situ hybridization, we have mapped the mouse SRC-1, SRC-2, and SRC-3 genes to chromosomal locations 12A2-A3, 1A3-A5, and 2H2-H4, respectively. By screening a mouse genomic DNA library, performing long-range polymerase chain reaction and sequencing, we have cloned and characterized the mouse SRC-3 gene. The SRC-3 gene contains 19 exons and spans more than 38 kilobases (kb). Intron sizes are variable. Intron 1 (13.5 kb) and intron 15 (4.6 kb) contribute to almost half the total length of the gene. Among 20 exons identified, exon 10 is the largest (869 bp) and encodes the receptor interaction domain. The start and stop codons for translation are in exon 2 and 20, respectively. The relationship between SRC-3 gene structure and its functional protein domains suggests that many functional domains or subdomains are encoded by individual exons. The correlation between gene structure and alternative splice variants is also discussed. In summary, we have defined the structure of mouse SRC-3 gene and found that the genes in the SRC family are located in different mouse chromosomes. This information is important for developing valuable animal models harboring multiple disruptions of the SRC gene family to study their biological functions.

Animals↗
PubMed1981

ICR-191 and ethyl methanesulfonate induced mutagenesis at the immunoglobulin locus in the Y5606 cultured myeloma cell line.

The Y5606 mouse tumor synthesizing an IgG3, lambda immunoglobulin (Ig) was adapted to continuous growth in tissue culture. The spontaneous mutation rate at the Ig locus (approximately 3 X 10(-5)/cell/generation) in this cell line was found to be less than that in other cultured mouse myeloma lines. Treatment with either ICR-191 or ethyl methanesulfonate (EMS) increased the mutation rate approx. 100-fold. Spontaneous and ICR-191 induced mutants were synthetic variants that is they synthesized either heavy (H) or light (L) chains alone instead of the H and L chains synthesized by the parent. Following EMS treatment assembly variants which were synthesizing structurally altered H chains were isolated in addition to synthetic variants. The assembly variants appear to be a unique consequence of EMS mutagenesis.

Aminacrine↗
PubMed1999

Ponsin/SH3P12: an l-afadin- and vinculin-binding protein localized at cell-cell and cell-matrix adherens junctions.

We recently isolated a novel actin filament (F-actin)-binding protein, afadin, that has two isoforms, l- and s-afadins. l-Afadin is ubiquitously expressed and specifically localized at zonula adherens (ZA) in epithelial cells and at cell-cell adherens junction (AJ) in nonepithelial cells, whereas s-afadin is abundantly expressed in neural tissue. l-Afadin has one PDZ domain, three proline-rich regions, and one F-actin-binding domain, whereas s-afadin lacks the third proline-rich region and the F-actin-binding domain. To understand the molecular mechanism of the specific localization of l-afadin at ZA in epithelial cells and at cell-cell AJ in nonepithelial cells, we attempted here to identify an l-afadin-binding protein(s) and isolated a protein, named ponsin. Ponsin had many splicing variants and the primary structures of two of them were determined. Both the two variants had three Src homology 3 (SH3) domains and turned out to be splicing variants of SH3P12. The third proline-rich region of l-afadin bound to the region of ponsin containing the second and third SH3 domains. Ponsin was ubiquitously expressed and localized at ZA in epithelial cells, at cell-cell AJ in nonepithelial cells, and at cell-matrix AJ in both types of cells. Ponsin furthermore directly bound vinculin, an F-actin-binding protein localized at ZA in epithelial cells, at cell-cell AJ in nonepithelial cells, and at cell-matrix AJ in both types of cells. Vinculin has one proline-rich region where two proline-rich sequences are located. The proline-rich region bound to the region of ponsin containing the first and second SH3 domains. l-Afadin and vinculin bound to ponsin in a competitive manner and these three proteins hardly formed a ternary complex. These results indicate that ponsin is an l-afadin- and vinculin-binding protein localized at ZA in epithelial cells, at cell-cell AJ in nonepithelial cells, and at cell-matrix AJ in both types of cells.

Amino Acid Sequence↗
PubMed1993

Simple mucin-type carbohydrate antigens in pleomorphic adenomas.

Simple mucin-type carbohydrate structures, T, Tn and sialosyl-Tn, are regarded as general markers of carcinomas in several epithelial tissues as a result of incomplete synthesis with precursor accumulation. The structures have a very limited distribution in normal tissues and secretions, including saliva and salivary glands. The expression of simple mucin-type carbohydrate structures and ABH(O) variants was studied in paraffin-embedded and frozen tissue sections from 37 pleomorphic adenomas with associated normal parotid tissue, using immunohistology and a panel of MAbs with well-defined specificity for T, Tn, sialosyl-Tn, and blood group H and A variants hereof. The immature Tn and sialosyl-Tn antigen structures were expressed in the epithelial ductular structures of the tumors, whereas they were almost absent from normal parotid tissue, indicating aberrant glycosylation with accumulation of precursor structures. Furthermore, the tumors showed loss of A antigen. The prognostic significance of these results is discussed. The modified myoepithelial cells in periductular and solid areas expressed T and sialosyl-T antigens, similar to normal myoepithelial cells and basal cells. Thus these modified MEC seem to have retained their normal simple mucin-type glycosylation pattern, suggesting that T antigen may be used as a marker of MEC in salivary gland tumors.

Adenoma, Pleomorphic↗
PubMed2005

H2AX: tailoring histone H2A for chromatin-dependent genomic integrity.

During the last decade, chromatin research has been focusing on the role of histone variability as a modulator of chromatin structure and function. Histone variability can be the result of either post-translational modifications or intrinsic variation at the primary structure level: histone variants. In this review, we center our attention on one of the most extensively characterized of such histone variants in recent years, histone H2AX. The molecular phylogeny of this variant seems to have run in parallel with that of the major canonical somatic H2A1 in eukaryotes. Functionally, H2AX appears to be mainly associated with maintaining the genome integrity by participating in the repair of the double-stranded DNA breaks exogenously introduced by environmental damage (ionizing radiation, chemicals) or in the process of homologous recombination during meiosis. At the structural level, these processes involve the phosphorylation of serine at the SQE motif, which is present at the very end of the C-terminal domain of H2AX, and possibly other PTMs, some of which have recently started to be defined. We discuss a model to account for how these H2AX PTMs in conjunction with chromatin remodeling complexes (such as INO80 and SWRI) can modify chromatin structure (remodeling) to support the DNA unraveling ultimately required for DNA repair.

Amino Acid Sequence↗
PubMed2004

Studies on the interaction of Escherichia coli agmatinase with manganese ions: structural and kinetic studies of the H126N and H151N variants.

The H126N and H151N variants of Escherichia coli agmatinase (EC 3.5.3.11) were produced by site-directed mutagenesis, and their kinetic and structural properties were examined. About 51% and 30% of wild-type activity were expressed by fully manganese activated species of the H126N and H151N variants, respectively. Mutations were not accompanied by changes in the K(m) value for arginine (1.2+/-0.3 mM), K(i) value for putrescine inhibition (3.2+/-0.4 mM), molecular weight (M(r) 67,000+/-2000), tryptophan fluorescence properties (lambda(max) = 342 nm) or CD spectra of the enzyme. However, the interaction with the required manganese ions was significantly altered, as indicated by the effects of dialysis of the enzymes against metal-free buffer. We conclude that replacement of His151 with asparagine results in the loss of a catalytically essential Mn(2+) upon dialysis and concomitant reversible inactivation of the H151N mutant, and that the affinity of a more weakly bound Mn(2+) is decreased in the H126N variant.

Amino Acid Substitution↗