Effect of antler growth period on the chemical composition of velvet antler in sika deer (Cervus nippon)

The aim of this study was to provide basic information to allow improved scientific assessment of velvet antlers’ quality by investigating the change of chemical composition depending on antler growth period in sika deer (Cervus nippon). Twelve antlers harvested from sika deer stags (aged 4–5 years) by antler growth periods of 40 days (FDG) and 60 days (SDG) after the casting of antler buttons from the previous set were analysed to compare the chemical composition, such as crude protein, ash, ether extract, amino acid, collagen, glycosaminoglycans (GAGs), uronic acid and sialic acid. The weight of velvet antler in FDG was lower than that of the SDG (P<0.05). SDG had a higher (P<0.05) content of crude ash than FDG. FDG had a higher (P<0.05) content of crude protein than SDG. Amino acid composition was also higher in FDG than in SDG for all sections. The content of collagen was higher in SDG than in FDG for the middle and base (P<0.05) sections. However, collagen levels were exceptionally higher (P<0.05) in FDG than in SDG for the upper section. While the content of collagen was significantly higher (P<0.05) for the upper section than for the middle and base sections in FDG, this was significantly increased (P<0.05) downward from the upper to the base section in SDG. Uronic acid content was higher in FDG than SDG for all three sections but there was no significant difference between groups in the middle and base sections. The content of GAGs was significantly higher (P<0.05) in FDG than SDG for all three sections. The content of sialic acid was the same as that of GAGs. Consequently, in this study, velvet antler production was increased with the extension of antler growth period, but the contents of protein, total amino acid composition, GAGs, uronic acid and sialic acid were decreased and those of ash and collagen were increased. Therefore, it is expected that the quality of velvet antler may be decreased greatly by extension of antler growth period.     

The carbohydrate–polypeptide linkages, the amino acid sequences of the peptides adjacent to some of these bonds, and the composition and size of the carbohydrate units of α1-acid glycoprotein

1. The glycopeptides derived from a proteolytic digest of sialic acid-free α₁-acid glycoprotein were separated on a DEAE-cellulose column into five main fractions. 2. The average molecular weight of these glycopeptides was 2400, except for one fraction whose molecular weight was 3100. The average molecular weight of the sialic acid-free carbohydrate units was found to be 2200. From these data and the carbohydrate content of the native protein and the assumed molecular weight of 44000, it was concluded that α₁-acid glycoprotein probably possesses five carbohydrate units. The sialic acid-containing carbohydrate units of this glycoprotein have an average molecular weight of 3000, except for one unit the molecular weight of which is significantly higher. 3. The N-, non-N- and C-terminal amino acids of the main glycopeptides were determined. Aspartic acid and threonine occur in most peptides. Alanine, glycine, proline, serine and lysine were present in varying amounts. Traces of other amino acids were also found. 4. The amino acid sequence of three main glycopeptides was established and indicated that these glycopeptides are located at different positions of the polypeptide chain of the glycoprotein. These sequences are: Asp(NH₂)-Pro-Lys; Thr-Asp(NH₂)-Ala; Asp(NH₂)-Gly-Thr. 5. From the results of a series of chemical reactions (periodate oxidation, hydrazinolysis, dinitrophenylation, mild acid hydrolysis) it was shown that the hydroxyl group of the N-terminal threonine and the -amino group of lysine are free and that the β-carboxyl group of aspartic acid is present as amide. It was concluded that this amide group is involved in the carbohydrate–polypeptide linkages of at least four carbohydrate units of α₁-acid glycoprotein. 6. The carbohydrate composition of the sialic acid-free glycopeptides was determined in terms of moles of neutral hexoses, glucosamine and fucose/mole. 7. Fucose, at least to the larger part, is not linked to sialic acid, and its (glycosidic) linkage is significantly more stable toward acid hydrolysis than the bond of the sialyl residues. 8. Heterogeneity of the carbohydrate units of α₁-acid glycoprotein was found with regard to size and to content of fucose and sialic acid.     

Sialic acid-binding dwarf elder four-chain lectin displays nucleic acid N-glycosidase activity

Sialic acid-binding dwarf elder agglutinin (SEA) present only in rhizomes of the medicinal plant Sambucus ebulus L., was found to be a tetrameric glycoprotein consisting of two covalently-associated dimers of an enzymic A chain with rRNA N-glycosidase activity (EC 3.2.2.22) linked to a B chain with agglutinin properties. The lectin inhibited protein synthesis by a cell-free system and depurinated ribosomes. Cloning of the corresponding gene and molecular modeling of the deduced amino acid sequence demonstrated that SEA has a three-dimensional structure which resembles that reported for other two tetrameric type 2 RIPs from Sambucus (SNAI and SSA). The lectin agglutinated red blood cells and displayed sugar affinity for sialic acid residues apart from d-galactose, binding to the mucin-producing gut goblet cells. Since sialic acid is present in animal cells, especially in epithelial lining gut cells, but not in plants, SEA could play a role in the defense against insect attack.     

Combining ancestral sequence reconstruction with protein design to identify an interface hotspot in a key metabolic enzyme complex

It is important to identify hotspot residues that determine protein–protein interactions in interfaces of macromolecular complexes. We have applied a combination of ancestral sequence reconstruction and protein design to identify hotspots within imidazole glycerol phosphate synthase (ImGPS). ImGPS is a key metabolic enzyme complex, which links histidine and de novo purine biosynthesis and consists of the cyclase subunit HisF and the glutaminase subunit HisH. Initial fluorescence titration experiments showed that HisH from Zymomonas mobilis (zmHisH) binds with high affinity to the reconstructed HisF from the last universal common ancestor (LUCA‐HisF) but not to HisF from Pyrobaculum arsenaticum (paHisF), which differ by 103 residues. Subsequent titration experiments with a reconstructed evolutionary intermediate linking LUCA‐HisF and paHisF and inspection of the subunit interface of a contemporary ImGPS allowed us to narrow down the differences crucial for zmHisH binding to nine amino acids of HisF. Homology modeling and in silico mutagenesis studies suggested that at most two of these nine HisF residues are crucial for zmHisH binding. These computational results were verified by experimental site‐directed mutagenesis, which finally enabled us to pinpoint a single amino acid residue in HisF that is decisive for high‐affinity binding of zmHisH. Our work shows that the identification of protein interface hotspots can be very efficient when reconstructed proteins with different binding properties are included in the analysis. Proteins 2017; 85:312–321. © 2016 Wiley Periodicals, Inc.     

Identification of critical amino acids involved in α1-β interaction in voltage-dependent Ca channels

In voltage-dependent Ca²⁺ channels, the α₁ and β subunits interact via two cytoplasmic regions defined as the Alpha Interaction Domain (AID) and Beta Interaction Domain (BID). Several novel amino acids for that interaction have now been mapped in both domains by point mutations. It was found that three of the nine amino acids in AID and four of the eight BID amino acids tested were essential for the interaction. Whereas the important AID amino acids were clustered around five residues, the important BID residues were more widely distributed within a larger 16 amino acid sequence. The affinity of the AID_A GST fusion protein for the four interacting β_1b BID mutants was not significantly altered compared with the wild-type β_1b despite the close localization of mutated residues to disruptive BID amino acids. Expression of these interactive β mutants with the full-length α_1A subunit only slightly modified the stimulation efficiency when compared with the wild-type β_1b subunit. Our data suggest that non-disruptive BID sequence alterations do not dramatically affect the β subunit-induced current stimulation.     

Two Arginine Residues of Streptococcus gordonii Sialic Acid-Binding Adhesin Hsa Are Essential for Interaction to Host Cell Receptors

Hsa is a large, serine-rich protein of Streptococcus gordonii DL1 that mediates binding to α2-3-linked sialic acid termini of glycoproteins, including platelet glycoprotein Ibα, and erythrocyte membrane protein glycophorin A, and band 3. The binding of Hsa to platelet glycoprotein Ibα contributes to the pathogenesis of infective endocarditis. This interaction appears to be mediated by a second non-repetitive region (NR2) of Hsa. However, the molecular details of the interaction between the Hsa NR2 region and these glycoproteins are not well understood. In the present study, we identified the amino acid residues of the Hsa NR2 region that are involved in sialic acid recognition. To identify the sialic acid-binding site of Hsa NR2 region, we prepared various mutants of Hsa NR2 fused with glutathione transferase. Fusion proteins harboring Arg340 to Asn (R340N) or Arg365 to Asn (R365N) substitutions in the NR2 domain exhibited significantly reduced binding to human erythrocytes and platelets. A sugar-binding assay showed that these mutant proteins abolished binding to α2-3-linked sialic acid. Furthermore, we established S. gordonii DL1 derivatives that encoded the corresponding Hsa mutant protein. In whole-cell assays, these mutant strains showed significant reductions in hemagglutination, in platelet aggregation, and in adhesion to human leukocytes. These results indicate that the Arg340 and Arg365 residues of Hsa play an important role in the binding of Hsa to α2-3-linked sialic acid-containing glycoproteins.     

Sialic acid in hemolymph and affinity purified lectins from two marine bivalves

Sialic acids have been implicated in a variety of complex biological regulatory and signalling events and their functional importance is reflected by their presence in a wide variety of phyla. Potentially they may inhibit intermolecular and intercellular interactions. Lectins that exhibit specificity for sialic acid or sialoglycoconjugates are ubiquitous in the body fluids of invertebrates and this has supported the assumption that these lectins are involved in defense against microbes that express sialic acids on their surfaces. This biological function has also been inferred from the absence of sialic acids in lower invertebrates. However, most invertebrate lectins are heterogeneous and may also bind other ligands. The biological significance of the different carbohydrate specificities are not yet known. We have demonstrated the presence of sialic acids in hemolymph from two marine bivalves, the Pacific oyster Crassostrea gigas (≈15 μg ml⁻¹) and the horse mussel Modiolus modiolus (48–100 μg ml⁻¹) by several different assays. The sialic acid was mostly in free form. Affinity purified lectins from the horse mussel also contained bound sialic acids (2–5 μmol g⁻¹). Oyster hemolymph stimulated the in vitro phagocytosis of bacteria by oyster hemocytes. The stimulation by hemolymph is facilitated by a dialyzable component, that apparently is active irrespective of the binding to sialic acid (BSM). Addition of sialic acid had no significant effect on the in vitro phagocytosis of bacteria by oyster hemocytes.     

LOCALIZATION AND IDENTIFICATION OF GALACTOSE/N-ACETYLGALACTOSAMINE AND SIALIC ACID-CONTAINING PROTEINS IN CHINESE HAMSTER METAPHASE CHROMOSOMES

Four lectins were used to recognize galactose/N-acetyl-galactosamine (Gal/GalNAc) and sialic acid residues in proteins of Chinese hamster metaphase chromosomes. In situ binding pattern of a fluorescein isothiocyanate-labelled (Gal/GalNAc)-specific lectin Sophora japonica agglutinin (SJA) showed that chromosomal SJA-binding proteins are primarily localized to the helically coiled substructure of chromatids. Numerous SJA-binding proteins were identified in Western blots of chromosomal proteins, their molecular weights ranging from 26 to 200kDa. Another Gal/GalNAc-specific lectin, peanut agglutinin (PNA), with a slightly different sugar binding specificity, did not bind to Chinese hamster metaphase chromosomes, and in Western blots only two chromosomal protein bands were faintly stained. The in situ labelling patterns of two sialic acid-specific lectins, Maackia amurensis (MAA) and Sambucus nigra (SNA) agglutinins, both showed that the helically coiled substructure of chromatids is also enriched in sialylated proteins. In Western blot analysis 11 MAA-binding protein bands with molecular weights ranging from 54 to 215kDa were identified, while SNA only bound to one protein band of 67kDa. MAA and SNA are specific for α (2|ad3)- and α (2|ad6)-linked sialic acid residues, respectively. Thus, it is likely that α (2|ad3)-linked sialic acid residues are more common in chromosomal proteins than α(2|ad6)-linked sialic acid residues. These data suggest that Gal/GalNAc and sialic acid-containing glycoproteins exist in metaphase chromosomes and that these proteins may have a role in the formation of higher order metaphase chromosome structures.     

Comparative Studies of the Indoleamine Dioxygenase-like Myoglobin from the Abalone Sulculus diversicolor

The abalone Sulculus diversicolor contains abundant myoglobin in its buccal mass. The myoglobin is homodimeric and the molecular mass of the constituent polypeptide chain is 41,000 Da. The amino acid sequence and gene structure are highly homologous with those of a vertebrate tryptophan-degrading enzyme, indoleamine dioxygenase (IDO). Thus Sulculus myoglobin evolved from an IDO gene, and represents a typical case of functional convergence. The oxygen equilibrium properties of Sulculus myoglobin were examined and compared with those of myoglobins from other sources. It binds oxygen reversibly, and the P₅₀ was determined to be 3.8 mmHg at 20°C and pH 7.4, showing that the oxygen affinity of Sulculus myoglobin is significantly lower than those of usual 16 kDa myoglobins. It also displays no cooperativity (n_max: 1.02–1.06) and no alkaline Bohr effect between pH 7.0 and 7.9. The cDNA-derived amino acid sequences of vertebrate IDOs, molluscan IDO-like myoglobins and a homolog in the yeast Saccharomyces were aligned, and several amino acid residues were proposed as candidates for key residues to control the function of IDO or myoglobin.     

Carbohydrate-to-carbohydrate interactions between α2,3-linked sialic acids on α2 integrin subunits and asialo-GM1 underlie the bone metastatic behaviour of LNCAP-derivative C4-2B prostate cancer cells

Complex interplays among proteins, lipids and carbohydrates can alter the phenotype and are suggested to have a crucial role in tumour metastasis. Our previous studies indicated that a complex of the GSLs (glycosphingolipids), AsGM1 (asialo-GM1), which lacks α2,3-linked sialic acid, and α2β1 integrin receptors is responsible for the metastatic behaviour of C4-2B prostate cancer cells. Herein, we identified and addressed the functional significance of changes in sialylation during prostate cancer progression. We observed an increase in α2,3-linked sialic acid residues on α2 subunits of α2β1 integrin receptors, correlating with increased gene expression of α2,3-STs (sialyltransferases), particularly ST3GAL3. Cell surface α2,3-sialylation of α2 subunits was required for the integrin α2β1-dependent cell adhesion to collagen type I and the same α2,3-linked sialic acid residues on the integrin receptor were responsible for the interaction with the carbohydrate moiety of AsGM1, explaining the complex formation between AsGM1 and α2β1 integrin receptors. These results provide novel insights into the role of sialic acids in the organization and function of important membrane components in invasion and metastatic processes.     

Identification of the β-Lactamase Inhibitor Protein-II (BLIP-II) Interface Residues Essential for Binding Affinity and Specificity for Class A β-Lactamases

The interactions between β-lactamase inhibitory proteins (BLIPs) and β-lactamases have been used as model systems to understand the principles of affinity and specificity in protein-protein interactions. The most extensively studied tight binding inhibitor, BLIP, has been characterized with respect to amino acid determinants of affinity and specificity for binding β-lactamases. BLIP-II, however, shares no sequence or structural homology to BLIP and is a femtomolar to picomolar potency inhibitor, and the amino acid determinants of binding affinity and specificity are unknown. In this study, alanine scanning mutagenesis was used in combination with determinations of on and off rates for each mutant to define the contribution of residues on the BLIP-II binding surface to both affinity and specificity toward four β-lactamases of diverse sequence. The residues making the largest contribution to binding energy are heavily biased toward aromatic amino acids near the center of the binding surface. In addition, substitutions that reduce binding energy do so by increasing off rates without impacting on rates. Also, residues with large contributions to binding energy generally exhibit low temperature factors in the structures of complexes. Finally, with the exception of D206A, BLIP-II alanine substitutions exhibit a similar trend of effect for all β-lactamases, i.e., a substitution that reduces affinity for one β-lactamase usually reduces affinity for all β-lactamases tested.     

A new histochemical method for the selective periodate oxidation of total tissue sialic acids

Summary Evaluation of the intensity of the periodic acid—Schiff (PAS) staining produced following oxidation for 1 h at 4°C with 0.4mm periodic acid in approximately 1m hydrochloric acid indicated that this reagent completely oxidized all available sialic acid residues of either the sialo- or sialosulphoglycoproteins of human and rat colon or the sialoglycoproteins of rat sublingual gland. These conditions produced no visible Schiff staining of either neutral macromolecules orvicinal diols located on hexose, 6-deoxyhexose orN-acetylhexosamine residues (neutral sugars) of sialo- and sialosulphoglycoproteins. Furthermore, there was no extraction of epithelial glycoproteins or de-O-acylation of side chain substituted sialic acid residues. These data demonstrate that 0.4mm periodic acid in approximately 1m hydrochloric acid can be used as a specific reagent for the selective visualization of sialic acids in the PAS procedure.Studies of the mechanism of the oxidation of neutral sugars with 0.4mm periodic acid in approximately 1m hydrochloric acid indicated that their lack of PAS reactivity was not due to the production of Schiff unreactive hemiacetals or hemialdals. It is suggested that the selectivity of 0.4mm periodic acid in approximately 1m hydrochloric acid is a result of an increase in the rate of the oxidation of the sialic acid residues together with a decrease in the rate of oxidation of neutral sugars.     

Resonant recognition model of neuropeptide Y family: hot spot amino acid distribution in the sequences

The resonant recognition model is used to predict structurally and functionally important amino acid residues (so-called hot spots) in the neuropeptide Y (NPY) family. Thirty-three polypeptides belong to this family. All of them consist of 36 amino acids. The model predicts that residues 10 and 28 in the polypeptides are hot spots. In the 33 polypeptides, most of the amino acids at residue 10 are acidic amino acids, glutamic acid and aspartic acid. Other minor amino acids, serine, glycine, and proline, have high probabilities of -turn occurrence. Amino acids at residue 28 are all branched hydrophobic amino acids, isoleucine, leucine, and valine. The profile for predicting hot spots indicates repeating patterns of residues 1–18 and residues 19–36. Absolute values at residue i and residue i + 18 are the same, but these residues have opposite signs. Therefore the model of the NPY family predicts hot spots concerning a combination of residue i and residue i + 18.     

Histochemical analysis of glycoproteins in the unicellular glands in the epidermis of an Indian freshwater fish Mastacembelus pancalus (Hamilton)

Summary The unicellular glands in the epidermis of the Indian freshwater fish Mastacembelus pancalus consist of three types of mucous cells and sacciform cells. The histochemical properties of their secretory glycoproteins have been analysed by means of a battery of histochemical methods. These included methods for the identification and simultaneous visualization of oxidizable vicinal diols, O-acyl sugars, O-sulphate esters and sialic acid residues with or without side-chain O-acyl variants. Four general classes of glycoproteins (GPs) were identified. These included (i) GPs with O-sulphate esters and oxidizable vicinal diols, (ii) GPs with oxidizable vicinal diols and sialic acid residues with or without O-acyl substitution at C7, (iii) GPs mainly with O-sulphate esters, low moieties of GPs with oxidizable vicinal diols, O-acyl sugars and sialic acid residues with side-chain O-acyl variant predominantly at C8 (or which are di- or tri-substituted) or C9 and in traces of sialic acid residues without O-acyl substitution or with O-acyl substitution at C7, and (iv) GPs with traces of oxidizable vicinal diols, O-acyl sugars and sialic acid residues with O-acyl substitution at C8 (or which are di- or tri-substituted) or C9. The physiological significances of these GP classes and their release on the surface of the epidermis are discussed with special reference to their role in lubrication, protection and inhibition of the invasion and proliferation of pathogenic microorganisms in the epidermis, as adapted to the peculiar mode of life of the fish.     

Fine sugar specificity of the mistletoe (Viscum album) lectin I

The behaviour ofN-acetyllactosamine-type oligosaccharides and glycopeptides on a column of mistletoe lectin I (MLI) immobilized on Sepharose 4B was examined. The immobilized lectin does not show any affinity for asialo-N-glycosylpeptides and related oligosaccharides, which possess one to four unmaskedN-acetyllactosamine sequences. However, substitution of at least one of theN-acetyllactosamine sequences by sialic acid residues, either at O-3 or O-6 of galactose, slightly enhances the affinity of the lectin. Such sialylatedN-glycosylpeptides or oligosaccharides are eluted from the lectin column by the starting buffer as retarded fractions. Surprisingly, the affinity of the immobilized MLI is higher for P1 antigen-containing glycopeptide isolated from turtle-dove ovomucoid and for glycopeptides from bovine thyroglobulin containing terminal non-reducing Gal1–3Gal sequences. These structures are strongly bound on the lectin column and their elution is obtained with 0.15M galactose in the starting buffer.In memory of Hartmut Franz.     

The periodate oxidation of bovine bone sialoprotein, and some observations on its structure

1. Bovine bone sialoprotein (mol.wt. 23000) contains N-acetylneuraminic acid and N-glycollylneuraminic acid, fucose, galactose, mannose, N-acetylgalactosamine and N-acetylglucosamine residues in the form of a very small number, perhaps one, of highly branched oligosaccharide structures linked covalently to peptide. 2. Periodate oxidation of the sialoprotein results in quantitative destruction only of the sialic acid and fucose residue consistent with the earlier findings of their positions as terminal groups. 3. Terminal sialic acid residues are attached to galactopyranose residues by 2,3-linkages, and to some N-acetylgalactosamine residues (at C-6). 4. Sequential Smith degradation indicates that N-acetylgalactosamine residues may be present as points of branching (linked in C-1, C-3 and C-6) and N-acetylglucosamine residues are located in the inner part of the structure, adjacent to the carbohydrate–peptide bond(s). 5. Mannose residues appear to be linked in the 1,3-positions.     

Semi-rational site-directed mutagenesis of phyI1s from <Emphasis Type="Italic">Aspergillus niger</Emphasis> 113 at two residue to improve its phytase activity

Through alignment of amino acid sequences among different phytases, we found that the amino acid at residues 53 and 91 vary broadly. To prove that the amino acid at residues 53 and 91 were related to phytase specific activity, two single mutant phyI1s Q53R and K91D were obtained by site-directed mutagenesis strategy. None of the single amino acid residues in the two mutants was in a position reported to be important for catalysis or substrate binding. Kinetic analysis of the phytase activity of the two mutants (Q53R and K91D) indicated that the mutants were attributed to 2.2- and 1.5-fold increased specific activity, and a 1.47- and 1.16-fold increased affinity for sodium phytate. In addition, the overall catalytic efficiency (k _cat/K _m) of the two mutants was improved 4.08- and 2.84-fold compared to that of the wild type. Such mutants will be instrumental for the structure–function study of the enzyme and for industrial application.     

An ultra‐high affinity protein–protein interface displaying sequence‐robustness

Protein–protein interactions are crucial in biology and play roles in for example, the immune system, signaling pathways, and enzyme regulation. Ultra‐high affinity interactions (K _d <0.1 nM) occur in these systems, however, structures and energetics behind stability of ultra‐high affinity protein–protein complexes are not well understood. Regulation of the starch debranching barley limit dextrinase (LD) and its endogenous cereal type inhibitor (LDI) exemplifies an ultra‐high affinity complex (K _d of 42 pM). In this study the LD–LDI complex is investigated to unveil how robust the ultra‐high affinity is to LDI sequence variation at the protein–protein interface and whether alternative sequences can retain the ultra‐high binding affinity. The interface of LD–LDI was engineered using computational protein redesign aiming at identifying LDI variants predicted to retain ultra‐high binding affinity. These variants present a very diverse set of mutations going beyond conservative and alanine substitutions typically used to probe interfaces. Surface plasmon resonance analysis of the LDI variants revealed that high affinity of LD–LDI requires interactions of several residues at the rim of the protein interface, unlike the classical hotspot arrangement where key residues are found at the center of the interface. Notably, substitution of interface residues in LDI, including amino acids with functional groups different from the wild‐type, could occur without loss of affinity. This demonstrates that ultra‐high binding affinity can be conferred without hotspot residues, thus making complexes more robust to mutational drift in evolution. The present mutational analysis also demonstrates how energetic coupling can emerge between residues at large distances at the interface.     

Protein selectivity in immobilized metal affinity chromatography based on the surface accessibility of aspartic and glutamic acid residues

The interaction of different species variants of cytochrome c and myoglobin, as well as hen egg white lysozyme, with the hard Lewis metal ions Al³⁺, Ca²⁺, Fe³⁺, and Yb³⁺ and the borderline metal ion Cu²⁺, immobilized to iminodiacetic acid (IDA)-Sepharose CL-4B, has been investigated over the rangepH 5.5–8.0. With appropriately chosen buffer and metal ion conditions, these proteins can be bound to the immobilized M ⁿ +-IDA adsorbents via negatively charged amino acid residues accessible on the protein surface. For example, tuna heart cytochrome c, which lacks surface-accessible histidine residues, readily bound to the Fe³⁺-IDA adsorbent, while the other proteins also showed affinity toward immobilized Fe³⁺-IDA adsorbents when buffers containing 30 mM of imidazole were used. These studies document that protein selectivity can be achieved with hard-metalion immobilized metal ion affinity chromatography (IMAC) systems through the interaction of surfaceexposed aspartic and glutamic acid residues on the protein with the immobilized M ⁿ +-IDA complex. These investigations have also documented that the so-called soft or borderline immobilized metal ions such as the Cu²⁺-IDA adsorbent can also interact with surface-accessible aspartic and glutamic acid residues in a protein-dependent manner. A relationship is evident between the number and extent of clustering of the surfaceaccessible aspartic and glutamic acid residues and protein selectivity with these IMAC systems. The use of elution buffers which contain organic compound modifiers which replicate the carboxyl group moieties of these amino acids on the surface of proteins is also described.Abbreviations IDA iminodiacetic acid - IDA-Mⁿ⁺ iminodiacetic acid chelated to metal ion - IMAC immobilized metal affinity chromatography - DHCC dog heart cytochrome c - HHCC horse heart cytochrome c, THCC, tuna heart cytochrome c - HMYO horse skeletal muscle myoglobin - SMYO sheep skeletal muscle myoglobin - HEWL hen egg white lysozyme     

Cytochemical study of the effect of aluminium on cultured brain microvascular endothelial cells

Summary The cytotoxic effect of aluminium was studied on cultured goat brain microvascular endothelial cells used as an in vitro model of the blood—brain barrier. Confluent monolayers of these cells were exposed for 4 days to aluminium maltol and, for control purposes, to maltol alone, and also to cadmium chloride as a known cytotoxic substance. The localization of plasmalemma-bound enzymatic activities of 5-nucleotidase and Ca²⁺-ATPase and the distribution of sialic acid residues were studied at the ultrastructural level.It was observed that the reaction for 5-nucleotidase activity was only insignificantly affected, indicating its resistance to the cytotoxic action of both substances used. On the contrary, the activity of Ca²⁺-ATPase was evidently suppressed, especially in the interendothelial clefts where junctional complexes are presumably to be formed. Aluminium also affects the density of sialic acid residues, as shown by their redistribution, leading to the appearance of relatively long segments of unlabelled apical cell surface.The data obtained suggest that observed changes in the localization of Ca²⁺-ATPase and sialic acid residues can lead ultimately to impairment of the formation and maintenance of intercellular junctions and to disturbances in the negatively charged domains of the endothelial cell surface. Whether these alterations, induced in vitro, contribute to in vivo disturbances of blood—brain barrier function requires further experimental study.