Involvement Of Lysine Residues Research Articles

Atypical Ubiquitination and Parkinson's Disease.

Ubiquitination (the covalent attachment of ubiquitin molecules to target proteins) is one of the main post-translational modifications of proteins. Historically, the type of polyubiquitination, which involves K48 lysine residues of the monomeric ubiquitin, was the first studied type of ubiquitination. It usually targets proteins for their subsequent proteasomal degradation. All the other types of ubiquitination, including monoubiquitination; multi-monoubiquitination; and polyubiquitination involving lysine residues K6, K11, K27, K29, K33, and K63 and N-terminal methionine, were defined as atypical ubiquitination (AU). Good evidence now exists that AUs, participating in the regulation of various cellular processes, are crucial for the development of Parkinson’s disease (PD). These AUs target various proteins involved in PD pathogenesis. The K6-, K27-, K29-, and K33-linked polyubiquitination of alpha-synuclein, the main component of Lewy bodies, and DJ-1 (another PD-associated protein) is involved in the formation of insoluble aggregates. Multifunctional protein kinase LRRK2 essential for PD is subjected to K63- and K27-linked ubiquitination. Mitophagy mediated by the ubiquitin ligase parkin is accompanied by K63-linked autoubiquitination of parkin itself and monoubiquitination and polyubiquitination of mitochondrial proteins with the formation of both classical K48-linked ubiquitin chains and atypical K6-, K11-, K27-, and K63-linked polyubiquitin chains. The ubiquitin-specific proteases USP30, USP33, USP8, and USP15, removing predominantly K6-, K11-, and K63-linked ubiquitin conjugates, antagonize parkin-mediated mitophagy.

Enzymatic lysine oxidation as a posttranslational modification.

Oxidoreductases catalyze oxidation-reduction reactions and comprise a very large and diverse group of enzymes, which can be subclassified depending on the catalytic mechanisms of the enzymes. One of the most prominent oxidative modifications in proteins is carbonylation, which involves the formation of aldehyde and keto groups in the side chain of lysines. This modification can alter the local macromolecular structure of proteins, thereby regulating their function, stability, and/or localization, as well as the nature of any protein-protein and/or protein-nucleic acid interactions. In this review, we focus on copper-dependent amine oxidases, which catalyze oxidative deamination of amines to aldehydes. In particular, we discuss oxidation reactions that involve lysine residues and that are regulated by members of the lysyl oxidase (LOX) family of proteins. We summarize what is known about the newly identified substrates and how this posttranslational modification regulates protein function in different contexts.

Acetylation of Aβ40 Alters Aggregation in the Presence and Absence of Lipid Membranes.

A hallmark of Alzheimer's disease (AD) is the formation of senile plaques comprised of the β-amyloid (Aβ) peptide. Aβ fibrillization is a complex nucleation-dependent process involving a variety of metastable intermediate aggregates and features the formation of inter- and intramolecular salt bridges involving lysine residues, K16 and K28. Cationic lysine residues also mediate protein-lipid interactions via association with anionic lipid headgroups. As several toxic mechanisms attributed to Aβ involve membrane interactions, the impact of acetylation on Aβ40 aggregation in the presence and absence of membranes was determined. Using chemical acetylation, varying mixtures of acetylated and nonacetylated Aβ40 were produced. With increasing acetylation, fibril and oligomer formation decreased, eventually completely arresting fibrillization. In the presence of total brain lipid extract (TBLE) vesicles, acetylation reduced the interaction of Aβ40 with membranes; however, fibrils still formed at near complete levels of acetylation. Additionally, the combination of TBLE and acetylated Aβ promoted annular aggregates. Finally, toxicity associated with Aβ40 was reduced with increasing acetylation in a cell culture assay. These results suggest that in the absence of membranes that the cationic character of lysine plays a major role in fibril formation. However, acetylation promotes unique aggregation pathways in the presence of lipid membranes.

Synthesis of β-Diketone DNA Derivatives for Affinity Modification of Proteins

Diketone DNA derivatives have been proposed to modify the guanidine group of Arg in proteins. The β-diketo group at the C2' atom of the sugar phosphate moiety has been introduced in DNA by acylation of oligonucleotide precursors, i.e., DNA fragments containing 2'-amino-2'-deoxyuridine, which have been synthesized by the chemical automatic synthesis. Water-soluble N-[3-(dimethylamino)propyl]-N′-ethylcarbodiimide (EDC) and 4,6-dioxoheptanoic acid have been used in the reaction. The ability of oligodeoxyribonucleotides containing the 2'-β-diketo group to react with guanidine, Nα-Boc-L-arginine, and Nα-Dns-L-arginine has been demonstrated. The introduction of this modification into one of the strands of the 15-base pair DNA duplex has been shown to lead to its destabilization. The conjugate formation of MutS and MutL proteins from the E. coli mismatch repair system with 17-base pair DNA duplexes containing the 2'-deoxy-2'-(4,6-dioxoheptylamido)uridine residue has been detected for the first time. To increase the selectivity of the DNA ligands containing the β-diketo group in the reaction with the Arg residues of proteins, we have proposed to treat the reaction mixture with hydroxylamine. This treatment leads to the cleavage of Schiff bases, which are formed with the involvement of lysine residues.

Importance of amino acids Leu135 and Tyr236 for the interaction between EhCFIm25 and RNA: a molecular dynamics simulation study.

The CFIm25 subunit of the heterotetrameric cleavage factor Im (CFIm) is a critical factor in the formation of the poly(A) tail at mRNA 3' end, regulating the recruitment of polyadenylation factors, poly(A) site selection, and cleavage/polyadenylation reactions. We previously reported the homologous protein (EhCFIm25) in Entamoeba histolytica, the protozoan causing human amoebiasis, and showed the relevance of conserved Leu135 and Tyr236 residues for RNA binding. We also identified the GUUG sequence as the recognition site of EhCFIm25. To understand the interactions network that allows the EhCFIm25 to maintain its three-dimensional structure and function, here we performed molecular dynamics simulations of wild-type (WT) and mutant proteins, alone or interacting with the GUUG molecule. Our results indicated that in the presence of the GUUG sequence, WT converged more quickly to lower RMSD values in comparison with mutant proteins. However, RMSF values showed that movements of amino acids of WT and EhCFIm25*L135T were almost identical, interacting or not with the GUUG molecule. Interestingly, EhCFIm25*L135T, which is the only mutant with a slight RNA binding activity experimentally, presents the same stabilization of bend structures and alpha helices as WT, notably in theC-terminus. Moreover, WT and EhCFIm25*L135T presented almost the same number of contacts that mainly involve lysine residues interacting with the G4 nucleotide. Overall, our data proposed a clear description of the structural and mechanistic data that govern the RNA binding capacity of EhCFIm25.

Self-Assembled Peptide-Based System for Mitochondrial-Targeted Gene Delivery: Functional and Structural Insights.

Human mitochondrial dysfunction can lead to severe and often deadly diseases, for which there are no known cures. Although the targeted delivery of therapeutic gene to mitochondria is a promising approach to alleviate these disorders, gene carrier systems for the selective delivery of functional DNA into the mitochondria of living mammalian cells are currently unavailable. Here we rationally developed dual-domain peptides containing DNA-condensing/cell-penetrating/endosome-disruptive and mitochondria-targeting sequences. Secondary structures of the dual-domain peptides were analyzed, and variations in the physicochemical properties (stability, size, and ζ potential) of peptide/DNA complexes were studied as a function of peptide-to-DNA ratio and serum addition. An optimized formulation, identified through qualitative and quantitative studies, fulfills the fundamental prerequisites for mitochondria-specific DNA delivery, successfully transfecting a high proportion (82 ± 2%) of mitochondria in a human cell line with concomitant biocompatibility. Nuclear magnetic resonance studies confirmed the effectiveness of our bipartite peptide design with segregated functions: a helical domain necessary for mitochondrial import and an unstructured region for interaction with DNA involving lysine residues. Further analyses revealed that the lysine-specific interaction assisted the self-organization of the peptide and the DNA cargo, leading to a structural arrangement within the formed complex that is crucial for its biological efficiency. Thus the reported gene vector represents a new and reliable tool to uncover the complexity of mitochondrial transfection.

Surface-displayed glyceraldehyde 3-phosphate dehydrogenase and galectin from Dirofilaria immitis enhance the activation of the fibrinolytic system of the host

Cardiopulmonary dirofilariosis is a cosmopolitan disease caused by Dirofilaria immitis, a filaroid parasite whose adult worms live for years in the vascular system of its host. Previous studies have shown that D. immitis can use their excretory/secretory (ES) and surface antigens to enhance fibrinolysis, which could limit the formation of clots in its surrounding environment. Moreover, several isoforms of the glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and galectin (GAL) were identified in both antigenic extracts as plasminogen-binding proteins. The aim of this work is to study the interaction of the GAPDH and GAL of D. immitis with the fibrinolytic system of the host. This study includes the cloning, sequencing and expression of the recombinant forms of the GAPDH and GAL of D. immitis (rDiGAPDH and rDiGAL) and the analysis of their capacity as plasminogen-binding proteins. The results indicate that rDiGAPDH and rDiGAL are able to bind plasminogen and stimulate plasmin generation by tissue plasminogen activator (tPA). This interaction needs the involvement of lysine residues, many of which are located externally in both proteins as have been shown by the molecular modeling of their secondary structures. In addition, we show that rDiGAPDH and rDiGAL enhance the expression of the urokinase-type plasminogen activator (uPA) on canine endothelial cells in culture and that both proteins are expressed on the surface of D. immitis in close contact with the blood of the host. These data suggest that D. immitis could use the associated surface GAPDH and GAL as physiological plasminogen receptors to shift the fibrinolytic balance towards the generation of plasmin, which might constitute a survival mechanism to avoid the clot formation in its intravascular habitat.

Defined Conjugation of Glycans to the Lysines of CRM197 Guided by their Reactivity Mapping

Systematic characterisation of the reactivity of the lysine moieties in CRM197 towards N-hydroxysuccinimide linkers bearing alkynes or azides is described. This involves two-step conjugation of various glycans to CRM197 by click chemistry in a well-defined manner. By semiquantitative LC-MS/MS analysis of proteolytic digests of the conjugates formed, the reactivity of lysine residues in the protein was mapped and ranked. Computational analysis of the solvent accessibility of each lysine residue (based on the CRM197 crystal structure) established a correlation between reactivity and surface exposure. By this approach, conjugation involving lysine residues (normally a random process) can be controlled. It enables the preparation of lysine-mediated glycoconjugates with improved batch-to-batch reproducibility, thereby producing neo-glycoconjugates with more-consistent biological activity.

Glycation Accelerates Fibrillization of the Amyloidogenic W7FW14F Apomyoglobin

Neurodegenerative diseases are associated with misfolding and deposition of specific proteins, either intra or extracellularly in the nervous system. Advanced glycation end products (AGEs) originate from different molecular species that become glycated after exposure to sugars. Several proteins implicated in neurodegenerative diseases have been found to be glycated in vivo and the extent of glycation is related to the pathologies of the patients. Although it is now accepted that there is a direct correlation between AGEs formation and the development of neurodegenerative diseases, several questions still remain unanswered: whether glycation is the triggering event or just an additional factor acting on the aggregation pathway. To this concern, in the present study we have investigated the effect of glycation on the aggregation pathway of the amyloidogenic W7FW14F apomyoglobin. Although this protein has not been related to any amyloid disease, it represents a good model to resemble proteins that intrinsically evolve toward the formation of amyloid aggregates in physiological conditions. We show that D-ribose, but not D-glucose, rapidly induces the W7FW14F apomyoglobin to generate AGEs in a time-dependent manner and protein ribosylation is likely to involve lysine residues on the polypeptide chain. Ribosylation of the W7FW14F apomyoglobin strongly affects its aggregation kinetics producing amyloid fibrils within few days. Cytotoxicity of the glycated aggregates has also been tested using a cell viability assay. We propose that ribosylation in the W7FW14F apomyoglobin induces the formation of a cross-link that strongly reduces the flexibility of the H helix and/or induce a conformational change that favor fibril formation. These results open new perspectives for AGEs biological role as they can be considered not only a triggering factor in amyloidosis but also a player in later stages of the aggregation process.

Exploring the Mechanism of Selective Noncovalent Adduct Protein Probing Mass Spectrometry Utilizing Site-Directed Mutagenesis To Examine Ubiquitin

Mass spectrometry (MS) is emerging as an additional tool for examining protein structure by way of experiments where structurally related mass changes induced in solution are subsequently detected in the gas phase. Selective noncovalent adduct protein probing (SNAPP) is a recent addition to this type of experiment. SNAPP utilizes noncovalent recognition of lysine residues with 18-crown-6 (18C6) to monitor changes in protein structure. It has been observed that the number of 18C6 adducts that attach to a protein is a function of the structure of the protein. The present work seeks to examine the underlying chemistry which controls the differential attachment of 18C6 to lysine by using ubiquitin as a model system. Ubiquitin is a small protein with a structure that has been well characterized by multiple techniques. Site-directed mutagenesis was used to create a series of ubiquitin mutants where the lysine residues were exchanged for asparagine one at a time. These mutants were then evaluated by SNAPP-MS to determine the relative contribution of each lysine as a binding site for 18C6. It was found that attachment of 18C6 is largely controlled by the strength of intramolecular interactions involving lysine residues. Salt bridges provide the greatest interference, followed by hydrogen bonds. In addition to determining the mechanism for SNAPP, insights are provided about the structure of ubiquitin including confirmation of the existence of two dynamic states for the native structure. These results are discussed in relation to the biological functions of ubiquitin.

Interaction of fucoidan with the proteins of the complement classical pathway

Fucoidan inhibits complement by mechanisms that so far remain to be unraveled, and the objective of this work was to delineate the mode of inhibition by this sulfated polysaccharide. For that purpose, low molecular weight fractions of algal ( Ascophyllum nodosum) fucoidan containing the disaccharide unit [→3)-α- l-Fuc(2SO 3 −)-(1→4)-α- l-Fuc(2,3diSO 3 −)-(1→] n have been studied. Gel co-affinity electrophoresis and a new affinity capillary electrophoresis (ACE) method have been implemented to characterize fucoidan-complement protein complexes. Fucoidan binds C1q, likely to its collagen-like region through interactions involving lysine residues, and then prevents the association of the C1r 2–C1s 2 subunit, required to form the fully active C1. In addition to C1q, fucoidan forms a complex with the protein C4 as observed by ACE. The fucoidan inhibits the first steps of the classical pathway activation that is of relevance in view of the proinflammatory effects of the subsequent products of the cascade. This study shows that a high level of inhibitory activity can be achieved with low molecular weight carbohydrate molecules and that the potential applicability of fucoidan oligosaccharides for therapeutic complement inhibition is worthy of consideration.

Calpain Mutants with Increased Ca2+ Sensitivity and Implications for the Role of the C2-like Domain

The ubiquitous calpain isoforms (mu- and m-calpain) are Ca(2+)-dependent cysteine proteases that require surprisingly high Ca(2+) concentrations for activation in vitro ( approximately 50 and approximately 300 microm, respectively). The molecular basis of such a high requirement for Ca(2+) in vitro is not known. In this study, we substantially reduced the concentration of Ca(2+) required for the activation of m-calpain in vitro through the specific disruption of interdomain interactions by structure-guided site-directed mutagenesis. Several interdomain electrostatic interactions involving lysine residues in domain II and acidic residues in the C(2)-like domain III were disrupted, and the effects of these mutations on activity and Ca(2+) sensitivity were analyzed. The mutation to serine of Glu-504, a residue that is conserved in both mu- and m-calpain and interacts most notably with Lys-234, reduced the in vitro Ca(2+) requirement for activity by almost 50%. The mutation of Lys-234 to serine or glutamic acid resulted in a similar reduction. These are the first reported cases in which point mutations have been able to reduce the Ca(2+) requirement of calpain. The structures of the mutants in the absence of Ca(2+) were shown by x-ray crystallography to be unchanged from the wild type, demonstrating that the increase in Ca(2+) sensitivity was not attributable to conformational change prior to activation. The conservation of sequence between mu-calpain, m-calpain, and calpain 3 in this region suggests that the results can be extended to all of these isoforms. Whereas the primary Ca(2+) binding is assumed to occur at EF-hands in domains IV and VI, these results show that domain II-domain III salt bridges are important in the process of the Ca(2+)-induced activation of calpain and that they influence the overall Ca(2+) requirement of the enzyme.

Regulation of E2F1 activity by acetylation.

During the G(1) phase of the cell cycle, an E2F-RB complex represses transcription, via the recruitment of histone deacetylase activity. Phosphorylation of RB at the G(1)/S boundary generates a pool of 'free' E2F, which then stimulates transcription of S-phase genes. Given that E2F1 activity is stimulated by p300/CBP acetylase and repressed by an RB-associated deacetylase, we asked if E2F1 was subject to modification by acetylation. We show that the p300/CBP-associated factor P/CAF, and to a lesser extent p300/CBP itself, can acetylate E2F1 in vitro and that intracellular E2F1 is acetylated. The acetylation sites lie adjacent to the E2F1 DNA-binding domain and involve lysine residues highly conserved in E2F1, 2 and 3. Acetylation by P/CAF has three functional consequences on E2F1 activity: increased DNA-binding ability, activation potential and protein half-life. These results suggest that acetylation stimulates the functions of the non-RB bound 'free' form of E2F1. Consistent with this, we find that the RB-associated histone deacetylase can deacetylate E2F1. These results identify acetylation as a novel regulatory modification that stimulates E2F1's activation functions.

Structure and Mechanism of Formation of Human Lens Fluorophore LM-1: RELATIONSHIP TO VESPERLYSINE A AND THE ADVANCED MAILLARD REACTION IN AGING, DIABETES, AND CATARACTOGENESIS

Human lens crystallins become progressively yellow-brown pigmented with age. Both fluorescent and non-fluorescent protein adducts and cross-links are formed, many of which result from the advanced Maillard reaction. One of them, LM-1, is a blue fluorophore that was earlier tentatively identified as a cross-link involving lysine residues (1). A two-step chromatographic system was used to unequivocally identify and quantitatively prepare a synthetic fluorescent cross-link with lysine residues that had identical UV, fluorescent, and chromatographic properties with both acetylated and non-acetylated LM-1. Proton, (13)C NMR, and molecular mass of the synthetic compound were identical with vesperlysine A, a fluorescent cross-link discovered by Nakamura et al. (2). The fragmentation patterns of vesperlysine A and LM-1 were identical as determined by NMR/mass spectrometry. Lenticular levels of vesperlysine A increase curvilinearly with age and reach 20 pmol/mg at 90 years. Levels correlate with degree of lens crystallin pigmentation and fluorescence and are increased in diabetes, in contrast to N(epsilon)-(carboxymethyl)lysine and pentosidine. Ascorbate, D-pentoses, and D-threose, but neither D-glucose under oxidative conditions, DL-glyceraldehyde, methylglyoxal, glyoxal, nor glycolaldehyde, are precursors. However, addition of C-2 compounds greatly catalyzes vesperlysine A formation from ribose. Thus, vesperlysine A/LM-1 is a novel product of the advanced Maillard reaction in vivo and a specific marker of a diabetic process in the lens that is different from glyco- and lipoxidation.

Involvement of lysine residues in the gating of the ryanodine receptor/Ca2+-release channel of skeletal muscle sarcoplasmic reticulum.

In this study, the modification of skeletal muscle ryanodine receptor (RyR)/Ca2+-release channel with 7-chloro-4-nitrobenzo-2-oxa-1,3,-diazole (Nbd-Cl) demonstrates that lysyl residues are involved in the channel gating. Nbd-Cl was found to have a dual effect: stimulation and inhibition of ryanodine binding and single channel activities. Nbd-Cl, in a time-dependent manner, first stimulated and subsequently inhibited ryanodine binding to both membrane-bound and purified RyR. Incubation of sacroplasmic reticulum membranes with Nbd-Cl for 5-20 s resulted in enhanced ryanodine-binding activity by 2-4-fold due, to an increased binding affinity by about tenfold, with no effect on the total binding sites (Bmax). However, under prolonged incubation (5-20 min), Nbd-Cl strongly inhibited ryanodine binding by decreasing the Bmax with no effect on the binding affinity. Similar effects of stimulation and inhibition by Nbd-Cl were obtained with single channel activity of RyR reconstituted into planar lipid bilayer. Nbd-Cl initially (within a few seconds) activated the channel to a highly open state, then (within a few minutes) inactivated it to the completely closed state. Nbd-Cl-modified protein, as assayed by ryanodine binding or single channel activities, was stable against thiolysis by dithiothreitol, suggesting Nbd-Cl modification of lysyl residues. Evidence from absorption and fluorescence excitation and emission spectra also demonstrated that lysyl residues in RyR were modified by Nbd-Cl. Spectrophotometric data were used to estimate a ratio of up to 1 mol Nbd bound/mol RyR (tetramer) and up to 4 mol Nbd bound per mol RyR (tetramer) for Nbd-Cl stimulated and inhibited RyR activities, respectively. The results clearly indicate the involvement of two classes of lysyl residues in RyR activity. Modification by Nbd-Cl of the fast-reacting group led to stimulation of ryanodine binding and single channel activities, while modification of the slow-reacting group resulted in inhibition of these activities. Thus, the involvement of lysine residues in the gating of the RyR channel is proposed.

Characterization and regulation of a chloride channel from bovine tracheal epithelium.

1. The patch-clamp technique was used to characterize chloride channels from the apical membranes of bovine tracheal epithelial cells. Application of GTP gamma S or NaF to excised patches revealed the existence of a novel type of Cl- channel regulated by G-proteins in a membrane-delimited manner. 2. The channel had a linear current-voltage relationship, with a conductance of 100-120 pS. Its open probability was independent of voltage. 3. The channel was highly anion selective (permeability ratio, PNa/PCl = 0.06 +/- 0.04) and had the halide permeability sequence: I- > Br- > or = Cl- > F-, corresponding to the Eisenman I sequence. This suggested that neither ionic size nor diffusion rate determined ion permeation through the channel. 4. The mole fraction behaviour was studied using fluoride and chloride ions. Mixtures of ions produced currents that would be expected from the linear combination of the two ions acting independently, indicating relatively simple permeation through the pore and compatible with a single ion binding site. 5. The channel was inhibited by the stilbene disulphonates SITS (4-acetamido-4'-isothiocyanatostilbene-2, 2'-disulphonic acid) and DNDS (4,4'-dinitrostilbene-2,2'-sulphonic acid). SITS introduced voltage dependence to channel gating and indicated the possible involvement of lysine residues in the channel permeation pathway. 6. NaF was unable to activate Cl- channels in the presence of the aluminum chelator, deferoxamine mesylate. This indicates that Al3+ ions play an important role in chloride channel activation by fluoride. NaF activation was not dependent on the presence of calcium ions. 7. The channel was insensitive to alkaline phosphatase and to the specific inhibitors of protein phosphatase types I and 2A, okadaic acid and calyculin A. 8. The channels could be activated by GTP gamma S or by NaF in the presence of the phospholipase A2 inhibitor quinacrine, indicating that this enzyme is not involved in channel regulation.

Immunochemical characterization of two thyroid-stimulating hormone beta-subunit epitopes.

The epitopes of human thyroid-stimulating hormone (hTSH) recognized by two murine monoclonal antibodies (MAbs), designated MAb 279 and MAb 299, have been characterized. These MAbs are highly specific for the beta-subunit of TSH. The epitope recognized by MAb 279 appears to be completely conserved between bovine and human TSH and partially conserved in the porcine species. The TSH beta-subunit epitope recognized by MAb 299 is only partially conserved between the human, bovine and porcine species. Both MAbs are capable of inhibiting the binding of TSH to its receptor in a TSH radioreceptor assay, indicating that the epitopes either coincide or are located close to the TSH beta-subunit receptor-binding sites. The carbohydrate moieties of the TSH beta-subunit appear to play little or no role in the epitope recognition by MAb 279 or MAb 299 while the integrity of the disulphide bonds are essential. The epitopic recognition may also involve lysine residues, as determined by the immunoreactivity with both MAbs following citraconylation of TSH. In addition, the amino acid sequence region between residues bTSH beta 34-44 could be excised by trypsin digestion of bovine TSH beta (bTSH beta) without eliminating epitopic recognition by either MAb. These results provide further insight into the relationship between the structure of the TSH beta-subunit epitopes and location of the receptor-binding sites.

Shared active sites of fructose-1,6-bisphosphatase. Arginine 243 mediates substrate binding and fructose 2,6-bisphosphate inhibition.

The active site of pig kidney fructose-1,6-bisphosphatase (EC 3.1.3.11) is shared between subunits, Arg-243 of one chain interacting with fructose-1,6-bisphosphate or fructose-2,6-bisphosphate in the active site of an adjacent chain. In this study, Arg-243 was replaced by alanine using techniques of site-specific mutagenesis and the cloned pig kidney enzyme expressed in Escherichia coli. Compared with wild-type enzyme, kinetic parameters of the altered enzyme characterizing catalytic efficiency, magnesium binding, and inhibition by AMP differed but by less than an order of magnitude; affinity for substrate fructose 1,6-bisphosphate was 10-fold poorer, and affinity for inhibitor fructose 2,6-bisphosphate was 1000-fold poorer. Molecular dynamics simulations were undertaken to determine possible alterations in active sites of the enzyme due to replacement of Arg-243 by Ala and suggested that in the mutant enzyme loss of one cationic group leads to reorganization of the active site especially involving lysine residues 269 and 274. The differences in properties of the mutant enzyme indicate the key importance of Arg-243 in the function of fructose-1,6-bisphosphatase and confirm on a functional basis the shared active site in this important metabolic enzyme.

Polymorphic forms of Lp(a) with different structural and functional properties: cold-induced self-association and binding to fibrin and lysine-Sepharose

Two different Lp(a) polymorphs were isolated from the same individual and shown to have important differences both in their solution properties and in interaction with lysine Sepharose and fibrin. One Lp(a) particle (d-Lp(a)) with a large apo(a) isoform had a density of 1.087 g/ml and a molecular weight of 3.17 million, while the other Lp(a) particle with a small apo(a) isoform having a mobility faster than that of apoB was larger and had a molecular weight of 3.75 million and a density of 1.054 g/ml. D-Lp(a) underwent cold-induced self-association and also had a higher affinity for lysine Sepharose, whereas the other Lp(a) polymorph did not. Both Lp(a) particles bound fibrin via two different binding sites, one of which involved fibrin lysine residues which are also recognized by plasminogen. Lysine-mediated binding of d-Lp(a) by fibrin was ten times stronger than that of the other Lp(a) particle, whereas non-lysine-mediated binding of either Lp(a) species by fibrin was of equal strength. At saturation, 80% of d-Lp(a) bound fibrin at sites that did not involve lysine residues, whereas only 33% of the other Lp(a) polymorph bound to these sites. These findings indicate that the binding of Lp(a) to fibrin is more complex than previously thought and imposes another layer of difficulty on our understanding of how Lp(a) regulates and/or impairs fibrinolysis.

Bicarbonate uptake in the marine macroalga Ulva sp. is inhibited by classical probes of anion exchange by red blood cells

We demonstrate in this work that HCO inf3 sup− uptake in the marine macroalga Ulva sp. features functional resemblances to anion transport mediated by anion exchangers of mammalian cell membranes. The evidence is based on (i) competitive inhibition of photosynthesis by the classical red-blood-cell anion-exchange blockers 4,4′-dinitrostilbene-2,2′-disulfonate and 4-nitro-4′-isothiocyanostilbene-2,2′-disulfonate under conditions where HCO inf3 sup− , but not CO2, was the inorganic carbon form taken up; (ii) inhibition of HCO inf3 − uptake by pyridoxal phospate, indicating the involvement of lysine residues in the binding/translocation of HCO inf3 sup− ; and (iii) inhibition of HCO inf3 sup− (but not of CO2) uptake by exofacial trypsin treatments, indicating the functional involvement of a plasmalemma protein. It is suggested that HCO inf3 sup− uptake mediated by such a putative anion transporter can be a fundamental step in providing inorganic carbon for the CO2-concentrating system of marine marcoalgae in an environment where the HCO inf3 sup− concentration is high, but the CO2 concentration and rates of uncatalyzed HCO inf3 sup− dehydration are low.