An essential arginyl residue at the nucleotide binding site of creatine kinase.

Treatment of rabbit muscle creatine kinase (EC 2.4.3.2) with either butanedione in borate buffer or phenylglyoxal in Veronal buffer decreases enzymatic activity correlating with the modification of a single arginyl residue per subunit of the dimeric enzyme. Very little activity is lost when modification is performed in the presence of MgATP or MgADP. Nucleotide binding to the modified enzyme is virtually abolished as determined by ultraviolet difference spectroscopy. The data suggest that an arginyl residue plays an essential role in the enzymatic mechanism of creatine kinase, probably as a recognition site for the negatively charged oligophosphate moiety of the nucleotide.     

Essential arginyl residues in fructose-1,6-bisphosphatase.

Modification of pig kidney fructose-1,6-bisphosphatase with 2,3-butanedione in borate buffer (pH 7.8) leads to the loss of the activation of the enzyme by monovalent cations, as well as to the loss of allosteric adenosine 5'-monophosphate (AMP) inhibition. In agreement with the results obtained for the butanedione modification of arginyl residues in other enzymes, the effects of modification can be reversed upon removal of excess butanedione and borate. Significant protection to the loss of K+ activation was afforded by the presence of the substrate fructose 1,6-bisphosphate, whereas AMP preferentially protected against the loss of AMP inhibition. The combination of both fructose 1,6-bisphosphate and AMP fully protected against the changes in enzyme properties on butanedione treatment. Under the latter conditions, one arginyl residue per mole of enzyme subunit was modified, whereas three arginyl residues were modified by butanedione under conditions leading to the loss of both potassium activation and AMP inhibition. Thus, the modification of two arginyl residues per subunit would appear to be responsible for the change in enzyme properties. The present results, as well as those of a previous report on the subject (Marcus, F. (1975), Biochemistry 14, 3916-3921) support the conclusion that one arginyl residue per subunit is essential for monovalent cation activation, and another arginyl residue is essential for AMP inhibition. A likely role of the latter residue could be its involvement in the binding of the phosphate group of AMP.     

Essential arginyl residues in yeast enolase.

Yeast enolase is rapidly inactivated by butanedione in borate buffer, complete inactivation correlating with the modification of 1. 8 arginyl residues per subunit. Protection against inactivation is provided by either an equilibrium mixture of substrates or inorganic phosphate, a competitive inhibitor of the enzyme. Complete protection by substrates correlates with the shielding of 1. 3 arginyl residues per subunit, while phosphate protects 1. 0 arginyl residue per subunit from modification.     

Evidence of essential arginyl residues in rabbit muscle pyruvate kinase.

Rabbit muscle pyruvate kinase is inactivated by 2,3-butanedione in borate buffer. The inactivation follows pseudo-first-order kinetics with a calculated second-order rate constant of 4.6 m^?1 min^?1. The modification can be reversed with almost total recovery of activity by elimination of the butanedione and borate buffer, suggesting that only arginyl groups are modified; this result agrees with the loss of arginine detected by amino acid analysis of the modified enzyme. Using the kinetic data, it was estimated that the reaction of a single butanedione molecule per subunit of the enzyme is enough to completely inactivate the protein. The inactivation is partially prevented by phosphoenolpyruvate in the presence of K⁺ and Mg²⁺, but not by the competitive inhibitors lactate and bicarbonate. These findings point to an essential arginyl residue being located near the phosphate binding site of phosphoenolpyruvate.     

Preparation, characterization and biocompatibility of electrospinning heparin-modified silk fibroin nanofibers

In this study, the electrospun silk fibroin nanofibrous scaffolds were modified with heparin by grafting after plasma treatment and blending electrospinning. Morphology, microstructure, chemical composition and grafting efficiency of the heparin-modified silk fibroin nanofibrous scaffolds were characterized to evaluate the effect of modification by means of scanning electron microscopy (SEM), Fourier transform infrared spectra (FTIR) and X-ray photoelectron spectrometer (XPS). The results showed that the heparin was successfully introduced to the silk fibroin nanofibrous scaffolds by both the two kinds of modification, and there was a hydrogen bonding between the silk fibroin and heparin. Moreover, the hydrophilicity, O-containing groups and negative charge density of the heparin-modified scaffolds were enhanced. In vitro coagulation time tests showed that the activated partial thromboplastin time (APTT), prothrombin time (PT) and thrombin time (TT) of the heparin-modified scaffolds were much higher than those of the pure silk fibroin scaffolds. L929 fibroblasts and EVCs spread and proliferated better on the heparin-modified scaffolds than on the pure silk fibroin scaffolds. Macrophages, neutrophils and lymphocytes were not observed in the heparin-modified scaffolds, which indicated that the modified scaffolds could induce minor inflammation in vivo. The results indicated that the electrospun heparin-modified silk fibroin nanofibrous scaffolds could be considered as ideal candidates for tissue engineering scaffolds.     

Role of arginyl residues in yeast hexokinase PII.

Yeast hexokinase PII is rapidly inactivated (assayed at pH 8.0) by either butanedione in borate buffer or phenylglyoxal, reagents which are highly selective for the modification of arginyl residues. MgATP alone offers no protection against inactivation, consistent with low affinity of hexokinase for this nucleotide in the absence of sugar. Glucose provides slight protection against inactivation, while the combined presence of glucose and MgATP gives significant protection, suggesting that modified arginyl residues may lie at the active site, possibly serving to bind the anionic polyphosphate of the nucleotide in the ternary enzyme:sugar:nucleotide complex. Extrapolation to complete inactivation suggests that inactivation by butanedione correlates with the modification of 4.2 arginyl residues per subunit, and complete protection against inactivation by the combined presence of glucose and MgATP correlates with the protection of 2 to 3 arginyl residues per subunit. When the modified enzyme is assayed at pH 6.5, significant activity remains. However, modification by butanedione in borate buffer abolishes the burst-type slow transient process, observed when the enzyme is assayed at pH 6.5, to such an extent that after extensive modification the kinetic assays are characterized by a lag-type slow transient process. But even after extensive modification, hexokinase PII still demonstrates negative cooperativity with MgATP and is still strongly activated by citrate when assayed at pH 6.5.     

Evidence for an essential arginyl residue in bovine milk gamma-glutamyltransferase

Treatment of bovine milk gamma-glutamyltransferase with 2,3-butanedione in borate buffer markedly inactivates its gamma-glutamyltransferase activity. Inactivation is prevented by a combination of the gamma-glutamyl donor and acceptor substrates, glutathione, and glycylglycine, but less effectively by only one of them. Serine plus borate of maleate provides no protection against the inactivation. Amino acid analysis of the enzyme treated with butanedione in the presence and absence of the protecting substrate combination indicates that complete inactivation correlates with the modification of a single arginyl residue per molecule. The residue modified is associated with the smaller subunit of the two equal subunits which comprise the enzyme. The butanedione-treated enzyme retains a hydrolytic activity, another but less significant catalytic function of the enzyme. The results indicate that the arginyl residue is involved in recognizing the anionic moiety of the acceptor and in binding it to the acceptor site located on the smaller subunit of the enzyme.     

Essential arginyl residues in yeast phosphoglyceromutase.

Inactivation of yeast phosphoglyceromutase (tetramer) with 1,2-cyclohexanedione correlates with the modification of six arginyl residues per mole of the enzyme. Protection experiments using 3-phosphoglycerate suggest that four arginyl residues (one residue per subunit) are involved in the binding of the substrate to the enzyme. The modified enzyme reversibly regained its activity upon incubation with hydroxylamine. The reactivity of lysyl residues which have been shown to be involved in the active site is markedly reduced in the enzyme inactivated with 1,2-cyclohexanedione, indicating that the lysyl and arginyl residues are in close proximity in the active site.     

Functional consequences of modifying highly reactive arginyl residues of fructose 1,6-bisphosphatase. Loss of monovalent cation activation.

Modification of pig kidney fructose 1,6-bisphosphatase with 2,3-butanedione (in the presence of AMP) results in the loss of activation of the enzyme by monovalent cations. Under these conditions about 8 arginyl residues per mole of enzyme were modified. No other residues were modified. No loss of monovalent cation activation occurs when modification with 2,3-butanedione is carried out in the presence of AMP plus the substrate fructose 1,6-bisphosphate and 3.2 less arginyl residues were modified. Since fructose 1,6-bisphosphatase contains 4 subunits, it is suggested that one arginyl residue per subunit plays an essential role in monovalent cation activation of the enzyme. Studies on sulfhydryl group reactivity toward 5,5'-dithiobis(2-nitrobenzoic acid) explain the protection exerted by fructose 1,6-bisphosphate against the loss of monovalent cation activation in terms of an enzyme conformational change induced by substrate, which makes unreactive the essential arginyl residue. The results of the present paper, as well as previous evidence, are discussed in terms of the mechanism of monovalent cation activation of fructose 1,6-biphosphatase.     

New silk protein: modification of silk protein by gene engineering for production of biomaterials.

The interest in silk fibroin morphology and structure have increased due to its attractiveness for bio-related applications. Silk fibers have been used as sutures for a long time in the surgical field, due to the biocompatibility of silk fibroin fibers with human living tissue. In addition, it has been demonstrated that silk can be used as a substrate for enzyme immobilization in biosensors. A more complete understanding of silk structure would provide the possibility to further exploit silk fibroin for a wide range of new uses, such as the production of oxygen-permeable membranes and biocompatible materials. Silk fibroin-based membranes could be utilized as soft tissue compatible polymers. Baculovirus-mediated transgenesis of the silkworm allows specific alterations in a target sequence. Homologous recombination of a foreign gene downstream from a powerful promoter, such as the fibroin promoter, would allow the constitutive production of a useful protein in the silkworm and the modification of the character of silk protein. A chimeric protein consisted of fibroin and green fluorescent protein was expressed under the control of fibroin in the posterior silk gland and the gene product was spun into the cocoon layer. This technique, gene targeting, will lead to the modification and enhancement of physicochemical properties of silk protein.     

Phosphoglycerate mutase has essential arginyl residues

Phosphoglycerate mutase is inactivated by butanedione in borate buffer. Inactivation by 0.13 mM reagent correlates with the modification of one arginyl residue per subunit, and is prevented by either 2, 3-diphosphoglycerate or 3-phosphoglycerate. With 0.50 mM butanedione, inactivation is accompanied by the modification of three arginyl residues per subunit, two of which are protected by the combined presence of cofactor and substrate.     

Synthesis, characterization and immunogenicity of silk fibroin-L-asparaginase bioconjugates

L-asparaginase (ASNase) is one basic drug in the treatment of acute lymphoblastic leukemia (ALL). Because its half-life time is too short and it is easy to arouse allergic reaction, use in practical clinic is considerably limited. Silk fibroin (SF) with different molecular mass from 40 to 120 kDa is a natural biocompatible protein and could be used as a novel bioconjugate for enzyme modification to overcome its usual shortcomings mentioned above. When the enzyme was bioconjugated covalently with the water-soluble fibroin by glutaraldehyde, the enzyme kinetic properties and immune characteristics in vivo of the resulting silk fibroin-L-asparaginase (SF-ASNase) bioconjugates were investigated in detail. The results show that the modified ASNase was characterized by its higher residual activity (nearly 80%), increased heat and storage stability and resistance to trypsin digestion, and its longer half-life (63 h) than that of intact ASNase (33 h). The abilities of intact and modified ASNases to arouse allergic reaction are 2(4) and 2(1) antibody titers, respectively. Bioconjugation of silk fibroin significantly helps to reduce the immunogenicity and antigenicity of the enzyme. The apparent Michaelis constants of the modified ASNase (K(m(app))=0.844 x 10(-3)mol L(-1)) was approximately six times lower than that of enzyme alone, which suggests that the affinity of the enzyme to substrate l-asparagine elevated when bioconjugated covalently with silk fibroin. SF-ASNase bioconjugates could overcome the common shortcomings of the native form. Therefore, the modified ASNase coupled with silk fibroin has the potential values of being studied and developed as a new bioconjugate drug.     

Localization of arginyl residues modified with butanedione in glyceraldehyde-3-phosphate dehydrogenase from pig muscle

Glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.12) from pig muscle was inactivated by incubation with butanedione in triethanolamine buffer, pH 8.3. The inactivation was reversible after short treatment with butanedione; it became irreversible after 12-15 h, with a concomitant loss of two arginyl residues per subunit. The modified enzyme was digested with TPCK-trypsin and the peptides were purified by chromatography and electrochromatography. Two new peptides were obtained as the result of modification. From their partially determined sequence the modified arginyl residues were identified as Arg-13 and Arg-231 in the primary structure of pig muscle enzyme.     

Phosphorylation of silk fibroins improves the cytocompatibility of silk fibroin derived materials: A platform for the production of tuneable material

Silk fibroin demonstrates great biocompatibility and is suitable for many biomedical applications, including tissue engineering and regenerative medicine. Current research focuses on manipulating the physico‐chemical properties of fibroin, and examining the effect of this manipulation on firobin's biocompatibility. Regenerated silk fibroin was modified by in vitro enzymatic phosphorylation and cast into films. Films were produced by blending, at several ratios, the phosphorylated and un‐phosphorylated fibroin solutions. Fourier transform infra‐red spectroscopy was used to determine the specific P–OH vibration peak, confirming the phosphorylation of the regenerated silk fibroin solution. Differential scanning calorimetry showed that phosphorylation altered the intra‐ and inter‐molecular interactions. Further experiments demonstrated that phosphorylation can be used to tailor the hydrophylicity/hydrophobicity ratio as well as the crystalinity of silk fibroin films. Release profiling of a model drug was highly dependent on silk modification level. Cytotoxicity assays showed that exposure to lixiviates of phosphorylated films only slightly affected cellular metabolism and proliferation, although direct contact resulted in a strong direct correlation between phosphorylation level and cell proliferation. This new method for tuning silk biomaterials to obtain specific structural and biochemical features can be adapted for a wide range of applications. Phosphorylation of silk fibroins may be applied to improve the cytocompatibility of any silk‐based device that is considered to be in contact with live animals or human tissues.     

New silk protein: modification of silk protein by gene engineering for production of biomaterials

The interest in silk fibroin morphology and structure have increased due to its attractiveness for bio-related applications. Silk fibers have been used as sutures for a long time in the surgical field, due to the biocompatibility of silk fibroin fibers with human living tissue. In addition, it has been demonstrated that silk can be used as a substrate for enzyme immobilization in biosensors. A more complete understanding of silk structure would provide the possibility to further exploit silk fibroin for a wide range of new uses, such as the production of oxygen-permeable membranes and biocompatible materials. Silk fibroin-based membranes could be utilized as soft tissue compatible polymers. Baculovirus-mediated transgenesis of the silkworm allows specific alterations in a target sequence. Homologous recombination of a foreign gene downstream from a powerful promoter, such as the fibroin promoter, would allow the constitutive production of a useful protein in the silkworm and the modification of the character of silk protein. A chimeric protein consisted of fibroin and green fluorescent protein was expressed under the control of fibroin in the posterior silk gland and the gene product was spun into the cocoon layer. This technique, gene targeting, will lead to the modification and enhancement of physicochemical properties of silk protein.     

Yeast 3-phosphoglycerate kinase. Essential arginyl residues at the 3-phosphoglycerate binding site.

Yeast 3-phosphoglycerate kinase (ATP:3-phospho-D-glycerate 1-phospho-transferase, EC 2.7.2.3) is inactivated by phenylglyoxal. Loss of activity correlates with the modification of two arginyl residues, both of which are protected by all of the substrates. The modification is not accompanied by any significant conformational change as determined by optical rotatory dispersion. Ultraviolet difference spectrophotometry indicates that the inactivated enzyme retains its capacity for binding the nucleotide substrates whereas the spectral perturbation characteristic of 3-phosphoglycerate binding is abolished in the modified enzyme. The data suggest that at least one of the two essential arginyl residues is located at or near the 3-phosphoglycerate binding site. A likely role of this residue could be its interaction with the negatively charged phosphate or carboxylate groups of 3-phosphoglycerate.     

Implication of arginyl residues in mRNA binding to ribosomes

Modification of Escherichia coli robosomes with phenylglyoxal and butanedione, protein reagents specific for arginyl residues, inactivates polypeptide polymerization, assayed as poly(U)-dependent polyphenylalanine synthesis, and the binding of poly(U). Inactivation is produced by modification of the 30-S subunit. Both the RNA and the protein moieties of 30-S subunits are modified by phenylglyoxal, and modification of either of them is accompanied by inactivation of polypeptide synthesis. Modification of only the split proteins released from 30-S subunits by prolonged dialysis against a low-ionic-strength buffer, which contain mainly protein S1, produces inhibition of poly(U) binding and inactivation of polypeptide synthesis. Amino acid analysis of the modified split proteins showed a significant modifications of arginyl residues. These results indicate that the arginyl residues of a few 30-S proteins might be important in the interaction between mRNA and the 30-S subunit, which agrees with the general role assigned to the arginyl residues of proteins as the positively charged recognition site for anionic ligands.     

Arginyl and histidyl groups are essential for organic anion exchange in renal brush-border membrane vesicles

The effect of side chain modification on the organic anion exchanger in the renal brush-border membrane was examined to identify what amino acid residues constitute the substrate binding site. One histidyl-specific reagent, diethyl pyrocarbonate (DEPC), and 2 arginyl-specific reagents, phenylglyoxal and 2,3-butanedione, were tested for their effect on the specifically mediated transport of p-amino[3H]hippurate (PAH), a prototypic organic anion. The specifically mediated transport refers to the difference in the uptake of [3H]PAH in the absence and presence of a known competitive inhibitor, probenecid, and was examined in brush-border membrane vesicles isolated from the outer cortex of canine kidneys. The experiments were performed utilizing a rapid filtration assay. DEPC, phenylglyoxal, and 2,3-butanedione inactivated the specifically mediated PAH transport, i.e. probenecid inhibitable transport with IC50 values of 160, 710, and 1780 microM, respectively. The rates of PAH inactivation by DEPC and phenylglyoxal were suggestive of multiple pseudo first-order reaction kinetics and were consistent with a reaction mechanism whereby more than 1 arginyl or histidyl residue is inactivated. Furthermore, PAH (5 mM) did not affect the rate of phenylglyoxal inactivation. In contrast, PAH (5 mM) affected the rate of DEPC inactivation. The modification by DEPC was specific for histidyl residues since transport could be restored by treatment with hydroxylamine. The results demonstrate that histidyl and arginyl residues are essential for organic anion transport in brush-border membrane vesicles. We conclude that the histidyl residue constitutes the cationic binding site for the anionic substrate, whereas the arginyl residue(s) serves to guide the substrate to or away from the histidyl site.     

Role of arginine residues in the structure and biological activity of botulinum neurotoxin types A and E

When nicked types A and E as well as the unnicked (i.e., single chain) type E botulinum neurotoxins were treated with 1,2-cyclo-hexanedione, which specifically modifies the arginine residues in 0.2 M borate buffer, pH 8.0 i) both the nicked and unnicked neurotoxins were detoxified, ii) the unnicked single chain neurotoxin became resistant to nicking with trypsin, and iii) the serological reactivity of type A (type E was not tested) was altered. Reversal of the arginine modification partially restored toxicity. In the electroimmunodiffusion test the modified type A neurotoxin appeared as 2 cones; the height of one cone increased and the other decreased as the modification reaction progressed. These results indicate that i) at least one arginine residue is involved in maintaining the toxigenic structure of types A and E neurotoxins; ii) the site of nicking in type E is an arginyl bond; and iii) arginine residue is critical for at least one antigenic determinant of type A neurotoxin.     

Surface Modification and Characterisation of Silk Fibroin Fabric Produced by the Layer-by-Layer Self-Assembly of Multilayer Alginate/Regenerated Silk Fibroin

Silk-based medical products have a long history of use as a material for surgical sutures because of their desirable mechanical properties. However, silk fibroin fabric has been reported to be haemolytic when in direct contact with blood. The layer-by-layer self-assembly technique provides a method for surface modification to improve the biocompatibility of silk fibroin fabrics. Regenerated silk fibroin and alginate, which have excellent biocompatibility and low immunogenicity, are outstanding candidates for polyelectrolyte deposition. In this study, silk fabric was degummed and positively charged to create a silk fibroin fabric that could undergo self-assembly. The multilayer self-assembly of the silk fibroin fabric was achieved by alternating the polyelectrolyte deposition of a negatively charged alginate solution (pH = 8) and a positively charged regenerated silk fibroin solution (pH = 2). Finally, the negatively charged regenerated silk fibroin solution (pH = 8) was used to assemble the outermost layer of the fabric so that the surface would be negatively charged. A stable structural transition was induced using 75% ethanol. The thickness and morphology were characterised using atomic force microscopy. The properties of the self-assembled silk fibroin fabric, such as the bursting strength, thermal stability and flushing stability, indicated that the fabric was stable. In addition, the cytocompatibility and haemocompatibility of the self-assembled silk fibroin fabrics were evaluated. The results indicated that the biocompatibility of the self-assembled multilayers was acceptable and that it improved markedly. In particular, after the self-assembly, the fabric was able to prevent platelet adhesion. Furthermore, other non-haemolytic biomaterials can be created through self-assembly of more than 1.5 bilayers, and we propose that self-assembled silk fibroin fabric may be an attractive candidate for anticoagulation applications and for promoting endothelial cell adhesion for vascular prostheses.