Isolation and characterization of a basic encephalitogenic protein from the central nervous system of sheep.

A basic protein has been purified from sheep brain. The purified protein sedimented in the analytical centrifuge at 56,000 r.p.m. as an homogenous product. This protein induced an allergic encephalitis when injected into guinea pigs. Some physiochemical properties of the protein were studied: the sedimentation coefficient was 1.52 and the molecular weight was 20,000 +/- 2,000, as estimated by electrophoresis in acrylamide gels containing SDS and urea; the specific extinction coefficient (see article) was 6.01 +/- 0.20. The aminoacid composition of the molecule was determined and its most prominent aspects are a high content of arginine and lysine, the presence of a single tryptophan, the total absence of cysteine and cystine and a blocked N-terminal residue. All these properties are very close to those of human and bovine encephalitogenic proteins.     

Surface lysine and tyrosine residues are required for interaction of the major herpes simplex virus type 1 DNA-binding protein with single-stranded DNA.

Modification of the herpes simplex virus type 1 major DNA-binding protein (ICP8) with reagents and conditions specific for arginine, lysine, and tyrosine residues indicates that surface lysine and tyrosine residues are required for the interaction of this protein with single-stranded DNA. Modification of either of these two amino acids resulted in a loss and/or modification of binding activity as judged by nitrocellulose filter assays and gel shift. Modification specific for arginine residues did not affect binding within the limits of the assays used. Finally, quenching of the intrinsic tryptophan fluorescence of ICP8 in the presence of single-stranded DNA either suggests involvement of this amino acid in the binding reaction or reflects a conformational change in the protein upon binding.     

柞蚕抗菌肽D中精氨酸、赖氨酸和色氨酸等氨基酸残基的化学修饰

用不同的化学试剂修饰了柞蚕抗菌肽D分子中的色氨酸、精氨酸和赖氨酸等氨基酸残基。NBS修饰抗菌肽D,以及氨肽酶M对抗菌肽D作用的结果表明色氨酸残基对抗菌肽D抑制E.coli D31的作用影响不大。CHD和MLH对精氨酸和赖氨酸残基的修饰,导致抗菌肽D失去抑制E.coli的作用,但可逆地消除CHD和MLH的修饰作用后,抗菌肽D恢复了对E.coli D31的抑菌作用。这些结果初步认为,抗菌肽D抑菌作用与分子中的荷电性有关,改变了分子的电荷,也就同时失去了其抑菌功能。 此外,对精氨酸残基修饰的结果还表明,抗菌肽D的免疫原性与精氨酸残基有关。但是,抗菌肽D的免疫决定簇与其生物活性中心并不完全平行。     

A study on the effect of surface lysine to arginine mutagenesis on protein stability and structure using green fluorescent protein

Two positively charged basic amino acids, arginine and lysine, are mostly exposed to protein surface, and play important roles in protein stability by forming electrostatic interactions. In particular, the guanidinium group of arginine allows interactions in three possible directions, which enables arginine to form a larger number of electrostatic interactions compared to lysine. The higher pKa of the basic residue in arginine may also generate more stable ionic interactions than lysine. This paper reports an investigation whether the advantageous properties of arginine over lysine can be utilized to enhance protein stability. A variant of green fluorescent protein (GFP) was created by mutating the maximum possible number of lysine residues on the surface to arginines while retaining the activity. When the stability of the variant was examined under a range of denaturing conditions, the variant was relatively more stable compared to control GFP in the presence of chemical denaturants such as urea, alkaline pH and ionic detergents, but the thermal stability of the protein was not changed. The modeled structure of the variant indicated putative new salt bridges and hydrogen bond interactions that help improve the rigidity of the protein against different chemical denaturants. Structural analyses of the electrostatic interactions also confirmed that the geometric properties of the guanidinium group in arginine had such effects. On the other hand, the altered electrostatic interactions induced by the mutagenesis of surface lysines to arginines adversely affected protein folding, which decreased the productivity of the functional form of the variant. These results suggest that the surface lysine mutagenesis to arginines can be considered one of the parameters in protein stability engineering.     

Specific chemical modifications of link protein and their effect on binding to hyaluronate and cartilage proteoglycan

Specific chemical modifications of amino acid residues were performed on purified, native link protein from bovine articular cartilage. The effects of these on link protein's interactions with hyaluronate and bovine articular cartilage proteoglycan were assayed by gel chromatography. Interaction with hyaluronate was significantly perturbed by modification of lysine, arginine, tyrosine and aspartic/glutamic acid residues, but not histidine and tryptophan residues. No free, accessible sulphydryl group was found on native link protein. The requirement for unmodified lysine and arginine residues resembles that of the hyaluronate-binding site of pig laryngeal cartilage proteoglycan (Hardingham, T.E., Ewins, R.J.F. and Muir, H. (1976) Biochem. J. 157, 127-143). In contrast, proteoglycan binding was only significantly perturbed by the loss of arginine residues. This resistance may reflect hydrophobicity of the binding site or masking of the site from chemical modification by link protein self-association. Amidation of carboxyl groups, which destroyed hyaluronate binding but left proteoglycan binding intact, provides a means of generating a monofunctional link protein molecule of potential use in proteoglycan aggregation studies.     

Replacement of lysine by arginine, phenylalanine and tryptophan in the reactive site of the bovine trypsin-kallikrein inhibitor (Kunitz) and change of the inhibitory properties.

The reactive-site sequence of a proteinase inhibitor can be written as . . . -P3-P2-P1-P'1-P'2-P'3- . . . , where-P1-P'1-denotes the reactive site. Three semisynthetic homologues have been synthesized of the bovine trypsin-kallikrein inhibitor (Kunitz) with either arginine, phenylalanine or tryptophan in place of the reactive-site residue P1, lysine-15. These homologues correspond to gene products after mutation of the lysine 15 DNA codon to an arginine, phenylalanine or tryptophan DNA codon. Starting from native (virgin) inhibitor, reactive-site hydrolyzed, still active (modified) inhibitor was prepared by chemical and enzymic reactions. Modified inhibitor was then converted into inactive des-Lys15-inhibitor by reaction with carboxypeptidase B. Inactive des-Lys15-inhibitor was reactivated by enzymic replacement of the P1 residue according to Leary and Laskowski, Jr. The introduction of arginine was catalyzed by an inverse reaction with carboxypeptidase B, while phenylalanine or tryptophan were replaced by carboxypeptidase A. The reactivated semisynthetic inhibitors were trapped by complex formation with either trypsin or chymotrypsin. The enzyme - inhibitor complexes were subjected to kinetic-control dissociation, and the semisynthetic virgin inhibitors were isolated. The inhibitory properties of the semisynthetic inhibitors have been investigated against bovine trypsin and chymotrypsin and against porcine pancreatic kallikrein and plasmin. The homologues with either lysine or arginine in the P1 position are equally good inhibitors of trypsin, plasmin and kallikrein. The Arg-15-homologue is a slightly more effective kallikrein inhibitor than the Lys15-inhibitor. The semisynthetic phenylalanine and tryptophan homologues, however, are weak inhibitors of trypsin and still weaker inhibitors of kallikrein, but are excellent inhibitors of chymotrypsin. Their association constant with chymotrypsin is at least ten times higher than that of native Lys-15-inhibitor. A dramatic specificity change is observed with the phenylalanine and tryptophan homologues, which in contrast to the native inhibitor do not at all inhibit porcine plasmin. Thus, the nature of the P1 residue strongly influences the primary inhibitory specificity of the bovine inhibitor (Kunitz).     

Chemical modification and amino terminal sequence of calotropin DI from Calotropis gigantea

The effect of chemical modification of histidine, lysine, arginine, tryptophan and methionine residues on the enzymatic activity of calotropin DI has been studied. 1,3-Dibromoacetone inhibited the enzyme completely, indicating that a single histidine residue and a cysteine residue are involved in its catalytic activity. Its second bistidine residue was modified with diethyl pyrocarbonate without loss of activity. Modification of seven of its 13 lysine residues with 2,4,6-trinitrobenzene sulphonic acid led to 90% loss of its activity, but no single lysine residue appears to be essential for its activity. Four of the 12 arginine residues by 1,2-cyclohexanedione can be modified with little loss of activity. Modification of a single tryptophan residue and two methionine residues did not inhibit enzymatic activity. The blocked amino-terminal amino acid residue of calotropin DI has been identified as pyroglutamic acid. Its amino-terminal amino acid sequence to residue 14 has been determined and compared with that of papain. They show an extensive homology in their amino-terminal amino acid sequences.     

Amino acid supplementation of methanol-grown dried microbial cells in diets for young chicks

Growth rates and efficiency of food conversion of young chicks fed on diets marginally limiting in total nitrogen and containing 150 g/kg diet of flash-dried microbial cells (MC) with and without amino acid supplements, were measured in three experiments. Performance of these animals was compared with that of groups fed on a methioninefortified soya bean meal (SBM) control diet. In all experiments, chicks fed on the SBM control diet grew faster and were more efficient than chicks fed on the basal unsupplemented MC diet. In Experiment 1, arginine supplementation markedly enhanced weight gain and efficiency of food utilisation of chicks offered the basal MC diet; methionine had no effect. The second experiment demonstrated that a supplement containing methionine, arginine and tryptophan was more effective in augmenting the nutritional value of MC than either methionine with arginine or tryptophan with arginine. In the final experiment, weight gain and food intake of chicks fed on MC with supplements of arginine, methionine and tryptophan were increased markedly by additions of lysine and glutamic acid but not by addition of lysine alone. In all experiments, performance of animals in MC supplemented groups was lower than that of animals fed on the SBM control diet.     

Intracellular changes of metal elements by fucoidan extracted from brown seaweed (Cladosiphon okamuranus)

This study was undertaken to elucidate the intracellular changes of metal elements after the administration of fucoidan extracted from Cladosiphon okamuranus. TRL1215 cells (normal rat liver cell line) were treated with 0, 0.1, or 1.0 mg/ml fucoidan and incubated in 5% CO2 at 37 degrees C. The cellular levels of Mg, Al, Fe, and Zn were significantly increased in the 1.0 mg/ml fucoidan-treated cells compared to those of the 0.1 mg/ml fucoidan-treated cells and the control. Next, TRL1215 cells were cultured on Mylar film overnight. At 24 h after 5-bromo-2'-deoxyuridine dosing, 0, 0.1, or 1.0 mg/ml fucoidan was treated for 9 h. The cellular distribution of elements was analyzed using in-air micro-micro-particle induced X-ray emission. The X-ray spectra showed that yields of Al, Mg, and Zn were high in order of the 1.0 mg/ml fucoidan-treated sample, the 0.1 mg/ml fucoidan-treated sample, and the control. Fe yield was mildly increased by fucoidan administration. In fucoidan-treated cells, the focal accumulation of Br was correlated spatially with phosphorous-rich region, suggesting that Br was localized within the nucleus. Al distribution provided a spatial association with Br map. These data suggest that fucoidan increases the accumulations of Al, Mg, Fe, and Zn in normal rat hepatocytes, and fucoidan-binding Al is postulated to be transferred into the nucleus.     

Cartilage proteoglycans. Structure and heterogeneity of the protein core and the effects of specific protein modifications on the binding to hyaluronate.

Purified proteoglycans extracted from pig laryngeal cartilage in 0.15 M-NaCl and 4 M-guanidinium chloride were analysed and their amino acid compositions determined. Selective modification of amino acid residues on the protein core confirmed that binding to hyaluronate was a function of the protein core, and was dependent on disulphide bridges, intact arginine and tryptophan residues, and epsilon-amino groups of lysine. Fluorescence measurement suggested that tryptophan was not involved in direct subsite interactions with the hyaluronate. The polydispersity in size and heterogeneity in composition of the aggregating proteoglycan was compatible with a structure based on a protein core containing a globular hyaluronate-binding region and an extended region of variable length also containing a variable degree of substitution with chondroitin sulphate chains. The non-aggregated proteoglycan extracted preferentially in 0.15 M-NaCl, which was unable to bind to hyaluronate, contained less cysteine and tryptophan than did other aggregating proteoglycans and may be deficient in the hyaluronate-binding region. Its small average size and low protein and keratan sulphate contents suggest that it may be a fragment of the chondroitin sulphate-bearing region of aggregating proteoglycan produced by proteolytic cleavage of newly synthesized molecules before their secretion from the cell.     

Temporal profiling of essential amino acids in developing maize kernel of normal,opaque - 2 and QPM germplasm

Maize, an important cereal crop, has a poor quality of endosperm protein due to the deficiency of essential amino acids, especially lysine and tryptophan. Discovery of mutants such as opaque-2 led to the development of nutritionally improved maize with a higher concentration of lysine and tryptophan. However, the pleiotropic effects associated with opaque-2 mutants necessitated the development of nutritionally improved hard kernel genotype, the present-day quality protein maize (QPM). The aim of present study was to analyze and compare the temporal profile of lysine and tryptophan in the developing maize kernel of normal, opaque-2 and QPM lines. A declining trend in protein along with tryptophan and lysine content was observed with increasing kernel maturity in the experimental genotypes. However, opaque-2 retained the maximum concentration of lysine (3.43) and tryptophan (1.09) at maturity as compared to QPM (lysine-3.05, tryptophan-0.99) and normal (lysine-1.99, tryptophan-0.45) lines. Opaque-2 mutation affects protein quality but has no effect on protein quantity. All maize types are nutritionally rich at early stages of kernel development indicating that early harvest for cattle feed would ensure a higher intake of lysine and tryptophan. Two promising lines (CML44 and HKI 1105) can be used for breeding high value corn for cattle feed or human food in order to fill the protein inadequacy gap. Variation in lysine and tryptophan content within QPM lines revealed that differential expression of endosperm modifiers with varying genetic background significantly affects nutritional quality, indicating that identification of alleles affecting amino acid composition can further facilitate QPM breeding program.     

Chemical treatments of the labellar sugar receptor of the fleshfly.

The effects of treatments with various reagents, such as proteases, group specific protein reagents, detergents, phospholipases, and antibiotics, were examined on the sugar receptor of the fleshfly. Certain group specific protein reagents together with pronase markedly affected its response to sucrose, which suggested important rôles for stimulation of protein components and at least four specific residues, i.e. cysteine, tryptophan, arginine and lysine in the sugar receptor system. The packed receptor components and their renewal mechanism are discussed.     

Protein intake regulation in the weanling rat: Effects of additions of lysine,arginine and ammonia on the selection of gluten and energy

The effects of adding lysine, arginine and ammonia to gluten on the self-selection of protein and energy by the weanling rat simultaneously offered a choice of two diets differing only in gluten concentration (15 and 55%) were tested. Previous studies have shown that while lysine (6 g/100 g) additions to gluten decreased the amount of gluten selected by the rat from 40 to 20 g per 100 g of food eaten, selection was not related to the nutritional quality of the gluten. When graded levels of arginine (1.8, 3.6 or 7.2 g/100 g) were added to the gluten with or without lysine (0 or 6 g/100 g) the dietary protein selection was unaffected. The addition of ammonia (1.4 g/100 g as NH₄Cl) to gluten had initially the same effect as lysine (6 g/100 g) but with time protein intake returned to control levels. This effect of ammonia was unaltered by arginine additions. It is concluded that the mechanisms which lead to decreases in gluten selection caused by lysine or ammonia are not similar, and that the effects of lysine on gluten selection are not caused by an increased arginine requirement for urea cycle activity.     

Propagation of protein glycation damage involves modification of tryptophan residues via reactive oxygen species: inhibition by pyridoxamine

Nonenzymatic modification of proteins is one of the key pathogenic factors in diabetic complications. Uncovering the mechanisms of protein damage caused by glucose is fundamental to understanding this pathogenesis and in the development of new therapies. We investigated whether the mechanism involving reactive oxygen species can propagate protein damage in glycation reactions beyond the classical modifications of lysine and arginine residues. We have demonstrated that glucose can cause specific oxidative modification of tryptophan residues in lysozyme and inhibit lysozyme activity. Furthermore, modification of tryptophan residues was also induced by purified albumin-Amadori, a ribose-derived model glycation intermediate. The AGE inhibitor pyridoxamine (PM) prevented the tryptophan modification, whereas another AGE inhibitor and strong carbonyl scavenger, aminoguanidine, was ineffective. PM specifically inhibited generation of hydroxyl radical from albumin-Amadori and protected tryptophan from oxidation by hydroxyl radical species. We conclude that oxidative degradation of either glucose or the protein-Amadori intermediate causes oxidative modification of protein tryptophan residues via hydroxyl radical and can affect protein function under physiologically relevant conditions. This oxidative stress-induced structural and functional protein damage can be ameliorated by PM via sequestration of catalytic metal ions and scavenging of hydroxyl radical, a mechanism that may contribute to the reported therapeutic effects of PM in the complications of diabetes.     

Chemical modification of lysine and arginine residues in the myosin regulatory light chain inhibits phosphorylation

The contribution of lysine and arginine residues to the substrate specificity of the myosin light-chain kinase has been studied using chemically modified myosin light chains. Succinylation or maleylation of the myosin light chains caused complete inhibition of their phosphorylation. Modification of 50% of the lysine residues resulted in 90% inhibition of phosphorylation and this was accompanied by a 25-fold increase in the apparent Km. In contrast, phosphorylation of the myosin light chains by the cAMP-dependent protein kinase was relatively insensitive to lysine modification, with only a 15% reduction in phosphorylation following succinylation of 50% of the lysine residues. Treatment with either cyclohexane-1,2-dione or camphorquinone-10-sulfonic acid resulted in between 90 and 98% inhibition of myosin light-chain phosphorylation. These reagents caused modification of both lysine and arginine residues, and accordingly only part of the inhibition can be attributed to arginine modification. Modification of all of the cysteine and methionine residues caused only a 40% inhibition of phosphorylation. The results of this study support the concept that lysine and arginine residues act as essential specificity determinants for the myosin light-chain kinase in protein substrates.     

Polymerization of Lysozyme and Impairment of Its Amino Acid Residues Caused by Reaction with Glucose

In order to reveal the mechanism of the Maillard reaction between proteins and reducing sugars, unmodified and chemically modified lysozymes were incubated with and without glucose at 50°C and 75% relative humidity in the solid state. Incubation of unmodified lysozyme with glucose resulted in browning and polymerization of the protein, and noticeable losses of arginine, lysine, and tryptophan residues. Those changes were little affected by the presence of an oxygen adsorber. Acetylation of lysozyme almost completely prevented those changes, indicating that the reaction of free amino groups of the protein with glucose is essential at the initial stage of these changes.

Incubation of lysozyme the arginine residues of which were masked with 1,2-cyclohexane-dione (CHD) resulted in almost the same changes as above even in the absence of glucose. Those changes could be explained as caused by the action of CHD released from the arginine residues. This similarity in the effects on protein of CHD and glucose implies that dicarbonyl compounds are key components at the secondary stage of the Maillard reaction between proteins and reducing sugars.     

Trimethyllysine and protein function. Effect of methylation and mutagenesis of lysine 115 of calmodulin on NAD kinase activation

Unmethylated calmodulins have been enzymatically methylated at lysine 115, and a direct effect of this methylation on NAD kinase activation has been shown. Similar to naturally occurring calmodulins with trimethyllysine 115, the enzymatically methylated calmodulins activated an NAD kinase preparation to a maximal level that was at least 3-fold lower than the level of activation obtained with the corresponding unmethylated calmodulins. Methylation did not alter the cyclic nucleotide phosphodiesterase activator properties of these calmodulins. A genetically engineered calmodulin containing an arginine at position 115 instead of a lysine was produced by site-specific mutagenesis of a cloned synthetic calmodulin gene. The arginine derivative retained the higher maximal NAD kinase activator properties of the unmethylated calmodulins but was no longer susceptible to the effects of the methyltransferase. The data indicate that the reduction in the level of NAD kinase activation is the direct result of trimethylation of lysine 115 of calmodulin, provide a precedent for a functional effect of trimethyllysine in a protein, and raise the possibility that some of calmodulin's physiological activities may be affected by lysine methylation.     

Antiatherogenic effect of a low lysine: arginine ratio of protein involves alteration in the aortic glycosamihoglycans and glycoproteins

The effect of alteration of lysine: arginine ratio of the protein on the aortic glycosaminoglycans and glycoproteins was studied in rats fed cholesterol free and atherogenic diet. The concentration of total glycosaminoglycans and of individual fractions was significantly lower in the aorta in the case of diet with lysine: arginine ratio of 1.0, than the diet with a ratio of 2.0. Rats fed globulin fraction isolated from sesame seeds, which has a lysine: arginine ratio of 0.67 also showed significantly lower concentration of total and individual glycosaminoglycan fractions in the aorta than those fed casein (lysine:arginine ratio 2.0). Concentration of total hexose and fucose in the glycoproteins was also lower in the aorta in the case of lysine: arginine ratio 1.0. These results in the light of previous reports of increase in the aortic glycosaminoglycans in the early stages of atherosclerosis and increase in the total hexose and fucose in the glycoproteins in the atherosclerotic aorta indicate that the antiatherogenic effect of a low lysine: arginine ratio in the protein involves alteration in the aortic glycosaminoglycans and glycoproteins.     

Fluorescence and chemical studies on the interaction of Escherichia coli DNA-binding protein with single-stranded DNA.

Nanosecond and steady-state fluorescence spectoscopy were used to probe the environment of the tryptophan residues of Escherichia coli DNA-binding protein. A spectral shift and a change in quantum yield of the protein upon binding to DNA or oligonucleotides indicate that the tryptophan residues are near or at the DNA binding site. The observation of two excited-state lifetimes of the protein indicates that there is heterogeneity in the microenvironments of these tryptophan residues. The "short-lifetime" tryptophan residues are more sensitive to the interaction with DNA than the "long-lifetime" residues. The results of solute-perturbation studies with iodide or acrylamide indicate that there are tryptophan residues near the surface of the protein which are heterogeneous in their accessibility to these quenchers and that they become less accessible after DNA binding. Also, lysine residues of the protein have been shown to be essential to DNA binding by chemical-modification studies. Tyrosine, arginine, and cysteine residues appear not to be involved in this binding process. From studies of the decay of fluorescence anisotropy of the binding protein in the presence and absence of DNA, it has been concluded that (a) the tetrameric binding protein does not dissociate into subuniits upon binding to the oligonucleotide d(pT)16 and (b) the binding protein-fd DNA complex possesses "local flexibility" and, therefore, cannot be described as a continuous, rigid rod.     

The purification and characterization of a necrotoxin from tarantula, Dugesiella hentzi (Girard), venom

The major toxin, a necrotoxin, of the venom of Dugesiella hentzi (Girard) has been purified by gel filtration. The purified toxin was homogeneous by gel filtration, polyacrylamide gel electrophoresis, and an isoelectric focusing procedure. The molecular weight estimation was 6700 and the isoelectric pH was 10.0. The amino acid composition shows 16 lysine, 8 cysteine, and one tryptophan residues, with no tyrosine, methionine, alanine, arginine, or histidine residues. The purified protein is toxic to certain insects and mice with the primary site of action being muscle tissue in the mouse. Modification of the single tryptophan residue resulted in a loss of toxicity.A significant increase of serum creatine phosphokinase activity was observed in mice injected with the necrotoxin. Histological examination showed the primary lesions were acute focal areas of myocardial necrosis, and no histological differences in myocardial lesions were seen between mice injected with the purified necrotoxin or with the whole venom.