Modification Of Arginine Residues Research Articles

Natural plant enzyme inhibitors. Characterization of an unusual alpha-amylase/trypsin inhibitor from ragi (Eleusine coracana Geartn.).

An inhibitor I-1, capable of acting on both alpha-amylase and trypsin, was purified to homogeneity from ragi (finger-millet) grains. The factor was found to be stable to heat treatment at 100 degrees C for 1 h in the presence of NaCl and also was stable over the wide pH range 1-10. Pepsin and Pronase treatment of inhibitor I-1 resulted in gradual loss of both the inhibitory activities. Formation of trypsin-inhibitor I-1 complex, amylase-inhibitor I-1 complex and trypsin-inhibitor I-1-amylase trimer complex was demonstrated by chromatography on a Bio-Gel P-200 column. This indicated that the inhibitor is 'double-headed' in nature. The inhibitor was retained on a trypsin-Sepharose 4B column at pH 7.0. Elution at acidic pH resulted in almost complete recovery of amylase-inhibitory and trypsin-inhibitory activities. alpha-Amylase was retained on a trypsin-Sepharose column to which inhibitor I-1 was bound, but not on trypsin-Sepharose alone. Modification of amino groups of the inhibitor with 2,4,6-trinitrobenzenesulphonic acid resulted in complete loss of amylase-inhibitory activity but only 40% loss in antitryptic activity. Modification of arginine residues by cyclohexane-1,2-dione led to 85% loss of antitryptic activity after 5 h, but no effect on amylase-inhibitory activity. The results show that a single bifunctional protein factor is responsible for both amylase-inhibitory and trypsin-inhibitory activities with two different reactive sites.

An essential arginine residue at the substrate-binding site of p-hydroxybenzoate hydroxylase.

p-Hydroxybenzoate hydroxylase (EC 1.14.13.2) was rapidly inactivated by treatment with phenylglyoxal, by a process obeying pseudo-first order kinetics. The reaction with the reagent was also examined by amino acid analyses, radioactivity measurements, and spectrophotometric analyses. Results of these analyses were consistent with each other, which shows that the inactivation was due to modification of argiine residue(s). Addition of saturating amounts of p-hydroxybenzoate (or benzoate) during the treatment resulted in marked protection of the enzyme from the inactivation as well as a significant decrease in modification of arginine residues, while phenol showed no effect. Modification in the absence of p-hydroxybenzoate caused a spectral change in the flavin moiety of the enzyme similar to that due to the enzyme.substrate complex formation, and losses in both the overall activity and the substrate-binding ability accompanied the spectral change. On the other hand, such spectral change was not observed and the substrate-binding ability was retained even after the overall activity had decreased to a great extent when p-hydroxybenzoate as added during the modification treatment. These results suggest that phenylglyoxal (an analogue of the substrate) was incorporatd into the substrate-binding site and that an arginine residue is involved in the site, having an interaction with the carboxylate anion of the substrate.

Studies on the purification and structure-function relationships of murine lymphocyte activating factor (interleukin 1)

Lymphocyte activating factor [LAF; Interleukin 1 (IL 1)] obtained from the murine macrophage cell line, P388D 1, was partially purified by ammonium sulfate fractionation and chromatography on DEAE cellulose, Sephacryl S200 and phenyl Sepharose. Relative to the starting material (sp. act. = 17 U/mg protein), the LAF (IL 1) at the phenyl Sepharose stage of purification was 5–10% pure (sp. act. = 2 × 10 4 U/mg protein) with a yield of 7–30%, depending on the volume of starting material. Analysis of the LAF (IL 1) by isoelectrofocusing in polyacrylamide gels revealed that the LAF (IL 1) obtained after elution from the phenyl Sepharose column was associated specifically with one of eight bands stained with Coomassie Brilliant Blue. A specific activity of ≥3 × 10 5 U/ mg protein was estimated for a homogeneous preparation of LAF (IL 1). LAF (IL 1) activity was lost after modification of arginine residues by phenylglyoxal and was only weakly affected by modification of histidine residues with diethylpyrocarbonate. No active fragments were obtained after cyanogen bromide cleavage of LAF (IL 1).

Reversible modification of arginine residues in neocarzinostatin. Isolation of a biologically active 89-residue fragment from the tryptic hydrolysate.

Reaction of the antitumor protein neocarzinostatin with 1,2-cyclohexanedione in 0.25 M borate buffer, pH 9.0, resulted in complete modification of arginine residues in positions 66, 67, and 78. The arginine-modified protein lost its native structure and was biologically inactive in the inhibition of growth of HeLa cells, inhibition of DNA synthesis, and in vitro DNA strand scissions. Trypsin hydrolysis of 1,2-cyclohexanedione-modified neocarzinostatin resulted in selective cleavage of the Lys-Val (positions 20 and 21) bond of the primary structure yielding NH2-terminal 1-20 and the COOH-terminal 21-109 residue fragments. The latter contained modified arginine residues. Both peptide fragments were biologically inactive. Treatment of the arginine-modified neocarzinostatin and the arginine-protected 89-residue fragment with 0.25 M Tris-acetate buffer, pH 9.0, for 15 h resulted in the release of 1,2-cyclohexanedione, regenerating all three arginine residues. The regenerated protein and the 89-residue fragment were fully active biologically. Further, the regenerated 89-residue fragment possessed 70% of the reactivity of neocarzinostatin with antibody raised against the native protein. The conformation of the 89-residue fragment was almost identical with that of the native protein in CD spectral properties.

Inhibition of receptor-mediated clearance of lysine and arginine-modified lipoproteins from the plasma of rats and monkeys.

Reductive methylation of at least 30% of the lysine residues or 1,2-cyclohexanedione modification of 45% of the arginine residues prevented low density lipoproteins (LDL) from binding to cell surface receptors of fibroblasts in vitro, without significantly altering other physical or chemical properties of the LDL. When rat or human LDL with more than 30% of the lysine residues methylated were injected intravenously into rats, the clearance of these lipoproteins from the plasma was slowed considerably. The half-life of the reductively methylated LDL was approximately twice that obtained for control (unmodified) LDL, and the value for the fractional catabolic rate was approximately half that of the control. Furthermore, when human LDL modified by reductive methylation were injected into rhesus monkeys, the rate of clearance was similarly retarded, and the value for the fractional catabolic rate was reduced by approximately 50% as compared with the value for control LDL. A dual isotope labeling technique ((125)I and (131)I) was used to compare the disappearance of the control and modified LDL in the same animal. It was demonstrated that not only modification of lysine residues but also modification of the arginine residues with 1,2-cyclohexanedione retarded the plasma clearance of the rat LDL. However, the cyclohexanedione modification was spontaneously reversible at 37 degrees C, whereas reductive methylation of the lysine residues was stable. It is concluded that the selective chemical modification of lysine or arginine residues of LDL interferes with the normal uptake of these lipoproteins in vivo as well as by fibroblasts in vitro. These data provide an estimation of the level of receptor-mediated clearance of LDL from the plasma, a value that may be as high as 50% in rats and monkeys.

Identification of the C-1-phosphate-binding arginine residue of rabbit-muscle aldolase. Isolation of 1,2-cyclohexanedione-labeled peptide by chemisorption chromatography.

The arginine-specific reagent 1,2-cyclohexanedione reacts selectively with the arginine residue of the C-1-phosphate-binding site of aldolase and inactivates the enzyme. The labeled peptide isolated from tryptic digests of inactivated aldolase was found to correspond to the sequence Leu-43 to Arg-56, the residue modified by cyclohexanedione being Arg-55. This peptide was absent form digests of aldolase treated in the same way but protected from inactivation by the presence of substrate, thus correlating modification of Arg-55 with loss of activity. Selective isolation ofthe peptide containing the modified arginine residue was effected by chemisorption chromatography on boric acid gel, a procedure exploiting the specific interaction of matrix-bound boric acid groups with vicinal cis-hxdroxyl groups of cyclohexanedione-modified arginine side chains.

Sequence-specific endonuclease Bgl I. Modification of lysine and arginine residues of the homogeneous enzyme.

The sequence-specific endonuclease Bgl I from Bacillus globigii (RUB561) has been purified to homogeneity as determined by denaturing polyacrylamide gel analysis. The active form of the enzyme is a single polypeptide with a molecular weight of 32,000. The enzyme requires Mg2+ in the reaction mixture and displays a broad pH and monovalent cation requirement. Bgl I is not sensitive to sulfhydryl reagents but was affected by reagents that modify lysine and arginine residues. When lysine residues were modified by pyridoxal 5'-phosphate, both binding and catalysis were diminished while modification of arginine residues by 2,3-butanedione inhibited the enzyme activity but had no effect on its binding properties.

The synthesis of phenyl(2-3H)glyoxal

A simple inexpensive method has been developed for the synthesis of [2-3H]acetophenone, which has been converted into phenyl[2-3H]glyoxal. The latter compound has been used to modify arginine residues in alkaline phosphatase from two sources, and also a sialidase.

Essential arginine residues in the active sites of propionyl CoA carboxylase and beta-methylcrotonyl CoA carboxylase.

At least one arginine residue is essential for substrate binding in or near the active sites of propionyl CoA carboxylase (PCC) and beta-methylcrotonyl CoA carboxylase (beta MCC) in cultured human fibroblasts. This conclusion is based on studies of enzyme inhibition by phenylglyoxal, a reagent which specifically modifies arginine residues. Human fibroblast PCC both in extracts and in a 20-fold purified preparation was nearly completely protected from phenylglyoxal inhibition following incubation with propionyl CoA or ATP. It appears that a phosphate group from either ATP or the CoA moiety of propionyl CoA reacts with the essential arginine residue(s). beta MCC which was similarly inhibited by phenylglyoxal was protected by beta-methylcrotonyl CoA and ATP. Thus phenylglyoxal may be used to label specific arginine residues within the active sites of previously sequenced carboxylases.

Inactivation of purine nucleoside phosphorylase by modification of arginine residues

Treatment of purine nucleoside phosphorylase (EC 2.4.2.1), from either calf spleen or human erythrocytes, with 2,3-butanedione in borate buffer or with phenylglyoxal in Tris buffer markedly decreased the enzyme activity. At pH 8.0 in 60 min, 95% of the catalytic activity was destroyed upon treatment with 33 mM phenylglyoxal and 62% of the activity was lost with 33 m m 2,3-butanedione. Inorganic phosphate, ribose-1-phosphate, arsenate, and inosine when added prior to chemical modification all afforded protection from inactivation. No apparent decrease in enzyme catalytic activity was observed upon treatment with maleic anhydride, a lysine-specific reagent. Inactivation of electrophoretically homogeneous calf-spleen purine nucleoside phosphorylase by butanedione was accompanied by loss of arginine residues and of no other amino acid residues. A statistical analysis of the inactivation data vis-à-vis the fraction of arginines modified suggested that one essential arginine residue was being modified.

Preparation, characterization, and properties of a novel triple-modified derivative of the Ca2+-dependent protein modulator

A novel, chemically modified derivative of the Ca2+-dependent protein modulator of cyclic nucleotide phosphodiesterase was prepared by the sequential treatment of the native protein with tetranitromethane, 1,2-cyclohexanedione, and diethylpyrocarbonate. Three derivatives were isolated during the synthesis, i.e., a single-modified derivative containing two modified tyrosine residues per mole; a double-modified derivative containing two modified tyrosines plus five modified arginine residues per mole; and a triple-modified derivative containing two modified tyrosines, five modified arginines, and one modified histidine residue per mole. Intermolecular cross-linking was observed to occur as a minor side reaction during nitration of the native protein with tetranitromethane. All the derivatives were examined for phosphodiesterase-stimulating activity and troponin C like activities. All derivatives were found to retain the capacity of the native protein to stimulate modulator-deficient phosphodiesterase; furthermore, in each case, the stimulation of phosphodiesterase was a Ca2+-dependent process. On the other hand, both the double- and triple-modified modulators lost the troponin C like activities, i.e., the Ca2+-dependent change in mobility in urea–polyacrylamide gels and the ability to interact with troponin I in the presence of Ca2+ to form a urea-stable complex, while the nitrotyrosyl modulator retained these properties of the native protein.

The reactive site of eggplant trypsin inhibitor.

The reactive site peptide bond of the eggplant inhibitor against trypsin [EC 3.4.21.4] was identified by chemical modifications with 1,2-cyclohexanedione, 2,4,6-trinitrobenzenesulfonic acid, acetic anhydride and glyoxal, and by sequential treatments with trypsin and carboxypeptidase B [EC 3.4.12.3]. The inhibitor was significantly inactivated by chemical modifications of arginine residues, but was not affected by lysine modifications. Free arginine was released from the trypsin-modified inhibitor by carboxypeptidase B digestion, accompanied by a marked loss of inhibitory activity. A serine residue was newly exposed at the N-terminal amino acid of the inhibitor after modification with trypsin. The reactive site of the inhibitor against trypsin was concluded to be an arginylseryl bond. The inhibitor was completely inactivated by full reduction of its disulfide bonds.

Purification and binding properties of human platelet factor four.

Methods are described for the purification of a heparin-neutralizing protein from human platelets. The protein, obtained by conventional or affinity chromatographic techniques, is homogeneous by disc and sodium dodecyl sulfate gel electrophoresis, immunoelectrophoresis, and gel electrofocusing and can be obtained in a final yield of 75%. The protein has a subunit molecular weight of 9600, an isoelectric point at pH 7.6, and 18% basic and 22% acidic amino acid residues. The purified heparin-neutralizing protein forms dissociable complexes with heparin as measured by electrophoretic and Millipore filtration techniques employing [3H]heparin. The ability of a series of sulfated glycosaminoglycans to displace [3H]heparin from the binding protein was compared. The mole ratios required were: heparin less than heparan sulfate less than dermatan sulfate less than chondroitin 6-sulfate less than chondroitin 4-sulfate. Although the degree of sulfation of the aminoglycans correlated with the ability to displace [3H]heparin, the conformation fo the carboxyl group of the uronic acid and the location of the sulfate groups on the amino sugar also influenced the affinity for the protein. Evidence is also presented that binding to aminoglycans occurs via ionic interactions between lysine residues on the protein and negatively charged groups on the aminoglycan. Chemical modification of lysines by guanidination decreased heparin-neutralizing and binding activity, while modification of arginine residues had no effect. Heparin could prevent lysine modification when specifically bound to the heparin-neutralizing protein, but did not prevent lysine modification of other proteins.

Modification of arginine and lysine in proteins with 2,4-pentanedione.

Primary amines react with 2,4-pentanedione at pH 6-9 to form enamines, N-alkyl-4-amino-3-penten-2-ones. The latter compounds readily regenerate the primary amine at low pH or on treatment with hydroxylamine. Guanidine and substituted guanidines react with 2,4-pentanedione to form N-substituted 2-amino-4,6-dimethylpyrimidines at a rate which is lower by at least a factor of 20 than the rate of reaction of 2,4-pentanedione with primary amines. Selective modification of lysine and arginine side chains in proteins can readily be achieved with 2,4-pentanedione. Modification of lysine is favored by reaction at pH 7 or for short reaction times at pH 9. Selective modification of arginine is achieved by reaction with 2,4-pentanedione for long times at pH 9, followed by treatment of the protein with hydroxylamine. The extent of modification of lysine and arginine side chains can readily be measured spectrophotometrically. Modification of lysozyme with 2,4-pentanedione at pH 7 results in modification of 3.8 lysine residues and less than 0.4 arginine residue in 24 hr. Modification of lysozyme with 2,4-pentanedione at pH 9 results in modification of 4 lysine residues and 4.5 arginine residues in 100 hr. Treatment of this modified protein with hydroxylamine regenerated the modified lysine residues but caused no change in the modified arginine residues. One arginine residue seems to be essential for the catalytic activity of the enzyme.

Mechanism of action of heparin through thrombin on blood coagulation

It has been suggested that heparin can affect blood coagulation through thrombin, i.e. the binding of heparin to thrombin induces a conformational change in the enzyme, facilitating a complex formation between thrombin and antithrombin (Machovich, R., Blaskó, Gy. and Pálos, L. (1975) Biochim. Biophys. Acta 379, 193–200). This hypothesis seems to have been proved. Modification of arginine residues in thrombin did not result in decreased thrombin activity and decreased sensitivity to antithrombin, whereas the heparin sensitivity of the enzyme and the thrombin-antithrombin reaction were diminished.

Functional arginine residues involved in coenzyme binding by glutamate dehydrogenases

Reaction of the NADP-dependent glutamate dehydrogenase of Neurospora with 1,2-cyclohexanedione results in a biphasic loss of enzyme activity. At the end of the rapid phase of the reaction (t1/2 = 1.5 min) the enzyme activity is diminished by approximately 60% with the simultaneous loss of 1 residue of arginine per subunit. After 60 min, the enzyme activity is completely lost with the modification of a total of 2 arginine residues per subunit. Reaction of bovine liver glutamate dehydrogenase with cyclohexanedione causes a rapid loss of approximately 45% of the enzyme activity and modification of about 1.5 residues of arginine per subunit. More prolonged treatment results in reaction of an additional 4 residues of arginine per subunit but is without further effect on the residual activity. The activity of the Neurospora enzyme is not protected by substrate, coenzyme, or a combination of both; however, the activity of the bovine enzyme is partially protected by high levels of NAD or NADP. Although the Km for alpha-ketoglutarate is unchanged by a limited modification of either enzyme with cyclohexanedione, the Km for coenzyme is increased about 2-fold for the Neurospora enzyme and about 1.5-fold for the bovine enzyme. The Ki of the Neurospora dehydrogenase for the competitive inhibitor 2'-monophosphoadenosine-5'-diphosphoribose is unchanged by the enzyme modification, but nicotinamide mononucleotide, a competitive inhibitor for the native Neurospora enzyme, does not inhibit the glutamate dehydrogenase with 1 modified arginine residue. This finding implies that the modified arginine is at or near the nicotinamide binding iste of the enzyme.

Identification of functional arginine residues in ribonuclease A and lysozyme

A specific color reaction has been developed for the detection of N-7, N-8-(1,2-dihydroxycyclohex-1,2-ylene)-L-arginine-containing peptides. The reaction is based on the fact that hydroxylamine converts the blocking group to cyclohexanedione dioxime, which forms a red nickel complex. N-7, N-8-(1,2-dihydroxycyclohex-1,2-ylene)-L-arginine-containing peptides can also be detected by diagonal electrophoresis from the change of electrophoretic mobility of these peptides on interaction of the blocking group with borate. Since the modified arginine residues are resistant to tryptic cleavate, changes in tryptic peptide patterns can also be utilized to identify the presence of modified arginine residues. A combination of these approaches was used to identify the arginine residues modified by cyclohexanedione treatment. Bovine panctreatic RNase A loses approximately 90% of its activity on cyclohexanedione treatment with the modification of 2 to 3 arginine residues. Arginine-39 reacts most rapidly and its modification contributes most to inactivation of the enzyme. Arginine-85 also reacts rapidly with cyclohexanedione. Arginine-10 reacts slowly and no reaction was observed with arginine-33. Removal of the blocking groups by hydroxylamine treatment resulted in complete recovery of enzyme activity in samples where arginine-39 and arginine-85 had been modified, whereas 80% of activity was regained from samples where arginine-10 had also been modified. With egg white lysozyme, all 11 arginine residues react with cyclohexanedione, resulting in partial inactivation of the enzyme. The fully modified enzyme retains 35% of its activity. Since arginine residues are important for electrostatic interaction between the enzyme and the negatively charges cell surface, even the modified, basic residues can provide the necessary positive charges. In the presence of borate, activity is almost completely abolished, since the modified arginine-borate complex has a reduced net positive charge. Upon removal of the blocking groups by hydroxylamine, even the fully modified lysozyme regains complete activity. With the exception of the most reactive arginine (residue 5), modification of all other arginine residues contributes equally to inactivation of the enzyme. The possible reason for the importance of arginine-5 in maintaining activity is discussed. Advantages of the present method for the selective reversible modification of arginine residues of proteins and for the identification of reactive arginine residues are evaluated.

Inactivation of bovine carboxypeptidase A by specific modification of arginine residues with phenylglyoxal.

Upon treatment of bovine carboxypeptidase A [EC 3.4.2.1] with phenylglyoxal, an arginine-specific reagent, at pH 8.0 and 25°C, the peptidase activity was lost very rapidly whereas the esterase activity was lost more slowly. The inhibitor, $phenylpropionic acid, showed some protection against loss of the esterase activity but not against loss of the peptidase activity. These changes in the activites following reaction with phenylglyoxal occurred with concomitant modification of 2 to 3 arginine residues in the enzyme. A preliminary attempt has been made to locate the modified arginine residues by analyzing the fragments obtained by cleavage with cyanogen bromide of phenylglyoxal-modified carboxypeptidase A.

Enzymic and immunochemical properties of lysozyme—VI Conformation, enzymic activity and immunochemistry of derivatives modified at arginine residues

Modification of the arginine residues in lysozyme with cyclohexanedione has been carried out under two different conditions. The first derivative (CHD-Lysoz I) was prepared by reaction in 0·1 N NaOH. However, it was found that control lysozyme preparations subjected to these reaction conditions lost all enzymic activity, although their antigenic reactivity was not affected. To avoid this complication, a second derivative (CHD-Lysoz II) was prepared by reaction in 0·1 M triethylamine. In fact, lysozyme pretreated with this solvent retained entirely its enzymic activity and its antigenic reactivity. In both CHD-Lysoz I and CHD-Lysoz II, 10 arginine residues were modified. The two derivatives possessed identical conformations, as revealed from their optical rotatory dispersion and circular dichroism behaviors and from the availability of the disulfide bonds to reduction and susceptibility to chymotryptic attack, but were greatly unfolded relative to lysozyme. The two derivatives had no enzymic activity and very little antigenic reactivity with antisera to lysozyme. Conversely, with antisera to CHD-Lysoz I, lysozyme reacted only partially (60 per cent), and these sera showed appreciable (28 per cent) cross-reaction with CHD-human serum albumin. Interpretation of the enzymic and immunochemical data was complicated by the fact that too many (10) arginine residues were modified and that appreciable conformational changes were present. Preparation of a derivative with a least number of modified arginine residues was therefore desirable, and this was afforded by reaction of lysozyme with phenylglyoxal (PG-Lysoz). In PG-Lysoz, one arginine residue was modified and it was located at position 61. The derivative had identical optical rotatory dispersion and circular dichroism parameters with those of lysozyme. However, minor conformational changes were present, and these were only revealed by the slight increase in disulfide reducibility (0·26 bonds) and by a small increase in susceptibility to tryptic attack (from 0·16 bonds in lysozyme to 0·97 bonds in PG-lysozyme). The enzymic activity of PG-Lysoz was suppressed slightly (to 83 per cent) but the pH optimum was unaltered. With antisera to lysozyme, PG-Lysoz showed equal antigenic reactivity to that of lysozyme. Conversely, with antisera to PG-Lysoz, the reactions of lysozyme and PG-Lysoz were identical. Also, the two proteins had equal reactivities with antisera to CHD-Lysoz I (58–60 per cent relative to homologous antigen). It was, therefore concluded that arginine-61 is not located in an antigenic site of lysozyme. Also, the findings demonstrate that not all conformational changes will influence antigenic reactivity. The change in the latter will be dependent on the protein and on the nature of the conformational reorganization.

The effect of the modification of arginine side chains in casein on the coagulation of rennin-altered casein

SummaryThe modification of arginine residues in casein by treatment with glyoxal at pH 8·6 resulted in an inhibition of the coagulation of rennin-treated casein. The effective residues are on the κ-casein fraction and inhibition of coagulation was complete when about 1·5 residues of arginine/mole of κ-casein had been altered. It is suggested that the arginine forms part of a positively charged region that also includes lysine and histidine residues, and that this region is important in the coagulation of the casein.