CRYSTALLINE ACETYL DERIVATIVES OF PEPSIN

Crystalline pepsin has been acetylated by the action of ketene in aqueous solution at pH 4.07–5.5. As acetylation proceeds the activity decreases, the decrease being more rapid at pH 5.0–5.5 than at 4.0–4.5. Three acetyl derivatives have been isolated from the reaction mixture and obtained in crystalline form. The crystal form of these derivatives is similar to that of pepsin. Fractionation and solubility determinations show that these preparations are not mixtures or solid solutions of the original pepsin with an inactive derivative. A compound which contains three or four acetyl groups and which has lost all of its original primary amino groups can be isolated after short acetylation. It has the same activity as the original pepsin. A second derivative containing six to eleven acetyl groups has also been isolated. It has about 60 per cent of the activity of the original pepsin. A third derivative having twenty to thirty acetyl groups and about 10 per cent of the activity of original pepsin can be isolated after prolonged acetylation. The 60 per cent active derivative on standing in strong acid solution loses some of its acetyl groups and at the same time regains the activity of the original pepsin. The compound obtained in this way is probably the same as the completely active three acetyl derivative obtained by mild acetylation. These results show that acetylation of three or four of the primary amino groups of pepsin causes no change in the specific activity of the enzyme but that the introduction of acetyl groups in other parts of the molecule results in a marked loss in activity. The solubilities, amino nitrogen content, acetyl content, isoelectric point, and the specific activity have been determined by a variety of methods and found to be different from the corresponding properties of crystalline pepsin. The pH-activity curves, acid and alkali inactivation, and titration curves were not significantly different from the same respective properties of pepsin.     

Pseudo-esterase activity of human albumin: slow turnover on tyrosine 411 and stable acetylation of 82 residues including 59 lysines

Human albumin is thought to hydrolyze esters because multiple equivalents of product are formed for each equivalent of albumin. Esterase activity with p-nitrophenyl acetate has been attributed to turnover at tyrosine 411. However, p-nitrophenyl acetate creates multiple, stable, acetylated adducts, a property contrary to turnover. Our goal was to identify residues that become acetylated by p-nitrophenyl acetate and determine the relationship between stable adduct formation and turnover. Fatty acid-free human albumin was treated with 0.5 mm p-nitrophenyl acetate for 5 min to 2 weeks, or with 10 mm p-nitrophenyl acetate for 48 h to 2 weeks. Aliquots were digested with pepsin, trypsin, or GluC and analyzed by mass spectrometry to identify labeled residues. Only Tyr-411 was acetylated within the first 5 min of reaction with 0.5 mm p-nitrophenyl acetate. After 0.5-6 h there was partial acetylation of 16-17 residues including Asp-1, Lys-4, Lys-12, Tyr-411, Lys-413, and Lys-414. Treatment with 10 mm p-nitrophenyl acetate resulted in acetylation of 59 lysines, 10 serines, 8 threonines, 4 tyrosines, and Asp-1. When Tyr-411 was blocked with diisopropylfluorophosphate or chlorpyrifos oxon, albumin had normal esterase activity with beta-naphthyl acetate as visualized on a nondenaturing gel. However, after 82 residues had been acetylated, esterase activity was almost completely inhibited. The half-life for deacetylation of Tyr-411 at pH 8.0, 22 degrees C was 61 +/- 4 h. Acetylated lysines formed adducts that were even more stable. In conclusion, the pseudo-esterase activity of albumin is the result of irreversible acetylation of 82 residues and is not the result of turnover.     

N-Acetylbenzotriazole as a protein reagent. Specific behaviour towards delta-chymotrypsin.

When N-[14C] acetylbenzotriazole, presented here as a new agent for the acetylation of proteins, reacted at pH 8 and 25 degrees C with delta-chymotrypsin, 15 amino groups (the epsilon-amino groups of lysing residues and the alpha-amino terminus of half-cystine-1) and two phenolic groups (those of the two exposed tyrosine residues) were acetylated with respective pseudo first-order constants of 0.056 +/- 0.003 and 0.15 +/- 0.03 min(-1). Surprisingly, in contrast with the acetic anhydride reaction, the alpha-amino group of Ile-16 was found to be not acetylated as shown by N-terminus determination and activity measurements: the modified delta-chymotrypsin (or acetylated delta-chymotrypsin) was fully active after neutral dialysis. Only a transient inactivation due to the incorporation of one [14C] acetyl group per mole of catalytic site was observed. The kinetic constant found for reactivation at pH 8.5 was 0.315 +/- 0.005 min(-1) at 25 degrees C. The enzyme-catalyzed hydrolysis of N-acetylbenzotriazole was described by a k(cat) value of 0.093 +/- 0.005 min(-1) at pH 7 and 25 degrees C. Circular dichroism changes observed at 230 nm during the reaction at pH 8.5, of acetylated delta-chymotrypsin with N-acetylbenzotriazole indicated a total conversion of the amount of enzyme molecules which were in the 'inactive' or 'alkaline' conformation at this pH, into the 'active' or 'neutral' one. Benzotriazole alone was unable to induce such a conformational change. The rate constant of the reverse structural process from the 'neutral' to the 'alkaline' conformation was 0.32 +/- 0.02 min(-1): identical to that of the deacetylation of the catalytic site. Thus, the unusual lack of acetylation of Ile-16 alpha-amino group during delta-chymotrypsin treatment with N-acetylbenzotriazole is interpreted as a stabilization of the enzyme 'neutral' conformation where the Ile-16 alpha-amino group is buried, thus inaccessible to the reagent. The properties of the delta-chymotrypsin modification using N-acetylbenzotriazole led to practical uses: direct spectrophotometric titration of chymotrypsin operational normality at pH 7 and rapid preparation of acetylated delta-chymotrypsin. As a protein reagent, N-acetylbenzotriazole is particularly interesting because of its reactivity towards amino and phenolic groups of amino acid residues, its stability at acid pH, i.e., k(hydrolysis=7.38 X 10(-3) min(-1) at 25 degrees C [Ravaux et al. (1971) Tetrahedron Letters, 4013-4015] and its aromaticity, responsible for optical properties.     

INACTIVATION OF PEPSIN BY IODINE AND THE ISOLATION OF DIIODO-TYROSINE FROM IODINATED PEPSIN

In the presence of iodine at pH 5.0–6.0 a solution of pepsin absorbs iodine and the specific proteolytic activity of the solution decreases. The activity is less than 1 per cent of the original activity when the number of iodine atoms per mol of pepsin is 35–40. If the pH is 4.5 or less, iodine reacts very slowly and there is a correspondingly slower loss in activity. Glycyl tyrosine reacts with iodine in a manner similar to pepsin. Experiments were performed to determine the extent to which oxidation of pepsin by iodine occurs during iodination, and if such oxidation were responsible for the loss in enzymatic activity. Although the results were not absolutely decisive, there seems to be no appreciable oxidation taking place during iodination and no relationship between the slight oxidation and loss in peptic activity. From a dialyzed preparation of completely iodinated pepsin which was inactive and contained 13.4 per cent bound iodine, 82 per cent of the iodine was obtained in a solution which analyzed as a solution of diiodo-tyrosine. Because of the presence of a material which contained no iodine and prevented quantitative crystallization, only 53 per cent of the iodine containing substance could be crystallized. This 53 per cent was, however, identified as diiodo-tyrosine. The part of the titration curve which in pepsin and most proteins represents the phenolic group of tyrosine was, in the curve for iodinated pepsin, shifted toward the acid region as expected. From these results, it appears that the loss in proteolytic activity of pepsin, when treated with iodine under the specified conditions, is due to the reaction of the iodine with the tyrosine in pepsin.     

Involvement of tyrosine and lysine residues of retinol-binding protein in the interaction between retinol and retinol-binding protein and between retinol-binding protein and prealbumin. Acetylation with N-acetylimidazole and alkaline titration.

The behavior of holo-retinol-binding protein (RBP) from human plasma at alkaline pH was examined by absorption and circular dichroism measurements. Between pH 7.5 and 11.7 the ionization of the phenolic hydroxyl groups is reversible. However, there is a gradual irreversible loss of retinol as the pH is raised. After 4 hours at pH 11.7, 13 percent of retinol is lost from retinol-RBP. Alkaline titration of apo-RBP was time-independent and reversible between pH 7.5 and 11.7. The titration data of the phenolic hydroxyl groups in apo-RBP could be fitted with a single theoretical ionization curve of 8.6 phenolic groups having an apparent pK of 11. Acetylation of retinol-RBP with 10-fold molar excess of N-acetylimidazole over tyrosine resulted in the acetylation of all lysine residues and in the acetylation of 0.9 to 1.3 tyrosyl residues per molecule (out of eight). Acetylation of retinol-RBP, APO-RBP, and retinol-RBP-prealbumin complex with 50-fold molar excess of N-acetylimidazole resulted, again, with all of the lysine residues being acetylated and between 1.8 and 2.8 tyrosyl residues per molecule being acetylated. The acetylation did not affect the interaction between retinol and RBP. However, acetylation disrupted the normal binding between retinol-RBP and prealbumin. Deacetylation of tyrosyl residues with hydroxylamine failed to restore the normal binding of retinol-RBP to prealbumin. This excludes the acetylated tyrosyl-residues from being involved in the binding between the two proteins.     

Establishment of the enzymatic protein acetylation independent of acetyl CoA: recombinant glutathione S-transferase 3-3 is acetylated by a novel membrane-bound transacetylase using 7,8-diacetoxy-4-methyl coumarin as the acetyl donor

The current knowledge on biological protein acetylation is confined to acetyl CoA-dependent acetylation of protein catalyzed by specific acetyl transferases and the non-enzymatic acetylation of protein by acetylated xenobiotics such as aspirin. We have discovered a membrane-bound enzyme catalyzing the transfer of acetyl groups from the acetyl donor 7,8-diacetoxy-4-methyl coumarin (DAMC) to glutathione S-transferase 3-3 (GST3-3), termed DAMC:protein transacetylase (TAase). The purified enzyme was incubated with recombinant GST3-3 subunit and DAMC, the modified protein was isolated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) in gel digested with trypsin and the tryptic digest was analyzed by mass spectrometry. The N-terminus and six lysines, Lys-51, -82, -124, -181, -191 and -210, were found to be acetylated. The acetylation of GST3-3 described above was not observed in the absence of either DAMC or TAase. These results clearly establish the phenomenon of protein acetylation independent of acetyl CoA catalyzed by a hitherto unknown enzyme (TAase) utilizing a certain xenobiotic acetate (DAMC) as the active acetyl donor.     

LOCALIZATION OF DNA AND PROTEIN IN TIPULA IRIDESCENT VIRUS (TIV) BY ENZYMATIC DIGESTION AND ELECTRON MICROSCOPY

De-embedded ultrathin sections of ethanol-fixed Tipula Iridescent Virus particles were incubated with pepsin at pH 1.8, trypsin at pH 7.7, and DNase at pH 7.7. The outer shell of the particles, but not an inner core, was removed by the action of pepsin. Conversely, the inner core, but not the outer shell, was removed by the action of trypsin and DNase in combination, but not by either enzyme acting alone. These results are taken to mean that the outer shell of the particles is protein in nature and the inner core is nucleoprotein. Whole virus particles were also exposed to the same 3 enzymes. Trypsin and/or DNase had no effect on the whole particles, while pepsin at pH 1.8 digested away the outer shell of the particles and released an intact core, resistant to pepsin. The protein nature of the digested outer shells and the nucleoprotein nature of the released cores were confirmed by ultraviolet absorption spectra. Chemical analyses showed that the cores contain 89 per cent of the whole virus phosphorus but only 35 per cent of the nitrogen, while the outer shells contain only 5 per cent of the phosphorus but 63 per cent of the nitrogen. On the basis of nitrogen: phosphorus ratios the composition of the cores is estimated to be about 30 per cent DNA and 60 to 65 per cent protein.     

Nonenzymatic acetylation of histones with acetyl phosphate and acetyl adenylate.

Nonenzymatic acetylation of calf-thymus lysine- and arginine-rich histones was demonstrated to occur when these proteins were incubated with [14C]acetyl phosphate and [14C]acetyl adenylate. The levels of acetylation depend on both pH and on reagent concentration. When acetyl [33P]phosphate and acetyl [3H]adenylate were used as reagents, we found neither histone phosphorylation nor adenylylation. Most of the radioactivity of 14C-labeled acetylated histones was recovered as Ne-acetyllysine. Furthermore, only a small amount of O-bound radioactivity was released by the 14C-labeled acetylated arginine-rich histone during treatment with hydroxylamine. Experiments on the acetylation of histones, in the presence of increasing salt concentration, gave different results for the two acetylating agents.     

INACTIVATION OF PEPSIN BY IODINE : II. ISOLATION OF CRYSTALLINEl-MONO-IODOTYROSINE FROM PARTIALLY IODINATED PEPSIN

1. Pepsin solutions were iodinated at pH 5.0–6.0 until 10–20 per cent of the activity was lost and 1/20 (0.7 per cent) of the saturating amount of iodine had been introduced into the protein molecule. After alkaline hydrolysis 65 per cent of the original iodine was accounted for as mono-iodotyrosine although only 42 per cent was isolated as a crystalline product. No evidence was obtained to support the possibility that any group other than tyrosine in pepsin was iodinated. 2. Some of the properties of the crystalline l-mono-iodotyrosine were determined and compared to those of di-iodotyrosine. 3. One iodinated pepsin preparation was crystallized. The crystal form was the same as that of the original pepsin. A solubility curve of the crystals demonstrated that it was very different from pepsin and had nearly constant solubility.     

Titration study of acetylated lysozyme.

To study the interaction between carboxyl groups and amino groups in native lysozyme [EC 3.2.1.17], and to identify the positions and the pK values of the abnormal carboxyl groups, N-acetylated lysozyme was prepared. The acetylation did not affect the molecular shape of the enzyme, but changed six amino groups to a non-ionizable form, leaving one amino group free; this was determined to be Lys 33. In addition, pH titration of the acetylated lysozyme in 0.2 or 0.02 M KCl aqueous solution indicated fewer titratable groups with pK(int) of 7.8 or 10.4 compared with the native protein, though the number of titratable carboxyl groups was not affected by the acetylation. From the pH titration results and structural considerations, the unititratable carboxyl groups were suggested to be Asp 48, Asp 66, and Asp 87. On the other hand, spectrophotometric titration in 0.2 M KCl showed that all three tyrosine residues are titratable in the acetylated protein, although an abnormal tyrosine residue exists in the native state. Tyr 20 was suggested to be untitratable in the pH range of 8-12.6.     

Acetylation of Amino Groups and Its Effect on the Structure of Soybean Glycinin

Purified soybean glycinin (11S globulin) was acetylated at three degrees; low (21-51%), middle (60-81%) and high (90-92%) acetylation in lysine residues. With increasing the acetyl content, the β-structure gradually decreased and the random structure increased resulting in the exposure of tyrosine residue. These were determined from the results of optical rotatory dispersion, intrinsic viscosity, ultraviolet and fluorescence measurement. Gel filtration, ultracentrifugation and gel electrophoresis studies showed drastic conformational changes of highly acetylated 11S (over 90%), in which most of the modified protein (75%) polymerized, and the other dissociated into 3S protein. The close relation between the conformation of acetylated 11S and its emulsifying properties was discussed.     

Mode of action of Fusarium moniliforme endopolygalacturonase towards acetylated pectin

Endopolygalacturonase from Fusarium moniliforme was used to degrade acetylated homogalacturonan previously prepared from sugar beet pulp. The initial velocity and the final percentage of hydrolysis decreased very rapidly with increasing degree of acetylation, showing that acetyl substitution markedly affected the enzymatic activity. MALDI-TOF mass spectrometry was used to analyse the reaction products and to show acetyl groups on the oligogalacturonates. The results demonstrated that the enzyme was able to accommodate acetyl groups in its active site cleft. The influence of acetyl groups on the mode of action of the enzyme was discussed and compared to the influence of methyl groups.     

Epigenetic characteristics of bovine donor cells for nuclear transfer: levels of histone acetylation

Donor cell type, cell-cycle stage, and passage number of cultured cells all affect the developmental potential of cloned embryos. Because acetylation of the histones on nuclear chromatin is an important aspect of gene activation, the present study investigated the differences in histone acetylation of bovine fibroblast and cumulus cells at various passages and cell-cycle stages. The acetylation was qualitatively analyzed by epifluorescent confocal microscopy and quantitatively by immunofluorescent flow cytometry. Specifically, we studied levels of histone H4 acetylated at lysine 8 and histone H3 acetylated at lysine 18; acetylation at these lysine residues is among the most common for these histone molecules. We also studied levels of linker histone H1 in donor cells. Our results show that stage of cell cycle, cell type, and number of cell passages all had an effect on histone content. Histone H1 and acetyl histone H3 increased with cell passage (passages 5-15) in G0/G1- and G2/M-stage cumulus and fibroblast cells. We also found that acetyl histone H4 was lower in early versus late cell passages (passage 5 vs. 15) for G0/G1-stage cumulus cells. In both cell types examined, acetyl histones increased with cell-cycle progression from G0/G1 into the S and G2/M phases. These results indicate that histone acetylation status is remodeled by in vitro cell culture, and this may have implications for nuclear transfer.     

ABSORPTION OF PEPSIN BY CRYSTALLINE PROTEINS

Crystalline proteins, such as edestin or melon globulin, remove pepsin from solution. The pepsin protein is taken up as such and the quantity of protein taken up by the foreign protein is just equivalent to the peptic activity found in the complex. The formation of the complex depends on the pH and is at a maximum at pH 4.0. An insoluble complex is formed and precipitates when pepsin and edestin solutions are mixed and the maximum precipitation is also at pH 4.0. The composition of the precipitate varies with the relative quantity of pepsin and edestin. It contains a maximum quantity of pepsin when the ratio of pepsin to edestin is about 2 to 1. This complex may consist of 75 per cent pepsin and have three-quarters of the activity of crystalline pepsin itself. The pepsin may be extracted from the complex by washing with cold N/4 sulfuric acid. If the complex is dissolved in acid solution at about pH 2.0 the foreign protein is rapidly digested and the pepsin protein is left and may be isolated. The pepsin protein may be identified by its tyrosine plus tryptophane content, basic nitrogen content, crystalline form and specific activity.     

Histone acetylation in rat liver nuclei

Rat liver nuclei were incubated for various lengths of time in the presence of increasing concentrations of acetyl CoA. The rate of acetylation varied strongly according to the acetyl CoA concentration. A very small part of the histone was acetylated. After incubation in the presence of increasing acetyl CoA concentrations, four apparent Km could be determined. Electrophoretic analysis showed that only histone H3 was acetylated which suggests that each Km corresponds to a sequential acetylation of its lysine residues. This could be correlated with the possible role of histone acetylation in the control of gene activity.     

Modification of isoleucine-16 acetylated delta-chymotrypsin.

Activation of acetylated chymotrypsinogen with trypsin leads to catalytically active acetylated delta-chymotrypsin containing NH2-terminal isoleucine. The importance of the cationic terminus to the control of the active conformation of acetylated delta-chymotrypsin has been demonstrated (Oppenheimer, H. L., Labouesse, B., and Hess, G. P. (1966) J. Biol. Chem. 241, 2720). Later studies appeared to suggest that the modification of isoleucine-16 of delta-chymotrypsin is not accompanied by the loss of catalytic activity as measured by the hydrolysis of N-acetyl-L-tyrosine ethyl ester (Agarwal, S. P., Martin, C. J., Blair, T. T., and Marini, M.A. (1971)Biochem. Biophys. Res. Commun. 43, 510; Blair, T. T., Marini, M. A., Agarwal, S. P., and Martin, C. J. (1971) FEBS Lett. 1486) or by the loss of active site content (Ghelis, C., Garel, J. R., and Labouesse, J. (1970) Biochemistry 9, 3902). In the present studies, controlled acetylation of the terminal alpha-aminogroup of acetylated delta-chymotrypsin with acetic anhydride led to a progressive loss of active sites of the enzyme. Determination of the catalytic and kinetic properties of the modified enzyme with the specific ester substrate N-acetyl-L-tyrosine ethyl ester or the nonspecific substrates p-nitrophenyl acetate and cinnamyol imidazole gave nearly identical results. With N-acetyl-L-tyrosine ethyl ester as substrate, the Km (app) values for acetylated delta-chymotrypsin (1.0 plus or minus 0.1 mM) and the modified enzyme (0.67 plus or minus 0.05 mM) are nearly identical and the kcat value is reduced to about 25% in the latter enzyme species. This value correlates well with about 20% of the active sites in this enzyme as measured by the rapid initial liberation of p-nitrophenol. With p-nitrophenyl acetate as substrate, the acylation rate constants (0.13 plus or minus 0.04 s(-1) at pH 6.0, 25 degrees, in 3.3% acetonitrile) and the deacylation rate constants (0.01 s(-1) at pH 8.5, 25 degrees, in 3.3% acetonitrile) are identical for the acetyl isoleucine-16 and the isoleucine-16 enzymes. Furthermore, the residual enzyme activity could be correlated well with the residual NH2-terminal isoleucine content and with the moles of [1--14C]acetyl groups incorporated per mol of the enzyme. The activity associated with the modified enzyme can be attributed to the enzyme species in which isoleucine-16 of acetylated delta-chymotrypsin is not acetylated. These data are in general agreement with the studies of Ghelis et al. (1970) but are in disagreement with the results of Blair et al. (1971) and of Agarwal et al. (1971) and confirm the hypothesis that the final conformation of acetylated delta-chymotrypsin containing an acetylated NH2 terminus is catalytically inactive and resembles acetylated zymogen in many of its physical properties.     

Acetylation of Lys-373 in porcine pancreatic lipase after reaction of the enzyme or its C-terminal fragment [corrected] with p-nitrophenyl acetate

The reactions of lipase (449 amino acid residues) and lipase fragment (336-449) with p-nitrophenyl acetate have been studied from 2 different angles. In previous papers it has been shown that lipase and lipase fragment enzymatically hydrolyze p-nitrophenyl acetate. The amino acid residue of the catalytic site that is temporarily acetylated has not yet been characterized in lipase or lipase fragment. Besides this very fast enzymatic hydrolysis, acetylation reactions may take place on nucleophilic amino acid side-chain groups. In the present report, acetylated amino acid residues whose acetyl linkages were not cleaved after pH 7.5-8.5 incubations have been investigated. Several residues were acetylated in very low proportion, whereas lysine 373 was stoichiometrically acetylated in lipase and in lipase fragment. This specific acetylation may have been favored by the presence of a hydrophobic reversible binding site for p-nitrophenyl acetate near Lys-373. This acetylation did not greatly change the specific activity of lipase towards an emulsion of tributyrylglycerol in the presence of colipase, but under certain conditions it had an effect on the enzymatic hydrolysis of p-nitrophenyl acetate by the lipase fragment.     

The role of acidic dissociation of substrate's phenol group in the mechanism of tyrosine phenol-lyase

pH dependencies of the main kinetic parameters for the reaction of tyrosine phenol-lyase (TPL) with L-tyrosine were studied earlier at pH from 6.0 to 9.5. At this range, L-tyrosine, whose pK(a) for the phenol hydroxyl is 10.5, exists as the zwitterion. It was concluded that zwitterion is the only "active" form for any tyrosine-like substrate. In the present work, we examined pH dependencies for 2-fluorotyrosine, 3-fluorotyrosine, 3,5-difluorotyrosine, 2,5-difluorotyrosine, 2,6-difluorotyrosine, and 3-chlorotyrosine which are more acidic than tyrosine. Respective pK(a)'s are 9.2, 8.7, 7.3, 7.9, 8,35, and 8.3. At higher pH, most of these substrates exist predominantly as anions, having two negative charges at the carboxylic and phenol groups, and one positive charge at the amino group. No decrease of k(cat)/K(m) values attributable to acidic dissociation of the phenol group was found. From comparison of theoretical curves with the experimental data, we conclude that most likely, both zwitterion and anion forms of 3-fluorotyrosine, 3,5-difluorotyrosine, 2,5-difluorotyrosine, 2,6-difluorotyrosine, and 3-chlorotyrosine may be bound and subsequently catalytically transformed by TPL. The reactivities of the two forms are quite comparable. The roles of catalytic groups in the active site, especially Arg381 and Thr-124, are discussed.