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Using automated stepwise Edman degradation of suitable overlapping peptides, the complete amino acid sequence of the 268 residue peptida α1-CB7 from calf skin collagen was determined. The preparation and ordering of the chymotryptic, tryptic and thermolytic peptides is described in the preceding paper. As shown previously for other peptides from the helical region of collagen, glycine appears in every third position and proline is present in high amount. Hydroxylation of proline to 4-hydroxyproline and lysine to hydroxylysine only occurred in the Y position of the tripeptide unit Gly-X-Y. Non-random distribution of other amino acids between the X and Y positions was also observed. [ABSTRACT FROM AUTHOR]

Chymotryptic cleavage of α1-CB6, the C-terminal CNBr peptide of the α1-chain from calf skin collagen, yields three peptides, designated C1 (positions 1-113), C2 (positions 114-196) and C3 (positions 197-217), respectively. The present paper describes the amino acid sequence of C2. It was determined exclusively by application of the automated Edman degradation. The entire peptide C2 as well as some of its tryptic and thermolytic peptides were subjected to this procedure. The helical portion of α1-CB6-C2 (positions 114-192) was found to consist of tripeptide units containing glycine in every third position, hence being typical of the collagen structure. The α1-chain of calf skin collagen contains a 3-hydroxyproline residue which was located in position 164 in the tripeptide unit Gly-3Hyp-4Hyp. This is the first case of a vertebrate collagen in which hydroxylation was found of a proline in position X of the tripeptide unit Gly-X-Y. The helical part of the α1-chain terminates in a five-fold repetition of the tripeptide Gly-Pro-Pro. The four C-terminal amino acids of C2 are already part of the nonhelical area at the end of the α1-chain which in addition comprises the entire peptide C3. [ABSTRACT FROM AUTHOR]

The complete sequence (153 amino acids) of horse heart myoglobin has been established. The sequence of a peptide isolated from chymotrypsin hydrolyzates of globin allowed to fill a gap in the tentative sequence previously proposed; this peptide was studied by means of pepsin hydrolysis, action of N-bromosuccinimide on histidyl bonds, and Edman degradation method associated with dansylation. After hydrolysis of globin by chymotrypsin treated by TLCK, the same major peptides were found as after hydrolysis by non-treated chymotrypsin. These peptides were identified by ionexchange resins and bidimensional paper chromatography, and also by the determination of their amino-acid composition. The suppression of secondary cuts explains the presence of new peptides, whose the composition and N-terminal Sequences were determined, it was then possible to confirm several sequences which could be previously deduced from the study of split products after cyanogen bromide treatment. The specificity of chymotrypsin towards certain types of peptide bonds is discussed. Horse myoglobin, as compared with sperm whale myoglobin, contains 18 differences: 17 substitutions and one inversion. The major part of the substitutions are located ih N- and C-terminal sequences: they are all punctiform, resulting from the replacement of only one base in each codon. [ABSTRACT FROM AUTHOR]

Peptide A, the C-terminal cyanogen bromide fragment of thioredoxin, was degraded with chymotrypsin and pepsin. Partial sequences of 12 chymotryptic and 6 peptic peptides and the N-terminal tryptic peptide of trifluoro-acetylated peptide A were determined. The results were used to establish the order of the previously described tryptic peptides of peptide A and lead to the complete amino acid sequence of peptide A. Previous experiments had established the amino acid sequence of peptide B, the N-terminal cyanogen bromide fragment of thioredoxin and the present results thus give the complete amino acid sequence of thioredoxin from Escherichia coli B. The molecule contains 108 residues in a single polypeptide chain with a molecular weight of 11,657 as calculated from the sequence. The functional group of the protein occurs in position 32 to 35 and consists of a disulfide bridge formed by two half-cystine residues separated by a glycine and a proline residue. No metals were found as part of the functional group. [ABSTRACT FROM AUTHOR]

Water-insoluble chymotrypsin, trypsin and papain have been synthesized by covalent fixation of the enzymes to beads of cross-linked dextran (Sephadex®), beads of agarose, and cellulose powder. The fixation procedure involves two simple steps: (a) activation of the polysaccharide polymer by a cyanogen halide; (b) coupling of the protein to the activated polymer in slightly alkaline medium. The activation process can be controlled so as to retain certain useful or essential properties of the carrier material, such as particle shape and swelling capacity. The enzyme conjugates of Sephadex and agarose are therefore especially suitable for use in the column format. The coupling process is so mild that a large portion of the inherent enzymic activity is conserved. However, the steric properties and other features of the carrier framework often prevents full manifestation of the fixed activity. Catalysts that are highly efficient toward small substrates are easily obtainable. The agarose-bound enzymes can even exhibit high activities toward large proteins. Conjugates of the latter type should therefore be particularly useful for the digestion of proteins. [ABSTRACT FROM AUTHOR]

1. The ethanol-active isoenzyme of horse liver alcohol dehydrogenase has been carboxymethylated with iodo[14C]acetate and different samples of the labelled protein treated with trypsin (both with and without previous maleylation of the substrate), chymotrypsin, pepsin or cyanogen bromide. In each case most of the resultant peptides have been purified and characterized. 2. Data are given for those peptides originating from an N-terminal region comprising about 40% of the protein chain and the amino acid sequence of the first 140 residues is deduced. 3. It is established that over this region the two chains of the enzyme have an identical sequence. The N-terminus is an acetylated serine residue and the "active site" cysteine residue occupies position no. 46. One of the two tryptophans of the chain, but none of the four tyrosines, is in the sequence given. The region around position 100 is rich in cysteine residues. Some serine residues were found to have reacted during maleylation of the protein. 4. The amino acid sequence is compared to the previously known sequences of glyceraldehyde 3-phosphate dehydrogenase from two species. Similarities strongly suggest a true homology, at least of an N-terminal region, and support theories of a common ancestral origin for these enzymes. The reactive cysteine residues are, however, at widely different positions in the primary structures. [ABSTRACT FROM AUTHOR]

The C-terminal fragment of hog pepsin, which had been obtained in the preceding study on the cyanogen bromide hydrolysate of this protein, was subjected to sequential studies. The 37-residue peptide was digested in independent experiments by trypsin, chymotrypsin, and pepsin, and the amino acid sequence of the arising peptides was determined. In parallel experiments the sequence of seventeen amino acid residues in the N-terminal region of the peptide was established by the Edman degradation technique. The obtained data permit the C-terminal peptide of hog pepsin to be ascribed the amino acid sequence Asp-Val-Pro-Thr-Ser-Ser-Gly-GluLeu-Trp-Ile-Leu-Gly-Asp-Val-Phe-Ile-Arg-Gln-Tyr-Tyr-Thr-Val-Phe-Asp-Arg-Ala-Asn-Asn-LysVal-Gly-Leu-Ala-Pro-Val-Ala. These results extend and pauly correct our previous knowledge of this region of the pepsin molecule as recorded in literature. The problem of the tryptophan content of pepsin is briefly discussed. [ABSTRACT FROM AUTHOR]

The reaction rates between dansyl chloride and water, amino acids or peptides are studied as a function of pH and temperature. The rate of hydrolysis of dansyl chloride is constant and low up to pH 9.5 and above this pH it increases rapidly. The various reactive groups of amino acids and peptides react with dansyl chloride in their unprotonated form. It is shown that a compromise for optima conditions of dansylation (pH and temperature) may be found, taking into account the rate of hydrolysis and the pk of the group to be dansylated. A complete chromatographie separation on Silica gel G thin layer plates of dansyl amino acids is proposed using 4 solvents. The quantitative recovery of the separated derivatives is described. The study of the acid hydrolysis of dansylated proteins shows that the release of the dansyl derivatives from the peptide chain is faster than that of free amino acids; it also shows that the destruction of dansyl amino acids occurs rather rapidiy. Therefore a hydrolysis time of 4 hours (110°) is suggested, except in the special cases of the release of dansyl valine, dansyl leucine or dansyl isoleucine which needs 18 hours of hydrolysis. For quantitative estimation of each dansyl derivative a correction factor is given, taking into account the loss during hydrolysis, the recovery from the plates and the individual fluorescence of any dansyl derivative as compared to the fluorescence of a reference compound. The general conditions described in this work require 1 nmole of material for qualitative determination of the N-terminal amino acids, and 5-10 nmoles for their quantitative estimation. Some examples (α-chymotrypsin, trypsinogen, ribonuclease, lysozyme aspartokinase and triose phosphate dehydrogenase) illustrate the efficiency of the method. [ABSTRACT FROM AUTHOR]

The role of a chymotrypsin-like enzyme in the release of histamine in anaphylaxis has been investigated using sensitized rat peritoneal mast cells, Inhibition of this enzyme did not inhibit histamine release and, conversely, when histamine release was blocked the esterase activity was unaffected. It is concluded that the active chymotrypsin-like enzyme in these cells is not involved in the anaphylactic reaction. One of the inhibitors used (DFP) produced a dual action on histamine release. A possible explanation is discussed. [ABSTRACT FROM AUTHOR]

1. Selective modification of lysine-15 at the active site of the basic pancreatic trypsin inhibitor strongly alters its association with trypsin. Ka, the equilibrium constant for the association, and kd, the first-order rate constant for the dissociation are 20 nM-1 and 1.0 × 10-4 s-1 for the trypsin · 15.guanidinated-inhibitor complex as compared to 16 pM-1 and 6.6 × 10-8 s-1 for the trypsin · native-inhibitor complex. Guanidination of lysine residues (Lys26, Lys41 and Lys46) outside the active site of the inhibitor does not affect the stability of the binding with trypsin. 2. The selective guanidination of lysine-15 in the pancreatic inhibitor does not alters its association with chymotrypsin. 3. Reduction and carboxamidomethylation of the disulfide bridge Cys14-Cys38 in the selectively guanidinated inhibitor decreases Ka by a factor of 4.6 and increases kd by a factor of 55. The same chemical modification of the Cys14-Cys38 bridge in the non-guanidinated inhibitor decreases Ka by a factor of 2700 and increases kd by a factor of 330. [ABSTRACT FROM AUTHOR]

A chymotrypsin-like endopeptidase has been purified by ion-exchange chromatography, affinity chromatography, and gel filtration. The enzyme preparation is homogeneous in the ultracentrifuge and disc electrophoresis. The enzyme is proved to be free from any other proteolytic activities. The molecular weight of the proteinase as determined with several techniques (ultracentrifugation, gel filtration and electrophoresis without dodecylsulfate) is in the range between 13000–14000; however, by dodecylsulfate gel electrophoresis a molecular weight of 23000 was obtained. The obtained physical constants of hornet chymotrypsin are: sedimentation coefficient s20,w0 = 1.96 ×10-13 s, diffusion coefficient D20,20 = 131 μm² s-1, partial specific volume &vline; = 0.737 ml g-1, frictional ratio ƒ/ƒmin = 1.04, and degree of hydration = 0.1 g per g protein. [ABSTRACT FROM AUTHOR]

The apparent first-order rate constant (kobs) for the deacylation of α-benzamido-transcinnamoyl-chymotrypsin, determined by direct observation at 310 nm, pH 7.9 (kobs = 0.102 s-1; K. Brockelhurst and K. Williamson [1967] Chem. Commun., 666), differs from Kcat determined under the same conditions with [S]0 ... [E]0 (kcat = 0.033 s-1). This result is contrary to other evidence indicating that kcat is a measure of the deacylation rate constant. The discrepancy has been shown to be due to the presence in commercial samples of α-chymotrypsin of impurities which catalyse the deacylation reaction under the conditions used for the determination of kobs ([E]0 [S]0). The impurities, which may be removed from enzyme solutions by gel filtration on Sephadex G-25 or G-50, have been shown to be autolysis products (presumably peptides) of α-chymotrypsin. Catalysis of deacylation results from attack of the free α-amino groups of these peptides on the acyl-enzyme, to give N-α-benzamido-trans-cinnamoyl-peptides. The consequences of these findings must be considered in interpreting any experiment in which deacylation is observed directly at high enzyme concentration, with [E]0 [S]0. Particularly susceptible to error are experiments designed to determine the effect of pH on the deacylation rate constant, and literature results of some such experiments are critically assessed. [ABSTRACT FROM AUTHOR]

A number of peptide substrates of the general structure Ac-Lxn … -Lx2-Lx1-Gly-N'H2, have been synthesized and their α-chymotrypsin-catalyzed hydrolyses studied. The acylation rate constants, k23 (= kcat), and the dissociation constants of the enzyme-substrate complexes, KEA (= Km), have been determined using a modified pH-stat and a numerical method for the acquisition and processing of the data. On the basis of these constants a quantitative relationship between the peptide structure N-terminal to the cleaved bond and reactivity has been determined. The results are shown to be consistent with the enzyme-substrate interaction scheme proposed in 1971 by Segal et al. (Biochemistry 10, 3728). A comparison of the k23/KEA values indicates that the influence of a single structural change on the overall reactivity is virtually independent of the nature of the remainder of the substrate. In addition a comparison of the k23 and KEA values shows that, in general, changes in substrate structure are mainly reflected by changes in k23 rather than in KEA. A few exceptions have been found: KEA or KEA and k28 change when glycine is introduced at the x2 position, when this glycine is replaced by alanine or when alanine is introduced at the x3 position. [ABSTRACT FROM AUTHOR]

Preliminary to detailed amino acid sequence analysis of the peptide α1-CB7 from calf skin collagen, the chymotryptic, tryptic and thermolytic peptides were isolated and their positions in the intact chain determined. The collagenase derived peptides α1(78), α1(125) and α1(78)-CBN were also used as source of peptides and aided considerably the positioning of smaller fragments. All peptides isolated were placed in correct order as found subsequently in the more detailed sequence analysis. [ABSTRACT FROM AUTHOR]

1. The association constant, Ka, of the 1:1 complex formed between α-chymotrypsin and the pancreatic trypsin inhibitor is 110 μM-1 at pH 8.0 and 25°C. The second-order rate constant for the association, ka, is 110 mM-1 s-1 and the first-order rate constant for the dissociation, kd, is 10-3 s-1 under the same conditions. Thermodynamic parameters for complex formation at 25°C, pH 8.0 are δGa0 = -11.0 kcal × mol-1, δHa0 = 3 kcal × mo1-1 and ΔSa0 = 48 cal. · mol-1 · K-1. 2. Temperature and pH-dependences of the rate constants ka and kd have been studied. 3. The difference in stability between the α-chymotrypsin. inhibitor complex and the trypsin · inhibitor complex (Ka = 16 pM-1, ΔGa0 = -18.1 kcal × mo1-1) is due essentially to differences of kd values. The dissociation of the α-chymotrypsin · inhibitor complex is 1.5 × 104 times faster than that of the trypsin · inhibiter complex. Differences in Ka and kd values are discussed in terms of differences in stabilizing interactions. 4. The Cys14-Cys38 disulfide bridge of the inhibitor, which is highly succeptible to reduction when the inhibitor is free, is masked in the α-chymotrypsin · inhibitor complex. Reduction of the Cys14-Cys38 bridge in the free inhibitor does not prevent association with α-chymotrypsin. Values of ka and kd for the formation of the 1:1 α-chymotrypsin · reduced-inhibitor complex and for the complex formed with the native inhibitor are very similar. This situation differs from that observed with the trypsin · pancreatic-inhibitor complex. In that case, reduction of Cys14-Cys38 bridge considerably decreases the stability of the complex formed with trypsin; Ka is decreased by a factor of 3 × 104 and kd is increased by a factor of 8.6 × 103. [ABSTRACT FROM AUTHOR]

Nerve-growth factor, a substance specifically promoting, in vitro, the growth of nerve fibres from explants of embryonic dorsal root and sympathetic ganglia, has been purified from the venom of Vipers russelli. The purification proved remarkably difficult and presented some unusual features. The final product, which was homogeneous to all the tests available to us, was found to the a glycoprotein of molecular weight 37000 and containing about 20 % of carbohydrate. From the values of the sedimentation and diffusion coefficients it may be shown thai the axil ratio is approximately 1:7; the molecule has, therefore, an unusual shape. The substance is highly basic (isoetectric point approximately pH 10.5). It is also relatively stable: neither trypsin nor chymotrypsin affect its biological activity. However, activity is completely destroyed on treatment with pepsin. Comparison with nerve-growth factor from other sources does not, with the data at present available, reveal any striking similarities. The difficulties involved in the isolation and characterization of this factor from snake venoms are briefly discussed. [ABSTRACT FROM AUTHOR]

Succinylated derivatives of bovine chymotrypsinogen A and α-chymotrypsin were prepared by treatment of the native proteins with succinic anhydride; 60% of the 6-amino groups of the 13 lysine residues form stable succinylated products. The large negative charge at neutral pH is reflected in electrophoretic and titration behavior but absorption spectra, rotatory-dispersion spectra and reversible unfolding behavior indicate that the succinylated species are conformationally similar to the parent proteins. Succinylated chymotrypsin is active as an esterase with N-acetyl-L-tryptophan ethyl ester with a small decrease in the maximum-velocity parameter. The pH dependence suggests that three ionizing groups influence catalytic behavior. The apparent pKa values analyzed in terms of three groups are shifted relative to α-chymotrypsin. Thermal-unfolding studies of succinylated chymotrypsinogen demonstrate the presence of substrates near pH 7 but is consistent with two-state behavior at other pH values. [ABSTRACT FROM AUTHOR]

1. The replacement of the phenolic proton of tyrosine by alkyl, aryl or acyl groups completely abolishes the chymotryptic hydrolysis of tyrosine esters. Similarly, chymotrypsin fails to hydrolyze the tyrosyl bonds of dinitrophenyladrenocorticotropin. 2. Several model substrates have been synthesized and characterized. The action of chymotrypsin was followed potentiometrically in buffer containing some alcohol. The hydrolysis of the analogous compounds unsubstituted at the phenolic group was normal. 3. We conclude that inhibition is due, not to an inductive effect, but to a blocking effect which is markedly affected by replacement of the phenolic proton, even with a group as small as a methyl group. [ABSTRACT FROM AUTHOR]

The combination of trypsin and chymotrypsin with the Bowman-Birk inhibitor results in characteristic changes in absorption and fluorescence emission spectra, indicating an alteration of the microenvironments of the enzyme chromophores as a consequence of the interaction. One of the two tyrosine groups of this inhibitor is accessible to nitration with tetranitromethane. The pK of the resultant nitrotyrosine group is shifted to lower values upon combination of the inhibitor with trypsin or chymotrypsin, suggesting a proximity to the zone of contact. The reaction of trypsin with Bownian-Birk inhibitor is biphasic, an initial second-order combination being followed by a slower first-order change. The rate constant for dissociation of the complex increases at acid pH. The dissociation has a high activation energy. Bowman-Birk Inhibitor which has been convened to the modified form by prolonged incubation with catalytic amounts of trypsin forms a complex with trypsin which has the same rate constant and activation energy for dissociation as the complex formed by the native inhibitor. [ABSTRACT FROM AUTHOR]

Single crystals of active and phenylmethanesulfonyl-treated γ-chymotrypsin and δchymotrypsin were iodinated. The sites of iodination and their degree of occupancy were determined erystallographically. It was found that the pattern of iodination depends upon the form of chymotrypsin being iodinated, the pH, and whether the enzyme has been irreversibly inhibited, The effect upon γ-chymotrypsin catalytic activity of the iodination of specific residues was observed toward acetyl tyrosine ethyl ester. Chymotrypsinogen was iodinated in solution under slightly varig conditions; it was impossible to iodinate chymotrypsinogen crystals in either native or glutaraldehyde crosslinked form. The iodochymotrypsinogens were activated by trypsin under slow and rapid conditions and the catalytic properties of the resulting chymotrypsins examined. The activity of the iodochymotrypsins toward acetyl tyrosine ethyl ester depends upon both the conditions of iodination and those of activation. The crystallographic results are compared with those obtained previously for γ- and α-chymotrypsins and possible explanations for observed differences are advanced. We propose from the kinetic data that the slow interconversion of α- and γ-chymotrypsins is controlled by the ionization of the tyrosine-146 sidechain. [ABSTRACT FROM AUTHOR]

1. Turkey ovomucoid and chicken ovoinhibitor which have been found to be inhibitors of bovine trypsin and α-chymotrypsin can inhibit the esterolytic, proteolytic and elastolytic activities of porcine elastase. The formation of the complex between the elastase and each inhibitor could be demonstrated by means of electrophoresis at pH 6.9. The complexes are formed in molar ratios of one to one. They are stable and no temporary inhibition could be observed. 2. The complexes of porcine elastase with turkey ovomucoid and chicken ovoinhibitor inhibit bovine trypsin at the same molar ration as free inhibitors. The complexes of trypsin with these inhibitors inhibit elastase as do the free inhibitors. To complex of porcine elastase with turkey ovomucoid does not inhibit bovine chymotrypsin and the complex of chymotrypsin with this inhibitor does no inhibit elastase. The complex of porcine elastase with chicken ovoinhibitor inhibits bovine chymotrypsin but not the alkaline proteinase does not. It was concluded, therefore, that turkey ovomucoid has two independent binding sites, one for trypsin and the other for chymotropsin or elastase and that chicken ovoinhibitor has three independent binding sites, one for trypsin, another for chymotrypsin and a third one for either elastase or alkaline proteinase. 3. The complexes between porcine elastase and turkey ovomucoid or chicken ovoinhibitor are not absorbed on elastin whereas elastase is. 4. Kunitz soybean and pancreatic trypsin inhibitors, Birk-Bowman soybean trypsin and chymotrypsin inhibitor, groundnut and lima bean trypsin and chymotrypsin inhibitors do not inhibit the esterolytic, proteolytic and elastolytic activities of porcine elastase, even in weight ratios in excess of 10:1. [ABSTRACT FROM AUTHOR]

1. A comparative structural study of chymotrypsinogens A and B at acidic pH has shown in both cases an isomerisation I'⇌ II' occurs due to the unmasking of an abnormal carboxylate ion. 2.Chymotrypsin Bπ does not bind the profiavine which is a competitive inhibitor for trypsin and chymotrypsin Aα The acidic transition observed for the zymogens also exists for the corresponding enzymes (I⇌II). The unmasking of the abnormal carboxylate ions involves a dissociation of the chymotrypsin Aα or trypsin-proflavine complexes 3. The thermal transitions of chymotrypsinogen B have been studied and compared with those of chymotrypsinogen A, trypsinogen and chymotrypsin Bπ. 3. The thermal transitions of chymotrypsinogen B have been studied and compared with those of chymotrypsingogen A, trypsingogen and chymotrypsin Bπ. 4. The kinetics of denaturation by urea of chymotrypsinogen B, neochymotrypsingogen B, chymotrypsins Bπ and Bα were studied at different pH. The transformations of chymotrypsinogen B into neochymotrypsingoen and of chymotrypsin Bπ into chymotrypsin Bα are accompanied by a decreased stability in urea. The activation of chymotrypsingogen B to chymotrypsin Bπ increases the stability in urea. The activation of chymotrypsingogen B to chymotrypsin Bπ increases the stability of the molecule. However, the rates of denaturation of states II' and II of chymotrypsinogen and chymotrypsing Bπ are much more similar than he rates of denaturation of state I' and I. The implications of this result are discussed. Ca2+ binds specifically to chymotrypsinogens A and B and to trypsinogen. (pKCa2+ = 3.0, 3.1, and 3.2 respectively), probably on identical sites. The protection afforded by this binding against urea denaturation is of the same magnitude for trypsinogen and chymotrypsinogen A; it is much lower for chymotrypsinogen B. This observation together with the experiments concerning proflavine binding shows that chymotrypsinogen A shares with trypsinogen certain functional similarities that it does not share with chymotrypsinogen B. 5. The reactivity of phenol side-chains has been studied by titration and specific modification with acetylimidazole. The localisation of the normal and abnormal tyrosines in the sequences of chymotrypsinogens A and B is discussed. 6. The reactivity of indole side-chains was examined with 2-hydroxy-5-nitrobenzyl bromide. The four indoles of trypsinogen and the eight indoles of chymotrypsinogens A and B are masked in the native forms (I'). [ABSTRACT FROM AUTHOR]

It is shown that the change in activity and structure of acetyl-δ-chymotrypsin in the pH 7-11 range is not correlated with the ionizations of tyrosine residues. The pKs of the two titratable tyrosines of acetyl-δ-chymotrypsin are found to be 10.2 and 11.5, at 15° (9.6 and 10.8 after correction for electrostatic interactions), and those of the starting acetylated zymogen are 10.5 and 12.1 (10.0 and 11.5 after identical corrections). The plot of log rate of deacetylation of the titratable tyrosines versus pH is linear between pH 9 and pH 12. At a given pH there is a linear relation between the log deacetylation rate and the pKs of the residues, The pH rate profile of acylation of acetyl-δ-chymotrypsin by 4-carboxy-3-nitrophenyl- N,N-diphenylearbamate is bell-shaped, with a maximum at pH 7.9 at 15°, in 0.14 M KCl. The right side of the curve shows a pKapp of 8.9, whether the exposed titrable tyrosines of the enzyme are acetylated or not. A similar PKapp (9.1) is found for the pH-dependent change in optical rotation of the acetylated enzyme, when the exposed tyrosines are acetated. After correction for electrostatic interactions, this apparent pK becomes 8.4 and can be assigned only to the α-aminogroup of the N-terminal isoleucine. [ABSTRACT FROM AUTHOR]

The thermostable ethanol-insoluble fraction of bovine adrenal tissue has been studied by physical and chemical procedures. Analysis in the ultracentrifuge revealed three sedimenting boundaries in such preparations; the corrected and extrapolated sedimentation coefficients were 5·7, 2·7 and 1·9 S. Free electrophoresis at pH 8.6 showed four to seven components; the major boundaries had (negative) mobilities of 12·3, 10·0, 5·6 and 1·2 × 10-5 cm² volt-1 sec.-1. Light absorption studies revealed a maximum in optical density, at a wavelength of 260 mμ, indicative of nucleic acid. The extinction coefficient at 280 m~ was found to be 35·5 at a concentration of 1 g. per 100 ml. The same parameter at 540 mμ (with biuret reagent) was found to be 2·5. To elucidate the chemical nature of the previously reported adrenal-specific antigen in these heterogeneous preparations, degradation studies were made, followed by complement fixation, haemagglutination and precipitation methods to measure the remaining activity. The antigen was not affected by periodate, but was readily destroyed by chymotrypsin. This indicated that it is part of a polypeptide structure. The thermostable preparations also contain a non-adrenal-specific antigen. This second antigen was not destroyed by chymotrypsin, but neither was it affected by periodate, suggesting that a protein may be implicated here also. [ABSTRACT FROM AUTHOR]

The presence of a chymotrypsin-like me in rat mast cells has been confirmed, and it has been shown that intact mast cells contain the fully activated enzyme without losing their histamine content, it has been shown by means of inhibitors that the chymotrypsin-like enzyme is distinct from the heat-labile SH enzyme required for the histamine releasing action of compound 48/80. The synthetic ester used as a substrate for chymotrypsin released histamine from rat mast cells probably by a non-enzymic reaction. [ABSTRACT FROM AUTHOR]