Kenzo Kurihara

States of amino acid residues in proteins: III. Histidine residues in insulin, lysozyme, albumin and proteinases as determined with a new reagent of diazo-i-H-tetrazole

Abstract A new coupling reagent, diazonium- i -H-tetrazole, for spectrophorometric determination of histidine residues in proteins was explored and applied to several proteins. The reagent has the following characteristics advantageous to the determination and is superior to common diazonium compounds. (a) Histidinebisazo- i -H-tetrazole, which is the reaction product to be determined spectrophotometrically, has a strong absorption band at 480 mμ, while tyrosinebisazo- i -H-tetrazole has a weaker band at a considerably longer wavelength, 550 mμ. (b) The coloration due to the formation of biazohistidine residues proceeds to completion before the bis coupling to tyrosine residues. (c) The reagent does not afford colored by-products during its reactions with amino acids or proteins at alkaline pH, so that one can use a high concentration of reagent necessary to determine the end point of coloration. Because of these characteristics, one can determined the molar histidine content simply by absorption photometry at 480 mμ of reaction mixtures of protein and the reagent. The reagent reacts uniformly with all of the histidine residues in bacitracin, lysozyme (EC 3.2.1.17), insulin, albumin and trypsin (EC 3.4.4.4). On the other hand, the reactions with the histidine residues in trypsinogen, chymotrypsinogen and α-chymotrypsin (EC 3.4.4.5) proceed in two steps, in which the reaction in the second step at higher reagent concentrations is due to the coupling to bound histidine residues. The moles of bound residues in the latter proteins were determined and discussed referring to the data obtained previously with other reagents.

High activity of adenyl cyclase in olfactory and gustatory organs

Abstract Bovine taste buds bearing papillae exhibit high adenyl cyclase activity comparable to the activity found in the brain, while the tongue epithelium without taste buds exhibits much lower activity than found in the taste buds bearing papillae. Rabbit olfactory epithelium also exhibits high adenyl cyclase activity. The results suggest that cyclic AMP may play an important role in the transducer process common to the sensory organs.

States of amino acid residues in proteins: V. Different reactivities with H2O2 of tryptophan residues in lysozyme, proteinases and zymogens

Abstract Hydrogen and organic peroxides were examined in a search for a reagent to differentiate between various states of tryptophan residues in proteins. Hydrogen peroxide in a solution of 0.5 M bicarbonate buffer (pH 8.1–9.4) containing 10% dioxane was found to possess a moderate oxidizing power to oxidize free and bound tryptophan residues at different concentrations of H2O2, and the strength of the tryptophan absorption band of the protein was lowered stepwise with increasing concentration. By the use of this reagent, tryptophan residues in several proteins were classified into various types with different oxidizabilities, and the moles, n, of each type per mole of proteine were determined; n=5 and 1 for lysozyme (EC 3.2.1.17), n=5, 1 and 1 for chymotrypsinogen and α-chymotrypsin (EC 3.4.4.5), and n=2, 1 and 1 for trypsinogen and trypsin (EC 3.4.4.4.) which are arranged in the order of decreasing oxidizability. On the activation of chymotrypsinogen, the oxidizability of the most strongly bound residue decreased greatly whereas, on the activation of trypsinogen, the oxidizability of the secondly oxidized residue increased slightly. The structural rearrangement on the activation was discussed in terms of these changes in state of tryptophan as well as those of other amino acids determined previously. The enzymic activity of lysozyme an proteinases was also measured as a function of H2O2 concentration, and the results were correlated with the degree of oxidation of tryptophan residues. All of the tryptophan residues in denatured proteins were oxidized at a low concentration of H2O2, so that the reagent is applicable for the determination of the molar tryptophan content in protein.

Mechanism of bitter taste reception: Interaction of bitter compounds with monolayers of Lipids from bovine circumvallate papillae

Abstract Monolayers of lipids from bovine circumvallate papillae were prepared as a model system for the gustatory receptor membrane. Interaction of the monolayers with bitter compounds was examined by measuring a surface pressure increase in the monolayers. It was found that there is a good correlation between the concentration of the bitter compounds to give an identical increase in surface pressure in the monolayers and their taste thresholds. From the results obtained in the present study it is proposed that bitter taste is induced by penetration of bitter compounds into the non-polar region of the lipid layer of the gustatory receptor membrane.

Inhibition of cyclic 3′, 5′-nucleotide phosphodiesterase in bovine taste papillae by bitter taste stimuli

In a previous paper [ 11, it was reported that bovine taste papillae and rabbit olfactory epithelium contain high adenyl cyclase activity comparable to the activity found in rat brain. This finding, together with the fact that photoreceptors exhibit high activities of adenyl cyclase [2,3] and cyclic 3’,5’-nucleotide phosphodiesterase [4], suggested that adenosine cyclic 3’,5’monophosphate (cyclic AMP) may play an important role in the transducer process common to the sensory organs. It is known that phosphodiesterase is inhibited by caffeine, theophylline, theobromine [S] and papaverine [4]. Considering the fact that these compounds are typical bitter taste stimuli, it may be conceivable that bitter taste stimulation is associated with inhibition of phosphodiesterase in the gustatory cells. In the present study, the effect of bitter compounds on the activity of phosphodiesterase in bovine taste papillae was examined and it was found that all the bitter compounds examined in the present study inhibit the phosphodiesterase.

Reactions of cyanuric halides with proteins I. Bound tyrosine residues of insulin and lysozyme as identified with cyanuric fluoride

Abstract The ultraviolet absorption band of tyrosine was found to be remarkably lowered and shifted toward shorter wavelengths by treatment with cyanuric fluoride, (CNF)3, whereas the bands of other amino acids were either unaffected by the treatment or affected below 295 mμ, the peak position in the difference spectrum of the ionization of tyrosine. By use of this phenomenon, the reactivities of the tyrosine residues of lysozyme and insulin with cyanuric fluoride were examined, and the following facts were established. Two of the three tyrosine residues in the lysozyme molecule react with cyanuric fluoride and the remaining one is non-reactive. Of the four tyrosine residues in the insulin molecule, two are reactive while the other two are non-reactive. Upon addition of alkali to an insulin solution, one of the two non-reactive residues is transformed rapidly and the other slowly into the reactive type. Of these two bound residues of the non-reactive type one is in the A the other in the B chains of insulin. The numbers and the positions of the bound residues of the same proteins, identified by other properties, are discussed.

States of amino acid residues in proteins VIII. Tyrosine, histidine and tryptophan residues in chymotrypsin in the presence of substrate and in diisopropylphosphoryl-chymotrypsin

Summary Tyrosine, histidine and tryptophan residues in α-chymotrypsin (EC 3.4.4.5) in the presence and absence of benzoylglycine methyl ester and those in diisopropylphosphoryl-chymotrypsin were analyzed by measuring their reactivities with cyanuric fluoride, diazonium-1-H-tetrazole and H2O2-dioxane, respectively, which are the reagents to differentiate between various states of these residues in protein. By the diisopropylphosphorylation of α-chymotrypsin, two of the total seven tryptophan residues, both of the two histidine residues and one of the four tyrosine residues in the molecule were bound more strongly or burried more deeply. These effects of phosphorylation were discussed from two different view points ; direct effects of the diisopropylphosphoryl group introduced into the specific serine residue and indirect effects of the conformational change induced by the phosphorylation. The addition of benzoylglycine methyl ester to a chymotrypsin solution lowered the reactivity of one of the tyrosine residues with cyanuric fluoride to the same extent as in the phosphorylation, but the histidine and tryptophan residues were unaffected by the presence of substrate. It was inferred from this highly specific effect of substrate that the tyrosine residue is masked by the substrate molecule and is involved in the specific binding of substrate to the enzyme. The activity measured with the same synthetic substrate dropped with pK = 9.6 slightly lower than pK = 10.2 obtained for the ionization of one of the tyrosine residues, and 2 moles of OH− were involved in the inactivation.

Dissociation of horse hemoglobin at high pH

Abstract 1. 1. A sedimentation study was made on the denaturation of horse hemoglobin by alkali or denaturation reagents: i.e., urea, formamide, and guanidine. It was found that two steps are involved in the denaturation at high pH; the first step from the native molecule to a component of 3.5 S , and the second step from the 3.5 S component to components of 2.0 S . The determination of the molecular weights and the frictional ratios of these components revealed that, in the first step, the native molecule is deformed with no change of the molecular weight, and, in the second step, the deformed molecule dissociates into halves. 2. 2. The denaturation by urea and formamide splits the native molecule also into halves, and the sedimentation constant of the split unit was 2.0 S , being the same as that of the half-size subunit formed by the action of alkali. Guanidine was most effective among the denaturation reagents tested and splits the native molecule into components of 1.2 S , which are presumably the quarter-size subunit. The molecular weights and the sedimentation and diffusion constants of the subunits of hemoglobin determined in this study are discussed, comparing them with the values reported previously for the subunits formed with acid or various denaturation reagents.

States of amino acid residues in proteins. VII. Hydrogen bonding of tyrosine residues in the insulin molecule.

Abstract Cyanuric fluoride, which is a reagent employed to differentiate between free and bound or buried tyrosine residues in proteins, reacts with one of the two tyrosine residues in each peptide chain, A or B, of insulin. By digestion of cyanuric fluoride-treated insulin with trypsin (EC 3.4.4.4) and with chymotrypsin (EC 3.4.4.5), the two tyrosine residues not reacting with cyanuric fluoride were located in the amino acid sequence. The A14 and B26 tyrosine residues were thus found to be the non-reactive type, and the A19 and B16 residues were the reactive type. The non-reactive B26 residue was transformed into the reactive type by tryptic digestion of native insulin, while the A14 residue remained non-reactive or bound after tryptic or chymotryptic digestion. From these effects of digestion on the reactivity as well as on the rate of ionization of the tyrosine residues, the intra-chain hydrogen bonding between the side chains of the B26 tyrosine and the B22 arginine and the inter-chain hydrogen bonding between the side chains of the A14 tyrosine and the B13 glutamica cid were suggested.

Peroxidase activity of hemoproteins. IV. Hematin complexes as model enzymes of peroxidase.

Abstract The process of activation to peroxidase due to the complexing between hematin and chelating reagents (pyridine, histidine, arginine, guanidine, formamide, acetate, and methanol) were studied by measuring both enzymic activity and spectral change as a function of reagent concentration. A large spectral change occurs when a hematin complex carrying two moles of a reagent is formed from hematin. On the other hand, the complexes active as peroxidase carry more than two moles of reagent; two moles on the ferric iron atom of hematin and others, probably, on the protoporphyrin ring. Histidine and arginine are effective in activating hematin, while other essential amino acids are ineffective. The values of k 4 (rate constant for the reaction with hydrogen donor) for the active nonprotein complexes are comparable to or one order less than those for common peroxidases, whereas the values of k 1 (rate constant for the reaction with H 2 O 2 ) are much smaller than the k 1 values for peroxidases. The role of the apoproteins of peroxidases is discussed in connection with the results obtained for the hematin complexes.