Some physical and enzymatic properties were studied on the purified fin whale anionic chymotrypsin which was homogeneous by polyacrylamide gel electrophoresis and ultracentrifugal analysis. The molecular weight was approximately 17, 000 (S_{20, W}=1.62) by the Archibald procedure, and the enzyme was found to be composed of approximately 126 amino acid residues. The isoelectric point was 5.2 (μ=0.1). The optimum pH for caseinolytic and N-acetyl-L-tyrosine ethyl ester hydrolyzing activities were about pH 8.0 and pH 7.5, respectively. The fin whale anionic enzyme was inhibited by DFP, like bovine pancreatic proteases, but not inhibited by N-tosyl-phenylalanine chloromethyl ketone. As far as the effects of bivalent cations, and natural trypsin inhibitors are concerned, no significant differences were found between fin whale and bovine anionic chymotrypsins. The substrate specificity was similar to that of bovine chymotrypsins B and α [EC 3. 4. 4. 6 and 5].

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1. Specific interactions of kinin releasing enzymes, pancreatic kallikrein [EC 3. 4. 4. 21] and snake venom kininogenase with bovine kininogen-II (low molecular weight kininogen), both native and modified, were examined. Venom kininogenase liberated bradykinin from native kininogen-II and its reduced and S-carboxymeth-ylated derivatives. Hog pancreatic kallikrein, on the other hand, did not release kinin from modified kininogen-II's, but liberated kallidin from the native bovine kininogen-II. Kallikrein and venom kininogenase cleaved the arginylseryl bond of the cyanogen bromide fragment containing the kinin moiety to liberate kallidin. From these results, it was assumed that a tertiary structure of the kininogen-II was required for the cleavage of the methionyl-lysyl bond to release kallidin by kallikrein, but not for the cleavage of the arginyl-seryl bond in the C-terminal part of the bradykinin sequence of kininogen-II.
2. Hog pancreatic kallikrein and venom kininogenase hydrolyzed poly-arginine, in addition to α-N-tosyl-derivatives of arginine and lysine methylesters, but did not hydrolyze α-N-tosylmethionine ethylester appreciably, Kallikrein also attacked poly-lysine, although the venom kininogenase did not hydrolyze poly-lysine appreciably. Thus, there are some differences in the actions of kallikrein and venom kininogenase. Trypsin [EC 3. 4. 4. 4] and plasmin [EC 3. 4. 4. 14] hydrolyzed all the substrates mentioned above, except α-N-tosylmethionine methylester.

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Suspensions of chromatophores from the blue-green mutant (G-9) of Rhodospirillum rubrum were illuminated by 592-nm actinic light, and the consequent steady-state absorbance changes in the near infra-red region were studied:
1) In chromatophores, there are two different types of pigments which undergo light-induced absorbance changes in the spectral region. The two pigments are called Liac-860 and Liac-890 in this paper.
2) Liac-860 is a major pigment responsible for the negative peaks (photo-bleaching) at 860nm and 810nm and the positive peak (photo-coloring) at 790nm in the light-minus-dark difference spectrum, whereas Liac-890 is a pigment responsible for the positive peak at 890nm in the spectrum. The midpoint oxidation-reduction potentials for these changes of Liac-860 and Liac-890 are +0.45 volt (n=1) and a value more negative than -0.1 volt, respectively.
3) These absorbance changes of Liac-860 and Liac-890 are influenced by the inhibitors of the electron transport system (antimycin A, 2-n-heptyl-4-hydroquinoline-N-oxide and phenylmercuric acetate), by the energy-trapping system (ADP plus P_{_??_}), and by the uncouplers (2, 4-dinitrophenol, atabrine dihydrochloride, carbonyl cyanide m-chlo-rophenylhydrazone and ADP plus arsenate), but not by the inhibitor of the energy conservation system (oligomycin).
4) The absorbance changes of Liac-860 and Liac-890 are influenced by proton concentration in the absence of antimycin A, but not in its presence.
5) It is discussed that the light-induced absorbance changes take place on the conversion of Liac-860 to Liac-860⁺ and of Liac-890 to Liac-890^-: Liac-860⁺ and Liac-890-are the primary electron acceptor and donor, respectively, for the electron transport system which involves a step influenced by proton concentration.

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1) In order to investigate the state of the tryptophan residue in RNase T₁ [EC 2. 7. 7. 26] and carboxymethyl RNase T₁ (CMRNase T₁), fluorescence emission spectra of these enzymes were measured in aqueous solutions and at various concentrations of urea and guanidine-HCl, using 295mμ excitation light. The maxima of emission spectra of RNase T₁ and CMRNase T₁ were at 320mμ which is different from 350mμ, the maximum wavelength for N-acetyl-tryptophan. The maxima of these enzymes in 8 M urea and 7 M guanidine-HCl were, however, located at about 350mμ. From these results, it was deduced that the tryptophan residue is buried in these protein molecules.
2) The tryptophan residue in RNase T₁ or CMRNase T₁ was resistant to the oxidation by NBS and to the modification by 2-hydroxy-5-nitrobenzyl bromide, while it was accessible to such chemical modifications at higher concentrations of urea or guanidine-HCl. The conversion to the accessible states took place in parallel with the shift of the fluorescence band.
3) Quenching of RNase T₁ fluorescence caused by addition of various nucleotides was studied and the dissociation constants of RNase T₁-nucleotide complexes were deter-mined.
4) Possible roles of the tryptophan residue in RNase T₁ were discussed.

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The amino acid sequences of three common Japanese strains of tobacco mosaic virus (TMV) protein were determined. Compared with the sequence of U-1 or Vulgare strain, the OM strain differs by two amino acid substitutions of valine for isoleucine at position 129 and asparagine for threonine at position 153. Structures of O and Kokubu strains differ only by a single substitution of valine for isoleucine at position 129.
A corrected amide group allocation at position 50 in all common strains of TMV including the Vulgare strain is also shown.

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1. Gas-liquid chromatography was applied to the analyses of N-acetylamino sugars and N-acetylamino sugar alcohols derived from glycol chitin. N-Acetyl-3-O-(2'-hydroxyethyl)-D-glucosamine, N-acetyl-6-O-(2'-hydroxyethyl)-D-glucosamine and the corre-sponding sugar alcohols were prepared as reference compounds. N-Acetyl-D-gluco-samine, N-acetvl-3-O-(2'-hydroxyethyl)-D-glucosamine and N-acetyl-6-O-(2'-hydroxyethyl)-D-glucosamine were identified as the constituents of glycol chitin.
2. The gas-liquid chromatographic analysis showed that by the digestion of glycol chitin with hen egg white lysozyme [EC 3. 2. 1. 17], only N-acetyl-D-glucosamine was found to be liberated as a monosaccharide fraction.
3. For the purpose of elucidating the attacking points of the lysozyme on glycol chitin, reducing end groups were analyzed by converting them into sugar alcohols by boro-hydride reduction. N-Acetyl-D-glucosamine and N-acetyl-3-O-(2'-hydroxyethyl)-D-glu-cosamine were identified as reducing ends.
4. Oligosaccharides in the enzyme digests were separated by gel filtration on Sephadex G-15, Bio-Gel P-2, and paper chromatography. The structure of the saccharides so far identified were: N-acetyl-D-glucosamine, N, N'-diacetyl-chitobiose, N, N'-diacetyl-3-O- (2'-hydroxvethyl)-chitobiose, N, N'-diacetyl-6'-O-(2'-hydroxyethyl)-chito-N, N'-diacetyl-3, 6'-di-O-(2'-hvdroxyethyl )-chitobiose.

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Ribonuclease T₁ [EC 2. 7. 7. 26] was photooxidized with and without protection by the substrate analog, a mixture of guanosine 2'-phosphate and guanosine 3'-phosphate. In the absence of the substrate analog, the enzymatic activity gradually decreased with loss of two to three histidine residues, while in the presence of the substrate analog, full activity was maintained and two histidine residues were protected against photo-oxidation. The tryptophan residue was oxidized with simultaneous O₂-uptake and the rate was scarcely affected by the presence of the substrate analog. These results demonstrate that two histidine residues are involved in the active centre of ribonuclease T₁ but that tryptophan is not involved.

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1. Photooxidation of ribonuclease T₁ [EC 2. 7. 7. 26] was studied to obtain information on the active center of the enzyme. The effect of pH on the photo-inactivation rate indicated the involvement of a group having a pKa value of 7.5 in the activity. This value is coincident with that for one of the functional groups which have been detected from kinetic studies.
2. Amino acid analysis of photooxidized RNase T₁, possessing only 2-3% of the original activity, showed that two histidine residues had been destroyed leaving the tryptophan residue mostly intact.
3. Photooxidation in the presence of 2', (3')-GMP resulted in the oxidation of only one histidine residue without any loss of the enzyme activity.
4. Alkaline titration of tyrosine residues, fluorescence emission spectrum, and difference spectrum occuring on exposure of the photooxidized enzyme to 6M guanidine hydrochloride all indicated that photooxidation does not significantly affect the gross conformation around the tyrosine and tryptophan residues of the enzyme.
5. The photooxidized enzyme could also combine with 2', (3')-GMP, though the affinity was about 4-fold lower than that for the native enzyme and was similar to that for the carboxymethylated enzyme.
6. The photooxidized enzyme could bind a substrate analog, guanylyl (2'→5') guanosine, as evidenced by the appearence of a difference spectrum which was similar to that induced by the nucleotide binding by the native enzyme. This suggested that the oxidized enzyme could also interact with the substrate, guanylyl (3'→5') guanosine.
7. The difference spectra caused by the binding of the inhibitor and substrate-analog nucleotides to the photooxidized enzyme were qualitatively similar to those observed with the native enzyme, suggesting that the two histidine residues sensitive to photooxidation are not involved in the interaction of the base moieties of these nucleotides with the enzyme protein.

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Lipopolysaccharides (LPS) and haptenic polysaccharides produced by intergroup recombinants of Salmonella were investigated.
1. The O-antigenic activities and contents of specific sugars were much less in LPS preparations from SR type recombinants than in those from the parent (S form). LPS of R-type recombinants was entirely devoid of the parental activities and sugars of the O antigens.
2. Recombinants between S. abony (donor) and S. milwaukee (recipient) produced defective LPS, lacking glucosamine at the terminal position of the R-core.
3. An antigenicity common to R recombinants was demonstrated in the SR-LPS of the progenies of a cross between S. riogrande (donor) and S. poona (recipient), suggesting that the R-core is exposed and can react with R-antibody owing to reduction in the number of O-side chains in the macromolecule.
4. Haptenic polysaccharides with O-antigenic specificities of the recipients were found in extracts of the SR recombinants. These substances were isolated and shown immunologically and biochemically to have polymerized O-side chains.

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The action of Taka-amylase A [EC 3. 2. 1. 1] on the several derivatives of phenyl α-maltoside was investigated and the comparative rates of the hydrolysis were deter-mined by analyzing the reaction products. The enzymatic reaction products were detected by thin layer- and gas liquid chromatography. Any of the hydroxyl groups in the reducing-end glucose residue of phenyl α-maltoside, when modified as described in the text, made the enzymatic reaction inaccessible. Some information on the role of 6-position of the non reducing-end glucose residue was also obtained. The steric requirement of the 6-position of the reducing-end glucose residue seemed to be rather strict in comparison with that of the non reducing-end glucose residue.

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The hydrolysis of p-nitrophenyl aceptate (NPA) was catalytically promoted by the synthetic copoly (DL-Trp, L-Glu 1:9). The pH and temperature dependence of the rate of hydrolysis were given as a bell-shaped curve, in which the optimum pH and the optimum temperature were observed at 5.95 and 40°C, respectively. Although copoly (DL-Trp, L-GIu 1:9) showed activity to NPA, copoly (L-Trp, L-Glu 1:9) did not show any activity. The apparent maximum velocity (V_max) and the apparent Michaelis constant (K_m) were ; V_max=1.5×10^-6 mole/min/mg, K_m=2.1×10^-3 M. The order of K_m was the same as that of enzyme, but the value of V_max was considerably low. So the capture of substrate by an active center of the copolymer may be the same order as that of enzyme, however, the copolymer-product complex may be too stable to be hydrolyzed into p-nitrophenol. The following process for the hydrolysis of NPA by copoly-(DL-Trp, L-Glu 1:9) may be speculated from the results of fluorescence titration, optical rotatory dispersion, rigidity measurement and infrared spectrum; 1) the orientation of substrate is given by tryptophanyl residues. 2) the substrate is hydrolyzed by acid-base interaction of COOH and COOH groups (27). 3) the removal of the products from active center is proceeded by the equilibrium of ENP_??_E+NPOH, where E is copolymer, ENP is copolymer-product complex and NPOH is product (p-nitrophenol).

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1. N-Acetyl-β-D-glucosaminidase [EC 3. 2. 1. 30] was isolated from Takadiastase Sankyo by means of several purification methods.
2. The purified enzyme was shown to be homogeneous by disc electrophoresis and ultracentrifugation.
3. The molecular weight of the enzyme was determined to be 140, 000-146, 000 by equilibrium ultracentrifugation.
4. The enzyme was a glycoprotein, and the amino acid and sugar composition of this enzyme was determined.
5. N-Acetyl-β-D-galactosaminidase activity was found in water extract of Takadiastase and the two activities were not separated from each other.
6. The K_m and pH optimum of the enzyme were determined by using p-nitrophenyl and phenyl N-acetyl-β-D-glucosaminides and phenyl N-acetyl-β-D-galactosaminide as substrates.

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A simple and reliable method for the quantitative determination of free tyrosine and tyrosine residues in proteins is described. The method involves alkali-denaturation of proteins in a boiling water bath, the color forming reaction between tyrosine and 1-nitroso-2-naphthol in 19 N H₂SO₄, and measurement of the absorbance at 520mμ.
The method is preferable because of its simplicity, high sensitivity, reproducibility and remarkable stability of the red color.
The procedure has been shown to be accurate with a wide variety of enzyme proteins.

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