1. Mouse erythrocytes were incubated with adenosine-u-¹⁴C or inosine-u-¹⁴C. Analyses on nucleotides and bases of the cells revealed that the cells incorporated adeno-sine-u-¹⁴C into their nucleotide pool by direct phosphorylation while deamination of adenosine-u-¹⁴C paralleled it as well. In contrast, only the base moiety of inosine-u-¹⁴C was incorporated into the nucleotide pool. Excretion of hypoxanthine into extracellular fluid was noted in both cases of incubation.
2. Red blood cells whose adenine nucleotides were labeled with adenosine-u-¹⁴C were thus prepared and used for the in vivo study on the metabolism of blood cell nucleotides. Base and ribose molecules seemed to disappear from the nucleotide pool of the circulating erythrocytes at a similar rate to each other. Purines moved from erythrocytes to tissue cells were incorporated into their RNA, but the radioactivity in ribose moiety of RNA-purine nucleotides was negligible.
3. Uptake of purines and their ribosyl derivatives by blood cells was compared in vivo with that by other tissues for the gross evaluation of the significance of dietary and circulating purines. Blood cells efficiently took up adenine administered orally, adenosine and adenine which had been infused into blood flow.

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Several aromatic amino compounds, which can be classified according to their aromatic nuclei into the three series, benzene, quinoline and naphthalene derivatives, have been examined as inhibitors of the α-chvmotrypsin [EC 3. 4. 4. 5]-catalyzed hydrolysis of N-acetyl-L-tyrosine ethyl ester at pH 8.0. All of the compounds were found to have the ability to interact with the enzyme in a competitive manner, aminonaphthalene derivatives being the most effective and aminobenzene derivatives being the worst inhibitors of the three series.
p-Aniino-N-acetyl-L-phenylalanine methyl ester was shown to be a good substrate of α-chymotrypsin (K_m=3.3×10^-3M, k_eat=201 sec^-1 at pH 8.0), one of whose merits is its relatively high solubility. It is approximately 25 times more soluble than N-acetyl-L-tyrosine ethyl ester in pH 8.0 aqueous buffer solution.
Kinetic constants for p-nitrophenylalanine ester derivatives were also evaluated, and it was shown that while their K_m values do not differ from the K_m value for N-acetyl-L-tyrosine ethyl ester, their catalytic rate constants are strikingly lower than those of esters of N-acetyl-L-tyrosine, p-amino-N-acetyl-L-phenylalanine and N-acetyl-L-phenylalanine.

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Modification of α-chymotrypsin [EC 3. 4. 4. 5] by the aromatic diazonium compounds, p-diazobenzenesulfonic acid, diazobenzene, 3-diazoquinoline and α- and β-diazonaph-thalene at 4°C was investigated. α-Diazonaphthalene, the most inhibitory of these compounds, when added in 40-fold molar excess caused 90% inhibition in 20 min at pH 8.0.
The inhibitory effects of these diazonium compounds increased with decrease in the K_i value of the corresponding aromatic amino compound from which they were prepared. When β-phenylpropionic acid, a competitive inhibitor of α-chymotrypsin, was added to the reaction mixture, marked retardation of the inhibitory reaction was observed. At a fixed ratio of diazonium reagent to α-chymotrypsin inhibition decreased with increase in the molar concentration of α-chymotrypsin. These aromatic diazonium compounds inhibited trypsin less than α-chymotrypsin. From these results the inactivation of α-chymotrypsin by these diazonium compounds may be interpreted in terms of an active-site-directed reaction.
The inhibitory effect of 3-diazoquinoline on α-chymotrypsin and trypsin differed from those of the other diazonium compounds tested.

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Thyroid-stimulating hormone (TSH) was purified from whale pituitary glands by a combination of acetone precipitation, gel filtration and stepwise elution chromatography on CM-cellulose and TEAE-cellulose. When the preparations were subjected to gradient elution chromatography on TEAE-cellulose, disc electrophoresis and isoelectric focusing, the multiplicity of whale TSH was always indicated. The most effective separation of each component was achieved by isoelectric focusing, and at least four hormonally active components with different isoelectric points were found. From isoelectric-focusing examination of extracts carefully obtained from single glands in the presence of protease inhibitors, it has been concluded that these multiple components are not artifacts produced during the isolation processes but present simultaneously even in a single whale pituitary gland.

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The ghost fraction of Bacillus megaterium KM is not appreciably soluble in water, but when it was dialyzed against 0.005M EDTA and then adjusted to pH 8.5, its turbidity decreased.
When this preparation at pH 8.5 was centrifuged at 105, 000×g for 40min, a reddish jelly precipitate (pellet A) was obtained. A suspension of Pellet A was ad-justed to pH 12. In this way some components became soluble and were separated from organella (Pellet B) by centrifugation at 105, 000×g for 60min.
Pellets A and B were investigated by sucrose density gradient centrifugations, electron microscopy and biochemical analyses. The sedimentation patterns of the samples showed that Pellet B was a single component, whereas Pellet A was polydispersed. An electron micrograph of Pellet B showed clearer figures of membraneous material than those of the ghost fraction, and “the unit membrane” structure was still visible.
The ratio of lipid to protein in Pellet B was much higher than in the ghost fraction.
These results suggest that treatment with strong alkali removes non-membraneous substances and that Pellet B consist of core membraneous organella.

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Homoserine kinase [EC 2. 7. 1. 39] was partially purified about 40-fold from sonic extracts of Brevibacterium flavum. A new assay method for homoserine kinase was examined in which ADP formed in the reaction was measured in a coupled spectrophotometric assay with pyruvate kinase [EC 2. 7. 1. 40] and with lactate dehydrogenase [EC 1. 1. 1. 27]. The optimum pH of the reaction was 7.4 with Tris buffer, and the K_m values at pH 7.5 for the substrates, homoserine and ATP, were 7.7×10^-4M and 1.2×10^-3M, respectively. Ammonium sulfate or KCl activated the enzyme about twice. Phosphohomoserine, one of the reaction products, inhibited the forward reaction competitively to both of the substrates, suggesting that the reaction mechanism was rapid equilibrium random Bi Bi, that is, random addition of two substrates and random release of two products with rapid equilibrium. Threonine, a metabolic end product, inhibited the activity competitively to homoserine and not competitively to ATP, and 50%, inhibition took place at about 8mM of threonine. A gel filtration experiment showed that the enzyme was neither aggregated with aspartate kinase nor with homoserine dehydrogenase and its molecular weight was about 5.5×10⁴. The properties of homoserine kinase of a threonine-analogue-resistant-mutant from B. flavum, which overproduces threonine in the culture medium, were essentially the same as those of the parental enzyme.

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Studies were done on kinetics of hemolysis of rabbit erythrocytes induced by partially purified streptolysin O (SLO).^***
1) An examination of the kinetics of hemolysis of rabbit erythrocytes by semilogarithmic plots of percent erythrocytes remaining versus time revealed the existence of lag phase and constant rate phase. The hemolytic rates observed at the constant rate phase were proportional to square of SLO concentration and decreased as the initial concentration of erythrocyte became higher.
2. Cholesterol inhibited hemolytic activity of SLO when added before the incubation of SLO with erythrocytes. However, when added after the initiation of hemolysis, neither cholesterol nor hemolysates prepared by SLO treatment inhibited hemolytic activity of SLO.
3) Independent of previous reduction with L-cysteine, SLO bound to cholesterol suspended in phosphate buffer and also to erythrocytes, while affinities of the reduced form of SLO for cholesterol as well as for erythrocytes were found higher than those of the unreduced form. Binding of the reduced form of SLO to erythrocytes was reversible at 4°C and SLO was transferable from erythrocyte to erythrocyte. However, once SLO had been bound to erythrocytes and hemolyzed them at 37°C, its hemolytic activity could not be detected with further addition of erythrocytes.
4) On the basis of these observations, mechanism of cytolytic activity of SLO was discussed.

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A simple method for linkage analysis of the reducing terminal of oligosaccharides was made feasible by Smith degradation combined with bromine oxidation. Hydroxy acids-glycolic, glyceric, threonic or erythronic acid produced from the aldonic acid terminal of oligosaccharides by Smith degradation were readily identified by gas-liquid chromatography. The usefulness of this method was demonstrated by analyses of 2-O-α-D-glucopyranosyl-D-glucose, 3-O-β-D-glucopyranosyl-D-glucose, 3-O-α-D-manno-pyranosyl-D-mannose, 4-O-α-D-glucopyranosyl-D-glucose, 6-O-α-D-glucopyranosyl-D-glucose, and several trisaccharides.

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A majority of guinea pig anti-ovalbumin sera obtained by immunization with oval-bumin emulsified in Freund's complete adjuvant were found to contain non-precipi-tating antibodies, while their contents varied depending on the groups of animals. For elucidating immunochemical properties of non-precipitating antibodies, 7S γ₁ and 7S γ₂-globulins were purified from several lots of antisera, and these antibodies were studied with regard to proportion of non-precipitating antibody, passive hemagglutination, complement fixation and passive cutaneous anaphylactic activities.
All the antibody preparations so far tested were found to contain non-precipi-tating antibody except one preparation of 7S γ₁-antibody. There was no evidence that a physico-chemically distinct type of immunoglobulin was responsible for the non-precipitating character of antiserum. Concerning other properties as antibody, any difference could not be observed between the non-precipitating and precipitating antibodies, when antibodies of the same immunoglobulin class were compared with each other.
On the basis of these observations, immunochemical properties of guinea pig non-precipitating antibodies to ovalbumin were discussed.

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A simple procedure is formulated to correct for the Johnston-Ogston effect which operates in the velocity sedimentation of mixtures comprising two components. The procedure is applied to the analysis of results obtained with proteoglycan mixtures from bovine nasal cartilage. It is shown that the method permits the evaluation of the true concentration ratio of the components from velocity sedimentation data obtained at two different total concentrations.

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A tryptophyl circular dichroic (CD) band at 305mμ with a small negative ellipticity of hen egg-white lysozyme [EC 3. 2. 1. 17] was found at neutral pH values. The effect of pH on this band and the positive band at 295mμ which had been observed previously was studied. The pH dependence of the both bands could well be interpreted in terms of the interaction of Trp-108 and Glu-35 with a pK value of about 6.
Di- and tri-N-acetylglucosamine and glycol chitin greatly enhanced the negative maximum at 305mμ. α- and β-Methyl glycosides of N-acetylglucosamine (NAG) enhanced this band slightly, while an equilibrium mixture of α- and β-NAG greatly reduced this band. All these reagents, however, enhanced the positive CD maximumm at 295mμ.
The negative CD maximum at 305mμ was also reduced or abolished in the presence of sucrose, ethanol, urea, or guanidine hydrochloride, which had been shown to interact with lysozyme and enhance the positive maximum at 295mμ. Glucose and KCl had no significant effect on the 305mμ-band.
These results suggest that the tryptophyl CD band at 305mμ of lysozyme is more useful than that at 295mμ for differentiating the interaction of various reagents with lysozyme.

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Auramine O [bis(p-dimethylaminophenyl)-methylenimine hydrochloride] had a very low fluorescence yield in water but the yield was markedly enhanced when bound to chromatophores of a photosynthetic bacterium, Rhodospirilluin rubrum (blue-green mutant). This binding was sensitive to pH, and type and concentration of cations in the suspending medium.
Fluorescence intensity of membrane-bound auramine O decreased moderately in the presence of monovalent cations (Na⁺, K⁺ or NH⁺₄) and more significantly in the presence of divalent cations (Mg²⁺, Ca²⁺ or Mn²⁺).
The fluorescence intensity of bound auramine O decreased reversibly by illumination of chromatophores. The time-course of the illumination-induced fluorescence change was separated into rapid and slow phases. The extent of the rapid phase was increased by addition of tetraphenylboron- or valinomycin plus K⁺. The relationship between the rapid phase of the fluorescence change and the light-induced H⁺ transport of chromatophores was suggested. The appearance of the rapid phase was also dependent on the redox level of chromatophores.
Auramine O fluorescence can be used as a probe for the survey of the physical parameters of chromatophore membranes (distribution and number of charged groups and ionic environment) and energetical level of membranes in energy transduction.

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Two cytochromes of the C-type, c-551.5 and c-555(550), were isolated from the soluble fraction of a green photosynthetic bacterium, Chloropseudomonas ethylica strain 2K, and highly purified by DEAE-cellulose and Amberlite CG-50 ion exchanger column chromatography.
Cytochrome c-551.5 has absorption maxima at 551.5, 523 and 418nm in the reduced state and at 528, 408 and 351nm in the oxidized state. This cytochrome is an auto-oxidizable, slightly basic protein having an isoelectric point of 7.77. It has a low mid-point redox potential of -209mV at pH 7.0. The molecular weight of this protein is close to 11, 500 and it contains two heme c groups per molecule of protein. These results indicate that cytochrome c-551.5 closely resembles cytochrome c₃ from Desulfovibrio vulgaris (strain Hildenborough).
Cytochrome c-555(550) has absorption maxima at 555, 523 and 417.5nm in the reduced state and at 525, 412 and 358nm in the oxidized state. This protein is very acidic, having an isoelectric point of 4.65. It has a mid-point redox potential of +103mV at pH 7.0. Its molecular weight is approximately 12, 000. Cytochrome c-555(550) is similar to algal and higher plant f-type cytochromes except for redox potential.
There is no evidence for the presence of a 13-type cytochrome in Cps. ethylica strain 2K.

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Intact mitochondria were prepared from mycelia of Aspergillus oryzae grown in the presence and absence of chloramphenicol (2mg/ml). To investigate the cytochroine components in normal and chloramphenicol mitochondria, ^** we measured difference spectra under various conditions at liquid nitrogen temperature and at room temperature. The low temperature difference spectrum (substrate-reduced minus oxidized) of normal mitochondria showed absorption peaks at 548.5, 554, 559.5 and 604.5mμ and a shoulder at 564mμ in the range from 540mμi to 650mμ. The peak at 604.5mμ was assigned to the α-band of cytochrome oxidase [EC 1. 9. 3. 1] (or a-type cytochrome(s)). The band at 554mμ was rather broad. On addition of dithionite to a mitochondrial suspension containing substrates (succinate plus α-ketogluta-rate) and antimycin A, the broad band at 554mμ split into two peaks at 553 and 556mμ. The peaks at 548.5 and 553mμ were assigned to the α-bands of cytochromes c andc₁, respectively. In the presence of intermediates of the tricarboxylic acid cycle and antimycin A three bands appeared at 556, 559.5 and 564mμ. They were ascribed to three b-type cytochromes, as in the case with mitochondria of higher plants. The difference spectrum (substrate-reduced minus oxidized) of normal mitochondria at room temperature showed an absorption band at 608mμ in addition to those at 551 and 560mμ. The α-band of this a-type cytochrome(s) was found at a longer wavelength than those in mitochondria of mammals (605mμ) and higher plants (602mμ).
The amounts of a-type cytochrome(s) and the three b-type cytochromes were markedly less in chloramphenicol mitochondria than in normal mitochondria, whereas the amount of c-type cytochromes in the former was much larger than in the latter mitochondria.

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Effects of divalent metal cations on the heavy meromyosin ATPase [EC 3. 6. 1. 3] reaction were studied by measuring the ATP-induced ultraviolet absorption difference spectrum at the pre-steady and steady states, and the P_i-liberation at the steady state.
Effects of MgCl₂ concentration on apparent values of the parameters, the maximum velocity and Michaelis constant of the ATPase, the velocity of formation of the difference spectrum, and the molar difference absorption coefficient were investigated. From the analysis of the results, the role of divalent cation on the ATPase reaction has been elucidated according to a reaction mechanism which involves two Michaelis complexes, ES and EMS, where M represents the divalent metal cation.
Rate constant of the step from EMS to give product, k₂', and the dissociation constant of EMS to EM and S, K_s', were determined. In the presence of any one of Mg²⁺, Co²⁺, Mn²⁺ or Ca²⁺, logk₂' vs. logK_s' plot was linear. The plot of logk₂' vs. reciprocal of ionic radius of the cation was also linear. Results indicate that the smaller the ionic radius of cation, the more strongly ATP is bound by EM and the more slowly product is formed from EMS.
The pH dependency of an apparent dissociation constant of EMS to ES and M yielded a second order dissociation curve of pKa=7.3. Two imidazole groups are suggested to participate in the binding of the divalent cation.

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Horseradish oxyperoxidase [EC 1. 11. 1. 7] is prepared by photolysis of an aerobic solution of the CO-peroxidase. Oxyperoxidase is decomposed to the ferric enzyme without appreciable intermediates. The decay of oxyperoxidase is greatly accelerated by the addition of dimethyl-p-phenylenediamine (DMPD). In the reduction of oxyperoxidase to the ferric enzyme, about one mole of DMPD is oxidized to its free radical in the presence of a limited amount of DMPD while three moles are oxidized in the presence of excess DMPD. Rate constants of the reaction between oxyperoxidase and various electron donors are measured.
The stability of Compound I is greatly affected by the contamination with “endogenous donor, ” but it has no significant effect on the stability of oxyperoxidase and the rates of cytochrome c peroxidation and hydrogen peroxide decomposition.
Oxyperoxidase is also decomposed oxidatively in the presence of peroxidase Compounds I and II. The mechanism of oxyperoxidase autodecomposition and the abnormal stoichiometry of reductive titration of oxyperoxidase by DMPD are discussed on the basis of oxidative decomposition of oxyperoxidase.

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1. Phosphorylase b kinase [EC 2. 7. 1. 38] from rabbit skeletal muscle required Ca ion for its activity. Little activity was shown in the presence of Ca ion less than 10^-10M. A significant activity, about 5% of the maximum, was observed at 5×10^-8M Ca ion. The concentrations of Ca ion which gave the half maximum activity and the maximum activity of the purified enzyme were about 10^-5M and 4×10^-4M, respectively.
2. Phosphorylase b kinase from rabbit cardiac muscle and chicken gizzard smooth muscle showed essentially the same responses to Ca ion as those of skeletal one.
3. One gram of rabbit skeletal muscle contained 9, 160 units of phosphorylase [EC 2. 4. 1. 1] and that amount of phosphorylase b kinase which could yield 1, 490 units of phosphorylase a per second from phosphorylase b in the presence of 4×10^-5M Ca ion. This indicates that the kinase in rabbit skeletal muscle can convert phosphorylase b to phosphorylase a within 6.1 sec. This value is compatible with the reported value needed for the conversion of phosphorylase b to phosphorylase a during the tetanus of frog skeletal muscle.
4. The stimulating effect of cyclic AMP on the kinase also required a minute amount of Ca ion.
5. The activation process of the kinase by a minute amount of Ca ion was reversible, either in the presence or absence of cyclic AMP.
6. The physiological significance of Ca ion as a triggering agent of glycogenolytic process was discussed with particular references to the role of Ca ion in excitationcontracticn coupling.

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Human red cell ghosts were prepared by a modification of the method of Dodge, Mitchell, and Hanahan, and shown to be reasonably pure from both chemical and morphological viewpoints. Digestion of ghosts with trypsin [EC 3. 4. 4. 4], chymotrypsin [EC 3. 4. 4. 5], bacterial protease, or their combinations caused the solubiliza-tion of protein only to a limited extent (35 to 49%, depending on the ghost preparations used). Hexosamines and sialic acid were also liberated partially, but no release of phospholipids and cholesterol occurred. Microscopic observations revealed that trypsin-treated ghosts consisted of two different profiles i.e. vesicular structures and membrane fragments, which could be separated from each other by sucrose density gradient centrifugation. It is concluded that human red cell ghosts, though homogeneous with respect to morphology and density, are actually composed of two types of population differing in the susceptibility to proteolysis.

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