Total lipid extracts from whole leaf and its chlorophyll lipoprotein of Sapium sebiferum Roxb. were separated and characterized, using column and thin-layer chromatography with other physical and chemical methods. It was found that there were more than thirty com-ponents in whole leaf lipids. Major compo-nents of the lipids were solanesol, plastoqui-none, sterol ester, tocopherol, chlorophyll a and b, lutein, monogalactolipid, diglycosyl glycerol lipid, some phospholipids, and other components. The lipids from chlorophyll lipoprotein were similar to those from whole leaf, but there were some differences in their proportions. The former contains more polar lipids such as di-, triglycosyl glycerol lipid and phosphatidyl glycerol, and also more α-toco-pherol, than the latter lipids.
The author wishes to express his gratitude to Dr. M. Nakazima and Dr. H. Fukami for measurements of infrared absorption spectra.

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Reduction of all of the four disulfide bonds in Taka-amylase A by β-mercaptoethanol in 8 M urea yields a randomly coiled polypeptide chain. Enzymatic activity can be recovered by removal of the reducing and denaturing reagents and by subsequent airoxidation. The present investigation was attempted to make clear what kinds of molecular species are involved as the intermediate during the reoxidation process, and whether the reformation of all the intact disulfide bonds of Taka-amylase A is necessary for the recovery of the enzymatic activity.
Reduced Taka-amylase A was partially oxidized to various levels and carboxy-methylated. Intermediates differing in their sulfhydryl content acquired different net charges by carboxymethylation, and could be successfully separated into four components by gel-filtration and subsequent DEAE-cellulose column chromatography. Among them, three components had enzymatic activity. One was fully renatured enzyme. The other two were the derivatives in which some sulfhydryl groups were partially reoxidized and the remaining residual sulfhydryl groups were carboxymethylated. These two compo-nents were found to have regained one and three disulfide bonds per molecule, respec-tively. They had 5 and 55 per cent of the original enzymatic activity, respectively. Partially reoxidized Taka-amylase A before carboxymethylation might have regained a higher enzymatic activity than the carboxy-methylated Taka-amylase A. It was thus demonstrated that reformation of all the original disulfide bonds is not necessary for the recovery of enzymatic activity of Taka-amylase A. A scheme of the reoxidation process was presented.
The authors are grateful to Sankyo Co. Ltd. for supplying CO “Taka-diastase Sankyo”, and to Dr. H. Kato for helpful suggestion and Mr. H. Murakami for amino acid analysis.

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1. As a convenient means for elucidation of the relationship between the structure and function of human placental chorionic gonado-tropin (HPCG), some proteolytic digestions of HPCG and fractionation of the gonadotropic-active fragments in the digests were investi-gated.
2. Gonadotropic activity was determined by the method of ovarian and uterine weight responses of immature female mice. The average value of the ovarian and uterine weights accounted for 10.1±0.2mg. and the probability that the value exceeded 10.8mg. was only 0.1 per cent in the case of 25 intact mice weighing 6 to 9g., so the principle increasing the ovarian and uterine weights of immature mice (7 to 8g. in body weight) above 11mg. was deter-mined to possess gonadotropic activity.
3. The digestions of HPCG were attempt-ed by following procedures: (a) by tryptic digestion in a test tube at pH 7.8 and 37°C for 30 minutes or at pH 7.8 and 20°C for 3 hours (fraction T-2), (b) by peptic digestion at pH 4.0 and 4°C for 22 hours in a test tube or in a Visking 18/32 tubing (fraction D-Pep) and (c) by pronase digestion at pH 7.8 and 4°C in a Visking 18/32 tubing. These digests were fractionated by chromatography on a column of Sephadex or Dowex 50-X8. Among the fractions obtained by column chromato-graphy, fractions T-2 and D-Pep were found to possess gonadotropic activities conveniently. Therefore, fractions T-2 and D-Pep further were partially purified on Dowex 50-X8 column. Thus fractions T-2-A and D-Pep-A possessed gonadotropic activities could be isolated.
4. The minimal effective dose of fraction T-2-A was determined to be between 0.09 and 0.19mg., and that of fraction D-Pep-A was determined to be less than 0.09mg.
5. The molecular weights of fractions T-2-A and D-Pep-A were presumed to be smaller than 6, 000 by their permeabilities-through the membrane of Visking 18/32 tubing.
The authors wish to express our thanks to Prof. M. Funatsu of Kyushu University and also to Prof. R. Sato of Osaka University for their help during preparation of the manuscript.

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The initial burst of Pi-liberation from the myosin-ATP system was measured under various experimental conditions after stopping the ATPase reaction by addition of trichloro-acetic acid and the following results were obtained.
1. The amount of the initial burst re-mained almost constant during purification procedures and repetition of the dilution-pre-cipitation procedure of myosin, and was unaf-fected by removal of the water-soluble fraction from myosin by DEAE-cellulose treatment.
2. The amount of the initial burst was usually 1 mole of Pi/mole (4×10⁵g.) of myosin over wide ranges of KCl concentration (0.04-1.1 M), pH (6.5-8.5) and temperature (0-30°C), if the Mg⁺⁺ concentration was higher than 1 mM. Although the amount of the initial burst was often less than the above value at low KCl concentration or at low temperature, it could always be restored to this value by raising the KCl concentration or temperature.
3. When the concentration of Mg⁺⁺ was higher than 1 mM, the amount of extra-libera-tion of Pi from the myosin-ATP system in-creased linearly with the ATP concentration until the amount of added ATP reached 1 mole/mole of myosin. At higher ATP con-centrations than this, the amount of the initial burst remained constant at the level of 1 mole/ mole of myosin. The time-course of Pi-libera-tion was unaffected by the addition of a large amount of ADP immediately after the com-pletion of the initial burst of Pi-liberation. The initial reaction of myosin ATPase was thus shown to be a stoichiometric and irrevers-ible interaction of ATP with myosin.
4. At low Mg⁺⁺ concentration, the timecourse of Pi-liberation consisted of 3 steps. In the i st step extra Pi was liberated instantane-ously after ATP addition. The velocity of Pi-liberation decreased gradually in the 2 nd step, and finally reached a constant steady value in the 3 rd step.
5. The amount of extra Pi-liberation in the 1 st step was 1 mole/mole of myosin when the Mg⁺⁺ concentration was higher than 0.05mM and it diminished when the Mg⁺⁺ con-centration was decreased to below 0.05 mM, and became zero in the absence of Mg⁺⁺. The amount of extra Pi-liberation in the 2 nd step was zero when the Mg⁺⁺ concentration was higher than 1 mM. When the Mg⁺⁺ con-centration was decreased below 1mM, it increased at first, showing a maximum at about 0.01mM Mg⁺⁺, and then decreased with decreasing Mg⁺⁺ concentration.
6. The amount of extra-liberation from synthetic actomyosin ATPase was large at low ionic strength. It decreased toward a constant value of 1 mole/mole of myosin when the ionic strength was increased. On the contrary, the amount of extra Pi -liberation of myosin ATPase used in the control experiment was small at low ionic strength. It approached the value of 1mole/mole of myosin as the ionic strength was increased.
7. It was confirmed that the initial burst was inhibited by p-chloromercuribenzoate and ethylenediaminetetraacetic acid under the present experimental conditions, as reported in our previous papers.

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1. The rate of liberation of total H⁺ by myosin-ATPase was measured using a Titri-graph. From the dependence of this rate on the ATP concentration, K_m and k₊₂ in 0.5 M KCl-1 mM MgCl₂ at pH 8.2 were found to be 3.8μM and 0.012 sec.^-1, respectively. K_m and k₊₂ in the presence of 0.1mM ethylene-diaminetetraacetic acid (EDTA) were 68 μM and 3.9 sec.^-1, respectively.
2. The time-course of the change in con-centration of free H⁺ in the myosin-ATP system was measured by a stopped-flow method, using cresol red as an indicator. In the presence of 1 mM MgGl₂, the change consisted of 3 steps. In the first step, 1 mole of H⁺ per 4×10⁵g. protein was rapidly .absorbed to myosin, and in the second step the H⁺ absorbed to the protein was again liberated and the concentration of H⁺ returned to the initial level about 2 seconds after the start of the reaction. In the third step which was the steady state, H⁺ was liberated linearly with time and simultaneously ATP was hydrolyzed. On the other hand, in the presence of 0.1 mM EDTA no rapid libera-tion-absorption of H⁺ in the initial stage was observed, and H⁺ was liberated at the same time as P_i3, after a lag period, which was about 0.21 seconds with 30 μM of ATP.
3. It was concluded from these two results that in the presence of MgCl₂ k_-1»k₊₂, while in the presence of ETDA k₊₂»k_-1, if -we adopt the reaction: E+S^k₊₁_??__k-1 ES k₊₂→E+ADP+Pi,
as the mechanism of the steady state ATPase activity.
4. The initial extra-liberation of P_i from the myosin-ATP system was measured under various conditions, together with the rapid absorption-liberation of H⁺ mentioned above. It was found that the amount of extra P_i-liberation is essentially equal to that of rapid H⁺-absorption under all the conditions in-vestigated. Notably both the phenomena were prevented by EDTA. Furthermore, the amount of rapid absorption of H+ in 0.5M KCl decreased in proportion to the decrease in extra-liberation of P_i, when the latter was reduced to less than 1 mole per 4 × 10⁵g. protein by changing the procedures used for isolation of the protein, and it in-creased to 1 mole 4×10⁵g. protein, as did the amount of extra-liberation of P_i in 1.0 M KCl.
5. From these results, the following reaction mechanism is proposed for the initial reaction of the myosin-ATP system in the presence of MgC1₂:
where k_-1»k₊₂, k₊₃>k₊₄»k₂+k₊₅ and E_??_P is the intermediate whose carboxyl group may be phosphorylated.

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1. p-Nitrothiophenyl myosin was prepared by the incubation of 1 mM p-nitrothiophenol (NTP) with myosin in the presence of 2mM ATP in 1.1 M KCl-22mM MgCl₂ at pH 6.5 and 0°C, in which the amount of the initial rapid liberation of P_i from the myosin-ATP system was 1mole/4 × 10⁵g. of myosin. The amount of initial extra-liberation of P_i decre-ased linearly with increase in the content of NTP bound to myosin and was reduced to 0.1mole/4×10⁵g. of myosin on the binding of 1.2 moles of NTP per 10⁵g. of protein. The ATPase activity in the steady state in-creased with increase in the content of NTP bound to myosin and was raised to about 5.5 times that of control myosin by the binding of 0.7moles of NTP to 10⁵g. of protein.
2. From analysis of the time-course of liberation of NTP from NTP-myosin at pH 7.0 and 25°C, it was concluded that myosin has at least two kinds of NTP-binding site in each molecule, one stable and the other labile. The quantity of the initial burst of P_i-libera-tion from the NTP-myosin-ATP system was not restored to 1 mole/4×10⁵g. of protein but remained at the low level of 0.15moles/4×l0⁵g. of protein, while the ATPase activity in the steady state decreased from 5.2 to 1.6 times the control value, on removal of NTP from the labile sites by incubating NTP-myosin for 4 hours at pH 7.0 and 25°C.
3. The rate constant of decomposition of the phosphoryl intermediate into myosin, Pi and H⁺ was measured from the time-course of H+-liberation after the addition of ATP to myosin at a molar ratio of 1:1. The rate constant remained at the constant value of 0.14 min^-1, between pH from 8 to 7.5. This value was about one-tenth of that of P_i-libera-tion from myosin ATPase in the steady state. However, the rate of decomposition of pho-sphoryl myosin in the actomyosin-ATP system was found to be more than 100 times that in the myosin-ATP system.
4. After the addition of ATP to myosin at a molar ratio of 1:1, the reaction was stopped by removing nucleotides by the char-coal treatment. Myosin, precipitated at a low ionic strength or at the isoelectric point, was separated with a Millipore filter and free P_i was washed out with buffer solution. The washed precipitate was extracted with trichlo-roacetic acid (TCA), and the amounts of TCA-labile phosphate in the precipitate were measured after various separation times, i.e., periods from the addition of charcoal to the end of the last washing with buffer solution. The content of TCA-labile phosphate and its decrease with increase in the separation time were consistent. with the assumption that TCA-labile phosphate was derived from the inter-mediate of the initial reaction of myosin with ATP.

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Single sarcomeres and myofibrillar fragments were isolated from pectoral muscle of chicken and the properties of their contraction induced by ATP were measured mainly in 0.04 M KCl at pH 7.0 and 25°C, in the presence of different concentrations of ATP, MgCl₂, CaCl₂, EDTA and EGTA. The following results were obtained.
1. The changes in the band patterns of myofibrillar fragments during contraction in-duced by ATP were as follows: in the first stage, a dense band appeared at the outer edge of the original H-zone. This band formation was accompanied by a gradual disappearance of the H-zone. In the second stage, the H-zone disappeared completely, the I-band shortened considerably and a dense band developed in the mid region of the sarcomere. Then, the so-called contraction band was formed at the Z-line and bands of lower density appeared at the outer edges of the original H-zone. At the next stage the latter bands fused, and then disappeared.
2. The minimum concentration of ATP required for contraction of myofibrillar frag-ments decreased and approached a constant value on increasing the concentration of MgCl₂. Contraction was unaffected by addi-tion of CaCl₂ or by treatment with EGTA. Contraction was inhibited by EDTA, and the concentration of this necessary for inhibition increased with increasing MgCl₂ concentration.
3. The rate of ATP-hydrolysis depended only on the concentration of MgATP^2-. It increased in proportion to the concentration of MgATP^2- at comparatively low concentra-tions, i.e., lower than about 10 μM, and ap-proached a constant value of 150-200 μmoles Pi/g./min when the concentration of MgATP^2-was sufficiently high.
4. On contraction of sarcomeres induced by a sufficiently high concentration of MgATP^2-, the intensity of light-scattering of a suspension of myofibrillar fragments de-creased to about 55 per cent of its original value the and degree of decrease was independent of the angle of scattered light. When the concentration of MgATP^2- was comparatively high, the rate of decrease in light-scattering intensity was observed to be in good propor-tion to the ATPase activity.
5. The average rate (V_s) of the initial stage of shortening of sarcomeres was measured under a microscope from the time-course of the increase in the number of contracted myofibrillar fragments. The relation of V_s to the rate of ATP-hydrolysis (V_h) was found to follow the equation, V_s=CV_h/(1+K_c/[S])², where K_c, is a constant, [S] is the concentra-tion of MgATP^2- and C is the “coupling” coefficient.
6. The average amount of hydrolysis of ATP necessary to cause contraction of myo-fibrillar fragments to the stage of disap-pearance of I-bands and formation of faint contraction bands was found to be 5.6-7.2μmoles Pi/g. of protein. This amount was independent of the temperature.

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1. The absorption spectra of the reduced and oxidized forms of the respiratory enzyme were calculated by correlating quantitatively the photochemical action spectrum with the difference spectra of cytochrome oxidase (a₃⁺⁺CO-a₃⁺⁺, and a₃⁺⁺CO-a₃⁺⁺⁺).
2. A quantitative correlation was achieved by taking advantage of the fact that the spectra of the cytochrome a₃⁺⁺-carbon mono-xide complex, cytochrome a⁺⁺⁺, and cyto-chrome oxidase (a⁺⁺⁺+a₃⁺⁺CO) had quite different ratios of absorption at 590mμ to that at 550mμ. This method of calculation gave the reduced spectrum more easily than the previous method.
3. The resulting spectra of the reduced and oxidized forms of the respiratory enzyme were in reasonable agreement with the results calculated previously.
4. The spectra of oxidized and reduced cytochrome a were also calculated from the spectra of the cytochrome oxidase-carbon monoxide complex and the photochemical action spectrum on the basis of the same principle.
The author expresses his cordial thanks to Prof. N. Shimazono for his kind advice and encouragement. He also wishes to thank Prof. K. Shibata of the Tokyo Institute of Technology for his valuable advice.

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1. Halophilic aldose dehydrogenase from halophilic Pseudonaonas No. 101 is predominantly associated with the cell envelope or wall-membrane complex and can be easily solu-bilized by a combination of cholate and deoxycholate. The solubilized enzyme has been partly purified by ammonium sulfate fractionation followed by column chromato-graphy on Sephadex G-200. The specific acti-vity of purified enzyme is 20 times higher than that of the particulate fraction or 150 times as high as that of the protoplasmic soluble preparations.
2. Upon purification, the enzyme looses the characteristic halophilism which is found in the crude preparations, but the addition of a protein from the particulate fraction restores the original halophilism.
3. The cytochrome involved in aldose oxidation system shows absorption band characteristic of a b-type cytochrome in the difference spectrum between the glucose-reduced minus oxidized state.

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Effects of blasticidin S, a nucleoside antibiotic of structural similarity with puromycin, on protein synthetic pathways was studied stepwise to determine the site of action of the antibiotic in a bacterial system. Blasticidin S markedly inhibited leucine incorporation into polypeptides under the direction of endogenous messengers in extracts of E. coli and B. megaterium. No inhibition by the antibiotic of oxidative phosphorylation was detected. Without interfering with formation of amino-acyl-sRNA, it blocked the transfer of leucine from leucyl-sRNA to polypeptides to the extent enough to account for the inhibition of free leucine incorporation into polypeptides. Unlike puromycin, blasticidin S reduced the release of peptides from the ribosome to the supernatant. It inhibited poly U-directed polyphenylalanine synthesis to the degree as high as that of the inhibition of leucine incorporation into polypeptides under the direction of natural messengers. It was suggested that the antibiotic attacked a certain steps following the formation of active complexes of ribosomes and messenger strands. Blasticidin S also inhibited leucine incorporation into poly-peptides in cell-free systems of mammalian cells, more highly in a system of mouse Ehrlich carcinoma than in that of normal rat liver.
This investigation was supported in whole by Public Health Service Researdh Grant CA05082-04, from the U. S. National Cancer Institute.

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A marked increase in phenylalanine ammonia-lyase and tyrosine ammonia-lyase activities was observed in sweet potato roots in response to wounding and infection. A rema-rkable rise of phenylalanine ammonia-lyase activity occurred during a period of 6-hour in both sliced and infected tissues. The change in activity of this enzyme with time after wounding showed a close parallel with the pattern of accumulation of polyphenols in the tissues. A similar picture was observed in case of the tyrosine ammonia-lyase, but the activity of this enzyme was very low compared to that of phenylalanine ammonia-lyase. DEAE-cellulose chromatography indicated that phenylalanine ammonia-lyase was separated into two components with phenylalanine am-monia-lyase activity by repeated DEAE-cellu-lose chromatography. Some properties of two types of phenylalanine ammonia-lyase were investigated, and a possible role of this enzyme in the biosynthesis of phenylpropane compounds in sweet potato roots was discussed.
The authors are grateful to Dr. T. Asahi for his helpful discussions during the course of this work. They are deeply indebted to Dr. T. Kosuge, Department of plant pathology, Universitp of California at Devis, for his cordial help in preparation of the manuscript.

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Pyocin, a bacteriocin from P. aeruginosa, was found to cause a rapid and complete cessa-tion of RNA, DNA, and protein synthesis following its adsorption on the surface of the sensitive bacteria. The incorporation of P³² into acid-soluble nucleotides was impaired to a smaller extent.
Unlike colicin, pyocin was not susceptible to the action of proteolytic enzymes, and its killing action was not reversed by the addi-tion of several proteolytic enzymes.
The authors are grateful to Dr. Norio Shimazono, in whose loboratory the present experiments were carried out, for his continous encouragement and in-terest throughout this work. Thanks are due to Drs. Fujio Egami and Kayoko Ikeda of the Depart-ment of Biophysics and Biochemistry, Faculty of Science, University of Tokyo, for their invaluable advices and helpful discussions.

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Using dispersed rat liver cells, C¹⁴-acetate incorporation into lipids was studied. The rate of lipid synthesis, as well as that of acetate oxidation in the cells, was linear with time, whereas the rates of both reactions in cell homogenates decreased with time. Distribu-tion of radioactivity in various fractions of lipid synthesized in the cells was comparable with the physiological pattern of the lipid fractions of normal rat liver. The type of medium used to suspend the dispersed cells significantly influenced the lipid synthesizing activity. Thus, 0.1 M sucrose medium was better than Lock e's or polyvinylpyrrolidone solution. ATP, Mg⁺⁺, citrate, GSH, and NADPH stimulated lipid synthesis.
With regard to a hormonal effect on lipid synthesis, epinephrine and dexamethasone (16α-methyl-9α-fluoro-l-dehydrocortisol) inhibit synthesis, while insulin stimulates it only in the presence of pyruvate. The inhibitory action of epinephrine is not caused by cyclic 3', 5'-AMP. These hormonal effects were not observed in a cell-free system.
The authors wish to thank Dr. K. Saito, Department of Biochemistry, Kansai Medical College, for his help with analyses of the lipid fractionations.

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