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The binding properties of dynein arms to the A- and B-tubules of outer doublets of cilia from Tetrahymena pyriformis were examined, with the following results: 1. When 30s dynein purified from Tetrahymena cilia was added to doublets deficient in dynein arms, it bound to both A- and B-tubules almost equally and formed arms along the edges. The overall length of arms bound to the A-tubule was 22 +/- 3 nm, and that of arms bound to the B-tubule was 24 +/- 3 nm. Each arm bound to the A- and B-tubules was pointed toward the base at angles of 55 degrees +/- 7 degrees and 48 degrees +/- 7 degrees, respectively. In the presence of sufficient amounts of dynein, the arms along the A- and B-tubules were located at intervals of 22.8 +/- 1.5 nm and 22.5 +/- 1.7 nm, respectively. 2. On adding ATP, only the arms bound to the B-tubule were dissociated from the doublet decorated with arms on both sides. The dissociated arms rebound themselves to the B-tubule after hydrolysis of the ATP. When several doublets decorated with arms along both A- and B-tubules were arrayed side by side, the interdoublet spacing increased from 14 +/- 2 nm to 17 +/- 2 nm on addition of ATP. 3. The turbidity of a suspension of trypsin [EC 3.4.21.4]-treated axonemes decreased rapidly on addition of ATP, then recovered partially. Observations by dark-field microscopy and electron microscopy showed that the doublets which had slid out from the axonemes on ATP addition formed large aggregates after hydrolysis of the ATP. The dynein arms were also solubilized from the axonemes upon addition of ATP, and rebound themselves to the B-tubule after hydrolysis of the added ATP. 4. The double-reciprocal plot for the ATPase [EC 3.6.1.3] activity of the trypsin-treated axonemes against ATP concentration was composed of two straight lines, from which the Km values were estimated to be 1.0 and 12.7 micrometer. The dependence of the decrease in turbidity of the axonemal suspension on ATP concentration indicated that the binding of ATP to sites with an apparent dissociation constant of 1 micrometer induced dissociation of the arms from the B-tubule.

The molecular weight of Streptomyces subtilisin inhibitor (SSI), a protein proteinase inhibitor, and that of the complex of SSI and subtilisin BPN' [EC 3.4.21.14] were determined by a sedimentation equilibrium method in 25 mM phosphate buffer, at pH 7.0, ionic strength 0.1 M (NaCl), 25.0 degrees C. The molecular weight of SSI was found to be 23,000 over a wide concentration range, 0.01-10 mg/ml, the range used for inhibitory, spectrophotometric, and kinetic measurements. Based on the amino acid sequence, the molecular weight of SSI has been calculated to be 11,500 (Ikenaka, T., et al. (1974) J. Biochem. 76, 1191-1209); therefore, the molecular weight of 23,000 obtained above suggests that SSI is in a dimeric form under usual conditions in the concentration range of 5 X 10(-7)-5 X 10(-4) M. The molecular weight of the subtilisin BPN'-SSI complex was determined to be 78,000 in the concentration range of 0.03-5.0 mg/ml by sedimentation equilibrium of the crystallized preparation and by that of a mixture of subtilisin BPN' and SSI treated as a multicomponent-polydisperse system. The molecular weight obtained here, combined with the results of binding stoichiometry (Inouye, K., et al. (1977) J. Biochem. 82, 961-967) that showed that one mol of SSI (molecular weight, 11,500) and one mol of the enzyme (molecular weight, 27,500) are tightly bound (Kd less than 1 nM), demonstrate that one mol of dimeric SSI binds two mol of the enzyme to form a stable complex, E2I2.

We previously reported (J. Biochem. 70,95--123 (1971) that the time course of Pi liberation in the reaction of Ca2+, Mg2+--dependent ATPase [EC 3.6.1.3.] of fragmented sarcoplasmic reticulum (SR) consists of a lag phase, a burst phase, and a steady phase. We also showed that the rate constant, kd, of decomposition of the phosphorylated intermediate (E approximately P) decreases during the initial phase, and suggested that the burst phase is due to transition of the kd value. Recently, Froehlich and Taylor (J. Biol. Chem. 250, 2013--2021 (1975)) claimed that the Pi burst is caused by the formation of an acid-labile intermediate containing phosphate (E.P) formed by rapid hydrolysis of E approximately P. In the present study, the transition of the kd value during the initial phase was measured precisely, and the results showed that the burst phase is due to a transition in the kd value, not to the existence of E-P. The main results obtained were as follows: 1. After the SR had been phosphorylated with [gamma-32P]ATP in the presence of Mg2+ and Ca2+ ions, further phosphorylated was stopped by the addition of EGTA. The concentration of E approximately 32P then decreased exponentially with time. 2. The first-order rate constants, kd, of decomposition of E aproximately 32P after adding EGTA decreased with increase in the interval, t, between the start of E approximately 32P formation and the time of adding EGTA...

Sarcoplasmic reticulum (SR) isolated from rabbit skeletal muscle was solubilized with a nonionic detergent, dodecyl octaethyleneglycol monoether (C12E8), at a weight ratio of detergent to protein of greater than 10, so that the Ca2+, Mg2+ dependent ATPase existed mainly in a monomeric form (7). The solubilized ATPase was reacted with 10 microM N-1-P or 5 microM DACM in the presence of 5 mM CaCl2, 0.4 M KCl, 20% glycerol and 50 mM TES at pH 7.5 and 20 degrees C. Under these conditions, about 1 mol of N-1-P was incorporated into 10(5) g SR protein on 10 min incubation and 1 mol of DACM was incorporated into the same amount of SR on 5 min incubation. Analysis of the tryptic digest of the N-1-P- or DACM-labeled. ATPase on SDS polyacrylamide gel revealed that almost all the fluorescence was associated with the 30K m.w. subfragment of the ATPase protein. Even when the amount of the probe incorporated into SR-ATPase was increased from 1 to 3 mol per 10(5) g SR protein, all was incorporated into the 30K subfragment. Both the activities of formation and decomposition of the phosphorylated intermediate (EP) were unaffected by these modifications. When the separately labeled ATPases were mixed together in the presence of C12E8 and the detergent was removed by incubation with Bio-Beads SM-2, a significant amount of fluorescence energy transfer was observed between N-1-P and DACM. However, energy transfer did not occur when the labeled ATPases were mixed after removal of C12E8.(ABSTRACT TRUNCATED AT 250 WORDS)

Electron energy loss spectra were measured for Y2O3 : Eu, Gd2O3 : Eu, Y2O2S : Eu, La2O2S : Eu, Gd2O2S : Eu, YVO4 : Eu and CaWO4 : Pb. Strong loss-peaks due to interband transitions are observed. Since no correlation is found between cathode-ray efficiencies and the energies of the loss-peaks which presumably originate in plasma resonance, the plasmon excitation is not important in the understanding of the efficiencies.

Glycerol-treated muscle fiber bundles were fixed at their rest length in 50 mM KC1, 2 mM MgC1(2), and 10 micron CaC1(2) at pH 7.8 and 0 degrees C in the presence of sufficient amounts of ATP, creatine kinase, and creatine phosphate. The fiber bundles were stretched linearly with time for 0.3 s at a constant amplitude, suddenly released, then fixed at the rest length for a constant time interval (alpha seconds). The stretch-release cycle was repeated, and the ATPase activity (the rate of ADP liberation) [EC 3.6.1.3] was measured. It was found that: 1. ATPase was activated by repeated stretch-release. As repetitive stretch-release of 1--2% of the rest length caused maximum activation, we usually selected a value of 2.5% of the rest length. The activation of ATPase was found to be a function of the duration, alpha, of the isometric phase after sudden release from stretching. The ATPase activity of fiber bundles was almost unaffected when they were oscillated by a simple stretch-release without an isometric phase after the sudden release (alpha=0). 2. The ATPase activity of oscillated muscle fibers increased with increase in the value of alpha, reached a maximal level, then decreased gradually with further increase of alpha to a value slightly larger than that of static fibers. At 0 degrees C, the value of alpha for the maximum activation was observed at about 2 s, and the maximum activity was about 2.5 times that of static fibers. At 20 degrees C, the alpha value for maximum activation was about 0.5 s, and the maximum activity was about 1.8 times that of static fibers. 3. The time course of ADP liberation after one stretch-release cycle could be easily calculated from the ATPase activity of the summed durations of the isometric phase, alpha, assuming that the ATPase activation was turned off and on by the stretching and release, respectively, and that the state of cross-bridges immediately after the stretch-release was independent of alpha of the cycle. The rate of ADP liberation after stretch-release thus obtained showed a short lag phase, a sigmoidal increase, a decrease to almost zero, then a return to nearly the original level (the rate of static fibers). About 1.3 mol of ATP per mol of myosin was hydrolyzed at both 0 degrees C and 20 degrees C during one cycle of the changes in the rate of ADP liberation.

The states of tyrosyl and tryptophyl residues of a dimeric protein proteinase inhibitor, Streptomyces subtilisin inhibitor (Sato, S & Murao, S. (1973), Agric. Biol. Chem. 37, 1067) were studies by solvent perturbation difference spectroscopy with methanol, ethylene glycol, polyethylene glycol, and deuterium oxide as perturbants, and by spectrophotometric titration at alkaline pH. It appeared that all three tyrosyl residues per monomer of the inhibitor were exposed on the surface of the molecule, and their apparent pK values were estimated separately to be 9.58, 11.10, and 12.42. The single tryptophyl residue per monomer of the inhibitor appeared to be partially buried in the protein molecule.

The dynamic properties of cross-bridge movement were investigated in glycerol-treated muscle fibers under various conditions by analyzing tension responses to two types of length change. First, the fiber bundles were stretched linearly with time for 0.3 s from the rest length (L0) by 2.5% of L0, suddenly released, then fixed at L0 (sudden release of the slow stretch). Second, they were stretched for 0.01 s by 2.5% of L0, then held at the plateau length (a quick stretch). 1. The transient tension responses following both length changes were divided into three phases: (i) very quick recovery of tension (0 approximately 0.05 s), (ii) quick recovery (0.05 approximately 0.3-0.4 s), and (iii) gradual recovery (0.3-0.4 s approximately several seconds). 2. The effects of activating conditions on the rates of the quick phases (0 approximately 0.3-0.4 s) were not associated with those on the nucleoside triphosphatase [EC 3.6.1.3] rates: the rates of the quick phases increased with increase in temperature and Mg2+-ATP concentration, with decrease in Ca2+ concentration, and also on replacement of Mg2+-ATP by Mg2+-ITP or Mn2+-ATP. Only a small amount of ADP, 0.07 mol per mol of myosin (Fig. 24 in the preceding paper), was liberated during the quick recovery phases. 3. The remaining slow tension recovery was concluded to be associated with one cycle of ATP splitting, and progressed very smoothly. This suggests that most of the cross-bridges do not exist in a synchronously dissociated state during one cycle of ATP splitting.

Our laboratory has presented strong evidence for the nonidentical two-headed structure of skeletal muscle myosin. We previously showed that each of the two kinds of heads, i.e., the burst head, which forms the myosin-P-ADP complex, and the nonburst head, which forms the myosin-ATP complex upon reaction with ATP, contains 1 mol of reactive lysine residue per mol which is modified rapidly with TNBS. We also found that in the presence of PPi only the reactive lysine residue in the burst head is modified with TNBS. Utilizing this phenomenon, we presented evidence [(1981) J. Biochem. 89, 831-839] indicating that the chemical structures around the reactive lysine residues in the burst and the nonburst head are different. In this study, we determined the amino acid sequence around the reactive lysine residues to demonstrate the nonidentical chemical structure of the two heads of skeletal muscle myosin. We found that the sequence around the reactive lysine residue in the burst head was ....Pro-Met-Asn-Pro-Pro-Lys-Tyr.... and the sequence in the nonburst head was ....Ser-Met-Asn-Pro-Pro-Lys-Tyr..... Thus, a proline residue located ner the reactive lysine residue in the burst head was found to be replaced by a serine residue in the nonburst head.

Microbial characteristics of two bacterial strains, of Alcaligenes and Acinetobacter capable of assimilating biphenyl and 4-chlorobiphenyl as a sole source of carbon have been studied with respect to polychlorinated biphenyl degradation. A variety of aromatic compounds were examined for the growth and O2 uptake of the organisms. It was observed that these organisms were able to adsorb 14C-2, 5, 2′-trichlorobiphenyl onto the cell surface in a short time and then gradually metabolized the adsorped one. The metabolites were moved into aqueous layer from the cells, Several spots including a yellow colored intermediate, i.e. meta cleavage product, and 2, 5-dichlorobenzoic acid as main products were detected on a thin-layer chromatography and subsequent autoradiography. However, neither incorporation of 14C-metabolites into the cell constituents nor 14CO2 evolution was observed.

Interactions of several amino acids and nucleotides with valyl-tRNA synthetase [EC 6.1.1.9] (VRS) from Bacillus stearothermophilus were investigated using as a probe the ligand-induced quenching of protein fluorescence (lambda ex = 295 nm, lambda em = 340 nm) of VRS. L-Valine, L-threonine, L-isoleucine, L-glutamic acid, L-leucine, and D-valine caused fluorescence quenching. Among them, L-threonine had a Kd value comparable to that for the cognate substrate, L-valine, but the other amino acids were bound more weakly as estimated by the fluorescence titration method. L-Alanine, L-histidine, and L-serine did not cause any fluorescence change. Among the nucleotides tested (ATP, ADP, AMP, GTP, ITP, CTP, and UTP), only ATP caused the fluorescence change. In the presence of an excess amount of ATP, only L-valine and L-threonine, among the tested amino acids, induced the fluorescence quenching, and the binding of L-valine was greatly favored under this condition. This is consistent with the results of the ATP-PP1 exchange reaction by VRS, in which only L-valine and L-threonine, of these 9 amino acids tested, could serve as substrates, and the Km value for L-valine was much smaller than that for L-threonine. Thus the binding of ATP to VRS enhances the substrate specificity of VRS towards amino acids.(ABSTRACT TRUNCATED AT 250 WORDS)

A method for the transformation of Zymomonas mobilis with plasmid DNA was developed by using shuttle vectors, pZA31, pZA32 and pZA33, as a source of transforming DNA. Partial spheroplasts of Z. mobilis were prepared by growing cells in a hypertonic medium supplemented with a drug which inhibits the biosynthesis of bacterial cell walls, such as penicillin G and d-cycloserine. They were transformed with plasmid DNA in the presence of 15% polyethylene glycol 6,000, after treated with 100 mm CaCl2. The frequency of transformation obtained was 104 to 105 transformants/μg of DNA for Z. mobilis IFO13756 (Z-6).

It has been found that the current through a Schottky barrier formed at the interface between palladium film and n-type titanium oxide (TiO 2 ) single crystal is sensitive to hydrogen or other reducing gases in the ambient. This effect is explained by taking account of the diminished barrier height at the Pd and TiO 2 interface caused by the action of gases, changing the work function of Pd metal. The change in the Pd work function estimated from the result is confirmed by the direct measurements of the metal surface potentials by use of a vibrating capacitor method. Similar electrical properties have been studied for junctions of TiO 2 with Pt, Au, Ni, Al, Cu, Mg and Zn, and of ZnO, CdS, GaP and Si w ith Pd.

A β-cyanoalanine (β-(-CNAla)-forming enzyme, which catalyzed the formation of β-CNAla from O-acetyl-L-serine and sodium cyanide, was isolated in crystalline form from Enterobacter sp. 10-1, a cyanide-resistant strain. The purification procedure involved protamine treatment, ammonium sulfate fractionation, column chromatography on DEAE-cellulose, DEAE-Sephadex A50, Sephadex G-100 and hydroxyapatite, and crystallization with ammonium sulfate. The crystalline enzyme was homogeneous by criteria of the ultracentrifugation and polyacrylamide gel electrophoresis. The enzyme was determined to have a molecular weight of about 68, 000, and consisted of two apparently identical subunits, each having a molecular weight of about 34, 000. The enzyme also catalyzed the formation of cysteine from O-acetyl-L-serine and sodium sulfide, and the cysteine-forming activity was 245 times greater than that of the β-CNAla-forming activity.

For trial use in the local chemotherapy of cancer by a combination of cytosine deaminase (EC 3.5.4.1) and 5-fluorocytosine (J. Biotechnol., (1985), 2, 13-21), 40 U of partially purified cytosine deaminase was obtained from 500 g of commercial compressed bakers' yeast. The enzyme, which is unstable, was immobilized to stabilize it by the use of commercial epoxy-acrylic beads (Eupergit C). The immobilized enzyme was made into enzyme capsules with cellulose tubing for dialysis to encapsulate it or urethane polymer to entrap it, which materials are biocompatible. The activity of the intact cellulose capsules thus made was 0.4% that of the immobilized enzyme inside. The enzyme capsules also were stable. Ten days after the cellulose capsules were implanted in rats, 25% of the starting activity remained. When the polyurethane capsules were tested in vitro for 9 mo for thermostability at 37 degrees C, the activity decreased rapidly (with a half-life of 28 d) during the first 4 mo, and then slowly (half-life, about 100 d) during the next 5 mo. A calculation to transform the biphasic decline into a sum of the exponential decline of two components of enzymic activities with different strengths and half-lives showed that the larger half-life was 5 mo.

Transfert de la capacite a degrader le raffinose chez Zymomonas mobiles par un plasmide construit in vivo entre R68.45 et le plasmide poD11.88 du raffinose

The oxidation of methanol and formaldehyde was investigated by using some combination systems of alcohol oxidase, catalase, which were purified from Candida N-16, and hydrogen peroxide. The activity of alcohol oxidase was irreversibly inhibited when the enzyme was incubated with 2.5 mm hydrogen peroxide for 15 min. However, the oxidation of methanol to formaldehyde by alcohol oxidase in the presence of catalase was extremely promoted by the addition of 30 mm hydrogen peroxide. Alcohol oxidase could oxidize not only methanol but also formaldehyde as follows: HCHO + 02 + H2O→HCOOH + H2O2. The formaldehyde oxidizing activity was inhibited by hydrogen peroxide. The system containing alcohol oxidase and catalase appears to be the entity of the oxygen-dependent oxidation system of formaldehyde previously found in the cell-free extract of the yeast.

1. 1. To clarify the chemical structures of the peptide chains of myosin A around the lysine residues, which are specifically attacked by trinitrobenzenesulfonate (TBS), myosin A, which contained 1–2 moles of trinitrophenyl group (TNP) per mole, was digested with subtilisin or pronase, and the TNP-peptides were isolated by column chromatography on talc or resin. 2. 2. The percentages of methionine and glutamic acid residues in the TNP-peptides were much lower, while those of proline and alanine were much higher, than those in the original myosin A. Notably, in the case of TNP-peptides which contained 23 moles of amino acid residues per mole of TNP-lysine, 7.4 moles were found to be proline residues. 3. 3. The TNP-peptides isolated by the above method were further purified by Dowex-50 X2 column chromatography and the finger-print technique after removal of TNP. Another method of purification of TNP-peptides was paper chromatography with n -butanol-amyl alcohol-pyridine-NH 4 OH-acetic acid-H 2 O as solvent, and the finger-print technique with 1.5 M phosphate buffer and with n -butanol-acetic acid-pyridine-H 2 O. In the first method 6 fractions, and in the second method 2 fractions, were isolated. 4. 4. The amino acid compositions and N-terminal amino acids of the former 6 fractions were readily explained as derived from SerTNPLys(Gly, Glu, Ser)(Gly, Ala). One fraction obtained by the latter method was TNPLys itself, and the amino acid sequence of the other fraction was determined to be Asp(NH 2 )ProProTNPLys by the Edman degradation and hydrazinolysis methods. 5. 5. The presence of the amide group of asparagine was deduced from the migration velocity of the peptide on ionophoresis. 6. 6. From the amino acid compositions of the TNP-peptides derived from myosin A before and after treatment with LiBr, it was concluded that the conformation of both these TNP-peptides is specifically altered by LiBr.

1. 1. The inhibition by excess substrate of myosin B-ATPase commences at a lower concentration of ATP as the ionic strength increases. 2. 2. A marked inhibition by substrate is observed in the presence of Mg ++ and a weak one in the presence of Mn ++ . In the presence of other bivalent cations, no inhibition can be observed. 3. 3. The inhibition by substrate disappears on the addition of a small amount of PCMB or Salyrgan. 4. 4. The inhibition by substrate is remarkably promoted by chelate compounds. Their promoting effect parallels closely with their relaxing effect of muscle model. 5. 5. It is demonstrated that the ATPase activity of superprecipitated myosin B is very much higher than that of a nonsuperprecipitated one, and it is concluded that the inhibition by substrate is due to the inhibition of superprecipitation by a high concentration of ATP. 6. 6. The ATP level, where the inhibition commences, is increased by an increase of the actin content of synthetic actomyosin, and at a sufficiently high ATP ATPase activity of synthetic actomyosin approaches that of myosin A.

The Michaelis constant ( K m ) and the maximum velocity ( v m ) of myosin A-ATPase (EC 3.6.1.3) and of myosin A-ITPase were measured in 0.6 M KCl, and in the presence of 7 mM CaCl 2 , over a wide range of temperature and pH. The following results were obtained: 1. 1. Both v m and K m of ITPase were increased proportionally to each other with increasing pH, and their half-saturation values were obtained at pH 6.8. The Arrhenius plots of v m and K m were readily represented by two reactions of different activation energies and the extrapolations of the linear portions intersected in each case at around 15°. The value of activation energies were 25.6 and 11.9 kcal/mole for v m and 7.9 and 2.1 kcal/mole for K m below and above 15°, respectively. 2. 2. At 20°, the K m of ATPase changed with changing pH in proportion to v m : both K m and v m showed a depression at neutral pH. The pH dependence of the ratio of v m of ITPase to that of ATPase was given by a bell-shaped curve having its maximum at pH 7.5. At 0°, the pH-activity curve of ATPase lacked the neutral depression. At pH 6, the Arrhenius plots of v m and K m were linear and the activation energies of v m and K m were 16.2 and −2.8 kcal/mole, respectively. At pH7, the activation energies of v m and K m , at temperatures higher than 10°, were noticeably different from those at temperatures lower than 10°; these values were for v m , 9.0 and 3.0 kcal/mole, and for K m , −1.9 and −10.4 kcal/mole, below and above 10°, respectively. 3. 3. These results were analyzed according to the mechanism proposed by one of the authors where by, in ATPase, two forms of the Michaelis complex, viz. , an active one (E 1 S) and an inactive one (E 2 S), can exist. It was concluded that the complex E 2 S is not formed in ITPase but in ATPase it is dominant at around pH 7.5, and at higher temperatures. It was further concluded that the rate-constant of dissociation of the Michaelis complex E 1 S into the enzyme and the substrate is much smaller than that of the breakdown of the complex into the products; the rate-constant of the formation of E 1 S is independent of pH.

Fundamental studies were carried out on the removal of polychlorinated biphenyls (PCB) contained in polluted source of supplying water. Activated carbon and coagulants such as aluminium sulfate and polyaluminium chloride (PAC) were used for removing PCB from water. In the present work, method for determination of PCB was also studied. Gaschromatographic determination of decachlorobiphenyl (DCB), which was obtained by complete chlorination of PCB with SbCl5, gave a good result. Among several preparations of PCB, Kanechlor-600 in water was determined as DCB in a good yield by gaschromatography, after extraction with n-hexane, concentration with Kuderna-Danish condenser, and chlorination with SbCl5 in a sealed glass tube at 200°for 1 hr. Removal of PCB from water was tested with Kanechlor-600 usnig the above method of determination. Two processes of treatment for aqueous solution containing 1ppm of Kanechlor-600 showed about 90% removal. One of them was the addition of activated carbon powder and coagulant at the same time, and the other was to pass the solution through a column of granular activated carbon. These processes were used in the purification system of water supplying work in some instances. It is considered that such higher purification system will be effective in removal of PCB from polluted water.

The p -nitrothiophenol-myosin A compound was produced by reaction of p -nitrothiophenol with myosin A in the presence of Mg 2+ and ATP, and was isolated by gel filtration through a Sephadex column. The change in absorption spectrum by the binding of p -nitrothiophenol to myosin A was similar to that of the formation of a thiolester bond between p -nitrothiophenol and succinic anhydride. The p -nitrothiophenol-myosin A compound was stable at acidic and neutral pH, but was unstable at alkaline pH. The stability was not affected by replacement of air with N 2 gas. The p -nitrothiophenol bound to myosin A was easily displaced by cysteine and H 2 S. p -Nitrothiophenyl-peptides were isolated with 24% recovery after proteolysis and chromatography on talc, since the proteolysis did not accelerate the liberation of p -nitrothiophenol. The hydromate was formed quantitatively by the addition of NH 2 OH to p -nitrothiophenyl-peptides. The amino acid composition of the p -nitrothiophenyl-peptides was determined, and Arg, Asp, Ser, Glu, Ileu and Leu were found as amino acid residues whose mole ratios to p -nitrothiophenol were nearly equal to or higher than 1. These results establish the covalent binding of p -nitrothiophenol to myosin A in the presence of Mg 2+ and ATP, and strongly suggest the binding of p -nitrothiophenol on a carboxyl side-chain of Glu or Asp in myosin A as an acylthiol bond. Effects of divalent cations, pH and modifiers of the ATPase activity of the p -nitrothiophenyl-myosin A were also investigated. The pH-activity curve of the p -nitrothiophenyl-myosin A was similar to those of the p -chloromercuribenzoate-myosin A compound and the trinitrophenyl-myosin A, and showed no depression at neutral pH. The ATPase (ATP phosphohydrolase, EC 3.6.1.3) of the p -nitrothiophenyl-myosin A was activated by p -chloromercuribenzoate or EDTA, whereas the ATPase of the trinitrophenyl-myosin A was not activated. Actomyosin reconstituted from the p -nitrothiophenyl-myosin A showed ATPase activity of the typical actomyosin type.

1. 1. In the presence of 0.6M of KCl, EDTA increases concomitantly the Vmax and Km of myosin B ATPase. Especially when concentration of EDTA is higher than 5mM, the line 1/v versus 1/[S] passes through the origin. 2. 2. EDTA decreases both grade and rate of the optical change of myosin B caused by ATP addition. 3. 3. The recovery step from the physically changed myosin B to the original myosin B follows strictly monomolecular kinetics. 4. 4. On the basis of these results the reaction mechanism of EDTA with the myosin B-ATP system is discussed.

The following compounds were synthesized as analogues of ATP:6-morpho 5′-triphosphate (III) and 2,6-dimethlymercapto-9-β- d -ribofuranosylpurine 5′-triphosphate (V). The interactions of these analogues with actomyosin system were investigated, together with those of 3′-deoxythymidine 5′-triphosphate (I), thymidine 5′-triphosphate (II)and 2′,3′-O-isopropylidene adenosine 5′-triphosphate (IV). The degrees of decrease in light-scattering of myosin B on addition of these analogues were similar to that induced by ATP, except in the case of Compound I. The rates of hydrolysis of analogues by myosin B in 0.6 M KCl and 7 mM Ca72+ were in the decreasing order of ATP > IV ≈ II > V > I ≈ III, while the order of hydrolysis in 0.075 M KC1 and 2mM Mg2+ was II > ATP > IV > I > III > V. Compounds II and IV, as well as ATP, induced contraction of myofibrils, while Compounds I, II and V did not. It was concluded that hydrogen bondings at the 6-N or O of the base and the 3′-O of ribose with myosin are necessary for the rapid hydrolysis fo an ATP analogue and for contraction of myofibrils by the analogue.

α-Amylase in Aspergillus oryzae is generally known to be present in mycelium as well as in medium, whether it is grown with or without shaking. The interesting fact is reported in this paper, when A. oryzae was grown in a phosphate deficient medium, however, α-amylase accumulation occurred only within the cell. From the results of pH-activity curve, starch gel zone electrophoresis and differential centrifugation, there seemed to be no distinctions between exo- and endocellular α-amylase. The liberation of α-amylase from the cell was observed to be dependent on pH and the concentrations of anions. The significance of the α-amylase accumulation and liberation in this system is discussed.

Pseudomonas 0–3 strain which was isolated from soil can grow on polyvinyl alcohol (PVA) as a sole carbon source. When 0.5 per cent of PVA (500, 1500 or 2000) was employed as the carbon source in the culture medium, PVA was almost completely lost from the culture fluid after a week and the concentration of total organic carbon measured by a TOC analyzer decreased from the initial value of about 2700 ppm to 250~300 ppm after 7~10 days culture. This bacterium was found to produce and secrete an inducible enzyme which degrade PVA. The way by which this enzyme degrades PVA was examined and the results were obtained which suggested that PVA was broken down oxidatively in a way of endowise splitting. However, the mechanism of PVA degradation has not been clarified yet. The optimum pH and temperature for enzyme activity were examined and they were 7.5~8.5 and 35~45°C, respectively. Morphological and biological characteristics of this bacterium were examined and it was similar to a strain of Pseudomonas boreopolis.

(1) 麹菌のα-アミラーゼ生成過程に於ける,菌体内遊離アミノ酸の検出を行なった. (2) α-アミラーゼの生成はエチオニン,グルタミン酸-γ-ヒドラチド及びノルロイシンにより阻害されたが,それぞれのホモローグアミノ酸により阻害は回復された. (3) 菌体内のグルタミン酸を,アイソトープ稀釈分析法で定量した結果,洗滌菌体によるα-アミラーゼの生成に於いては, lag phaseに於いてグルタミン酸量は増加し,α-アミラーゼの生成に伴って見かけ上減少したが,エチオニンを添加した時は初めから増加した. (4) Ageの古い菌体ではエチオニン阻害が少なく,又inducerの効果も少いことがわかった. (5) エチオニン添加により,核酸区分へのP32のincorporationが小さくなることを認めた. (6) 以上の結果から,少くとも若い菌体によるα-アミラーゼの生成には,遊離アミノ酸が何等かのかたちで関係しているように推定された.しかしながら,古いageの菌体の場合は,これらの関係に尚不明の点が残されている.