Buoyant-density centrifugation of unfixed chromatin has been performed in a newly devised medium containing 3-iodo-1, 2-propanediol and metrizamide. Chromatins were obtained from isotopically labeled mouse hepatoma cells in suspension culture, either grown normally or density labeled in a medium containing bromodeoxyuridine, by mild digestion of isolated nuclei with micrococcal nuclease. When a mixture of normal and density labeled chromatin, marked with [₁₄C] thymidine and [₃H] bromodeoxyuridine, respectively, was centrifuged in the medium, chromatin peaks represented by labeled DNA were resolved to the extent expected from their separate banding profiles. Centrifugation of an equivalent chromatin mixture labeled with [₁₄C] and [₃H] lysine, respectively, also yielded resolution of chromatin peaks represented by labeled proteins. Only small amounts of labeled proteins were dissociated from chromatin in the gradient medium. Labeled proteins recovered from the gradient fractions were analyzed by electrophoresis on sodium dodecyl sulfate-polyacrylamide gels. The results suggested that most of the histones remained associate) with the original stretches of DNA during the centrifugal fractionation period. Essentially all of the dissociated proteins were found to be non-histone proteins.

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1. The degree of calcium ion binding to chondroitin sulfate was larger than that of sodium ions. The association of calcium ions with the mucopolysaccharide cannot be ascribed solely to electrostatic forces.
2. No differences in the degree of ion binding of chondroitin sulfate isomers A and C were detectable.
3. The spacing between charged groups of chondroitin sulfate was found to be 0.48nm, which is consistent with the results of X-ray analysis.
4. The characteristic ratio was large. Therefore, rotation about the backbone bonds is highly restricted.

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1. The relation between intrinsic viscosity in 0.2M NaCI and molecular weight for samples of chondroitin sulfate isolated from shark cartilage could be expressed by [η]=6.5×10^-6M_r^1.14.
2. Their sedimentation coefficients were linear with respect to the square root of molecular weight, and the intercept was not zero but definitely positive, indicating that chondroitin sulfate in solution can be represented by a semi-flexible coil with a molecular diameter of about 1.2nm. This value was consistent with the results of X-ray analysis.
3. The molecular weight dependence of the sedimentation coefficient for chondroitin sulfate from ox nasal septa cartilage was similar to that for chondroitin sulfate isolated from shark cartilage.

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7-cis Retinal, one of the geometrical isomers of retinal, was prepared by the direct irradiation of all-trans retinal dissolved in ethanol and successive separation by high performance liquid chromatography. The procedures for its purification and identification are described.

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Glycyl-DL-norleucine methyl ester (GN), a diazoacetyl-DL-norleucine methyl ester (DAN) analog, in the presence of cupric ions was found to partially quench the protein fluorescence of acid protease from Cladosporium sp. No. 45-2, and cupric ions were also found to quench the fluorescence. These quenchings were pH-dependent. GN alone did not quench the fluorescence of the enzyme.
The interaction between the enzyme and GN in the presence of cupric ions was studied statically at pH 5.4 in terms of fluorescence change. The dissociation constant, K_d, of the enzyme-GN complex in the presence of a 20-fold molar excess of cupric ions (0.08mM) determined by fluorescence titration at 3°C (K_d=1.86mM) was in good agreement with that obtained for GN from kinetics of inhibition of DAN-induced inactivation in the presence of a 20-fold molar excess of cupric ions at 30°C (K_A=1.94mM) (Kanazawa, H. (1977) J. Biochem. 81, 1739-1744). At various concentrations of cupric ions, no change of K_d was found.
These results suggest that cupric ions are attracted to a negatively charged carboxyl group responsible for the formation of the enzyme-GN complex.

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The influence of the DTNB light chain of myosin on its enzymatic activities was examined by studying the superprecipitation of actomyosin and the actin-activated ATPase of heavy meromyosin (HMM) [EC 3. 6. 1. 3]. Although the Ca²⁺-, Mg²⁺-, and EDTA-ATPase activities of control and DTNB myosin were practically the same, the superprecipitation of actomyosin prepared from actin and DTNB myosin occurred more slowly than that of control myosin. The apparent binding constant obtained from double-reciprocal plots of actin-activated ATPase of DTNB HMM was lower than that of control HM. Recombination of DTNB myosin and HMM with DTNB light chains restored the original properties of myosin and HMM. The removal of DTNB light chain from myosin had no effect on the formation of the rigor complex between actin and myosin. These results suggest that the DTNB light chain participates in the interaction of myosin with actin in the presence of ATP.

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A new enzymatic method was developed for the assay of phospholipase D [phosphatidylcholine phosphatidohydrolase EC 3. 1. 4. 4] from cabbage leaves using choline oxidase from Arthrobacter globiformis cells. The method was based on the estimation of choline by the following series of enzymatic reactions after ending the phospholipase D reaction:
Choline+2O₂+H₂O^{Choline oxidase}-Betaine+2H₂O₂
2H₂O₂+Phenol+4-Aminoantipyrine^PeroxidaseQuinoneimine dye+4H₂O
The amount of choline was proportional to the amount of resulting quinoneimine dye with an absorbance maximum at 500nm. The phospholipase D reaction (choline liberation) was carried out at pH 5.5 in the presence of Ca²⁺ ions and ended by adding EDTA in conc. Tris-HCI buffer, pH 8, to give a final pH of around 8. The initial rate of the phospholipase D reaction was proportional to the enzyme concentration over the absorbance change range of O to 0.25 (equivalent to 0-21μM of choline) under the optimal reaction conditions.

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[Methyl-¹⁴C] methionine was supplied to yeast cells under aerobic and anaerobic conditions for the investigation of the pathway for ergosterol biosynthesis after the methylation of the side-chain. Under aerobic conditions, the incorporation of radioactivity into ergosterol was high. With a limited oxygen supply, in contrast, the radioactivity was first accumulated in ergosta-7, 24 (28)-dien-3β-ol and ergosta-8, 24 (28)-dien-3β-ol, and then transferred to ergost-7-en-3β-ol, ergost-8-en-3β-ol and ergosta-7, 22-dien-3β-ol with time. Under strictly anaerobic conditions, a double bond was introduced neither to Δ⁵ nor to Δ²². The results of the tracer experiments suggested the operation of several pathways in the late stages of ergosterol biosynthesis. It was also suggested that the main pathways varied depending on the conditions such as oxygen supply and other factors. The above conclusion was supported by the results of the analyses of the sterol compositions of the cells grown under various conditions.

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Dictyostelium discoideum is able to metabolize [1-¹⁴C] galactose to ¹⁴CO₂ despite the observation that galactose is inhibitory with respect to growth. Galactose-l-phosphate uridyl transferase activity is present throughout growth and development and varies in activity only slightly during the entire life cycle of D. discoideum, in contrast to the rapid increase in UDP-glucose 4-epimerase activity during development. Therefore, in D. discoideum, these two enzymes of the Leloir pathway are independently regulated, unlike E. coli where these enzymes are coordinately controlled.

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RNA from erythroid-enriched bone marrow cells of mouse was analyzed under two kinds of denaturing conditions (in 99% formamide and formaldehyde treatment), and globin mRNA sequence-containing RNAs larger than 18S in addition to 16S and lOS were detected by hybridization at a comparatively high Rot with complementary DNA (cDNA) to globin mRNA (10S).
The existence of precursor RNAs larger than 18S was confirmed by two different methods; (i) recentrifugation in 99% formamide of RNA collected from the corresponding regions of RNA fractions and following detection of globin mRNA sequences, (ii) the specific isolation of [3H]labeled precursor RNA with a cDNA-cellulose column and fractionation in a 99% formamide-sucrose gradient. Furthermore, pulse-chase experiments indicated that some large RNAs with globin mRNA sequences could be chased to lOS RNA during a 30 min chase in the presence of a high concentration of actinomycin D (10μg/ml).
From the results it was assumed that 35-37S RNAs with globin mRNA sequences were possible primary transcripts of globin genes and other RNAs with globin mRNA sequences in 16S, 20S, and 26S regions were intermediate precursors of lOS globin mRNA.

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OEP, a component consisting mainly of protein with small amounts of lipids and sugars, has been isolated from the autolysate of Pseudomonas aeruginosa and purified by physicochemical methods. It possesses remarkable biological properties, showing antitumor and interferon-inducing activities.
As regards the antitumor activity of the sample, the ED₅₀ value against ascites sarcoma-180 was 1μg/kg mouse/day, and its interferon-inducing activity amounted to 15 units at a concentration of 0.01μg/ml. Both activities increased after protease digestion, reaching about ten times those of the sample which had not undergone digestion.
The protease-treated OEP contained 17% protein, 14.5% total sugars, 31% lipids, 12.5% hexosamine, 3.8% KDO, and 2.7% phosphorus. Neutral sugars consisted of 12.4% rhamnose, 2.7% mannose, 66.9% glucose, and other unidentified material.
Total lipids derived from OEP consisted of 65% loosely-bound and 35% covalently-bound lipids: the former contained C_14:0, C_16:0, 0_16:1, C_18:0, and C_18:1 acids and the latter, β-OH C_10:0, C_12:0, α-OH C_12:0, β-OH C_12:0, C_16:0, and C_16:1 acids.
The antitumor and interferon-inducing activities of OEP remained after the removal of loosely-bound lipids from OEP.

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In the water-soluble fraction of rabbit skeletal muscle, at least two types of inorganic pyrophosphatase (PPase) are distinguishable on ion exchange column chromatography. One of them, pyrophosphatase-A (PPase-A), was isolated in an electrophoretically homogeneous form.
This enzyme catalyzed the hydrolysis of PP₁ but not that of other phosphate esters. Only Mg²⁺ was required for activity and stability. Other cations such as Ca²⁺, CO₂₊, Mn²⁺, and Zn²⁺ had no activating effect. The activity of this PPase was optimum at pH 7.4.
ATP, ADP, sodium imidodiphosphate (PNP), p-chloromercuribenzoate, and Ca²⁺ inhibited its enzymic activity. The enzyme was protected by dithiothreitol (DTT) against heat denaturation.
The molecular weight was estimated to be 67, 000 by gel filtration and the molecular size of the subunit was found to be 35, 000 by gel electrophoresis in the presence of sodium dodecyl sulfate (SDS). The enzyme probably consists of two identical subunits of 35, 000 daltons.

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A bacteriolytic enzyme, PR1-Lysozyme, has been purified from the lysate of mitomycin Cinduced pyocinogenic Pseudomonas aeruginosa, by acrinol treatment, Amberlite CG-50 chromatography, ammonium sulfate fractionation, Sephadex G-100 gel filtration and two cycles of SP-Sephadex C-50 chromatography. Homogeneity of the preparation was demonstrated by three electrophoretic techniques. PR1-lysozyme is a basic protein (pI, 9.4) and consists of a single polypeptide chain having a molecular weight of 24, 000. The amino acid composition of the protein was analyzed, and no cysteine residue was found among more than 210 amino acid residues. The optimum pH for enzymatic activity was 6.4 and the enzyme exhibited about 50 to 70 times greater specific activity than hen egg-white lysozyme when assayed with chloroform-killed P. aeruginosa as a substrate. By analyzing the products of enzymatic action on purified peptidoglycan of P. aeruginosa, the enzyme was identified as an N-acetylmuramidase, i.e., the same classification as hen egg-white lysozyme. PRl-Lysozyme did not show any activity towards intact cells of gram-positive and gram-negative bacteria tested. However, the enzyme was able to lyse chloroform-killed gram-negative and grampositive bacteria.

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Soybean inhibitor D-II is an inhibitor of bovine trypsin. Sequence analysis was carried out on the reduced and S-carboxymethylated protein by conventional methods to establish the complete amino acid sequence. The sequence of D-II indicated high homology with other legume inhibitors, but it was unique because of the occurrence of identical residues (arginine) at both of the reactive sites. This structure is thought to reflect that of a prototype doubleheaded inhibitor. The possible evolutionary process of the legume double-headed inhibitors is discussed on this basis.
Comparison with another soybean inhibitor C-II suggested that a single methionine (C-II)-glutamine (D-II) replacement at the P₂' position resulted in the loss of α-chymotrypsin inhibitory activity of D-II. The results of a hydrogen peroxide oxidation experiment on C-II supported this suggestion.
The sequence of the amino-terminal 21 residues of inhibitor E-I was determined using a sequenator. It was shown that this inhibitor lacks the amino-terminal nine residues of D-II.

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Soybean Bowman-Birk inhibitor, a double-headed inhibitor of trypsin and a-chymotrypsin, was treated with cyanogen bromide and then pepsin to yield two inhibitory active fragments. Structural investigation showed that one of the fragments was derived from the trypsin inhibitory domain and the other from the chymotrypsin inhibitory domain of the inhibitor.
In contrast to the unusual stability of the native inhibitor, the separated domains were less stable and could be inactivated with excess proteinases. These results suggest that the legume double-headed inhibitors acquired their unusual stability by duplicating an ancestral single-headed structure.

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Human low density lipoprotein (LDL) was dissolved in 0.3 to 2.0% Triton X-100 at pH 7.5 and apo-LDL (B protein) was extracted from LDL to form B protein-Triton complex. Sedimentation equilibrium study of this complex in a solvent nearly isopycnic to Triton X-100 showed that the molecular weight of the protein in the complex was 570, 000. The complex eluted almost at the void volume of a Sepharose 6B column, as would be expected for a complex with a total molecular weight of roughly 900, 000, on the assumption that 0.52g of Triton was bound to 1g of protein (Helenius, A. and Simons, K. (1972) J. Biol. Chem. 247, 3656-3661). The sedimentation coefficient of the complex gave f/fmin=2.2, indicating that the complex was either as asymmetric as a fibrinogen molecule or not compact. These results show that B protein exists in its complex with Triton X-100 as an elongated or a loosely expanded dimer based on the molecular weight of monomeric B protein of 270, 000. B protein may also exist in LDL as a dimer.

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The amino acid sequence of a ferredoxin (ferredoxin II, previously called minor component) from Aphanothece sacrum, a blue-green alga, was established by a combination of various conventional methods. The total number of residues was 99, giving a molecular weight of 10, 356, which is the largest reported for a chloroplast-type ferredoxin except for a halobacterial ferredoxin. This is the first time that the sequences of two ferredoxins isolated from one species of blue-green algae have been established. The ferredoxin contained one tryptophan and two methionine residues. The position of tryptophan in ferredoxin II was the same as in higher plant ferredoxins, but the methionines were in different positions from those in horsetail and Scenedesmus ferredoxins. No total charge difference was observed between ferredoxins I and II, though they were separated on a DEAE-cellulose column.
In an alignment of the sequences of chloroplast-type ferredoxins, Aphanothece ferredoxin II had two extra residues at positions 10 and 14; this feature is common in ferredoxins of other blue-green algae and a red alga, but ferredoxin I had no extra residues and showed features in common with ferredoxins of a green alga and vascular plants. The two ferredoxins are probably genetically independent isozymes and two phylogenetic models are postulated in terms of the molecular evolution of Aphanothece ferredoxins with special reference to the existence of extra residues in various ferredoxins.

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The single tryptophan residue in ribonuclease T₁ [EC 3. 1. 4. 8] was selectively oxidized by ozone to N'-formylkynurenine, which was then converted to kynurenine by acid-catalyzed deformylation in the frozen state. The two enzyme derivatives thus formed, NFK- and Kyn-RNase T₁, lost enzymatic activity at pH 7.5, at which native RNase T₁ most efficiently catalyzes the hydrolysis of RNA. At pH 4.75, the modified enzymes retained a decreased but distinct enzymatic activity toward RNA without alteration of substrate specificity, and Kyn-RNase T₁ was four times more active than NFK-RNase T₁. The binding of 3'-GMP to these modified enzymes decreased remarkably at pH 5.5, the optimum pH for binding to the intact enzyme. The γ-carboxyl group of glutamic acid 58 was still reactive to iodoacetic acid after modification of tryptophan 59. The amounts of the carboxymethyl group introduced into NFK- and Kyn-RNase T₁ were 0.36 and 0.59mol, respectively, under conditions such that quantitative esterification of native RNase T₁ takes place.
CD spectroscopy indicated that the tertiary structure of the molecule was disordered in NFK-RNase T1, but not significantly in Kyn-RNase T₁.
It is concluded that tryptophan 59 functions in maintaining the active conformation of the protein structure, particularly in constructing the active environment for a functionally important set of groups involved in the binding of the substrate at the active site, although direct participation of in tryptophan the catalytic function of ribonuclease T₁ is unlikely.

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Guanylyl-(2'-5')-guanosine binds to RNase T₁ in 1:1 stoichiornetry with a dissociation constant of 0.22mm at pH 5.0 and 25°C. This nucleotide, coupled to aminohexyl-Sepharose 4B, is able to serve as an affinity adsorbent for guanyloribonuclease [EC 3. 1. 4. 8]. The strength of interaction between the adsorbent and various guanyloribonucleases at pH 5.0 was found to decrease in the following order: RNase N₁>RNase F₁>RNase T_l>RNase St. The bound enzymes can be released from the adsorbent either by increase of ionic strength or by increasing the pH from 5.0 to 7.5. The interaction between RNase T₁ and the adsorbent is weakened by the presence of a low concentration of 2'-, 3'-, or 5'-GMP, which are competitive inhibitors of the enzyme. RNase F_l was purified to homogeneity by use of this affinity adsorbent.

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In order to investigate the nature of amino acid residues involved in the active site of a ribonuclease from Aspergillus saitoi, the pH-profiles of kinetic parameters of RNase Ms were measured. (1) The pH dependences of kinetic parameters were measured using 2', 3'-cCMP and 2', 3'-cUMP as substrates. Analyses of the results indicated the presence of three functional groups having pK_a values of ca. 2.7, 4.5, and 6.75 in the active site of RNase Ms. (2) The pH dependence of the inhibition constant of 2', (3')-AMP gave a pattern similar to the pK₁-pH profiles of the substrate described above, again indicating the involvement of three functional groups in the active site. (3) The inhibition constant of adenosine changed only slightly with change in pH, and the functional group having pK_a about 6.75 was not observed. The results indicate the interaction of this group with the phosphate moiety of the nucleotide. (4) A similar dependence of kinetic parameters of RNase Ms using three substrates of the cyclization step was obtained. (5) By analyzing the pH-profiles of pK_m and pK₁, it was concluded that the active site of RNase Ms contains at least three functional groups, a histidine (pK_a about 6.75), a histidine or carboxyl group (pK_a 4.0-4.5) and a carboxyl group (pK_a about 2.7).

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An improved method for the preparation of glycerinated Vorticella convallaria was investigated. The pretreatment of living vorticellas with a medium containing 0.1% saponins and subsequent treatment with an extraction medium containing 35% glycerol at about 0°C was satisfactory. The equilibrium average length of contractile stalks of glycerinated vorticellas was measured at various free calcium concentrations in the reaction medium. It was found that the contractile element in the spasmoneme of the stalk is contracted by a cooperative interaction involving at least two calcium ions.

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Two Lifschütz-positive C₂₄-bile acids were isolated from bladder bile of hens. One of these was identified by isotope dilution experiments after conversion to a ³H-labeled compound, and also by GLC after methoxylation, as 3β, 7α-dihydroxychol-5-enoic acid, a key intermediate of chenodeoxycholic acid biogenesis. The other, to which the structure 3β, 7α-dihydroxychol-4-enoic acid had been assigned previously, was proved to be its 3α-epimer by several experiments.
These findings favor the alternative pathway of chenodeoxycholic acid biogenesis proposed by Yamasaki and his associates.

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Nuclei and microsomes were prepared from the livers of normal, phenobarbital (PB)-treated and β-naphthoflavone (β-NF)-treated rats, and the contents of several enzymes in both subcellular fractions were examined. In normal rats, the enzyme activities in the nuclear fraction were about one-third of those of microsomes on a phospholipid basis. The induction of some particular enzymes by the drugs was observed with nuclei as well as with microsomes. Cytochrome P-450 and NADPH-cytochrome c reductase were increased by PB treatment and cytochrome P-448 was induced by β-NF treatment both in nuclei and in microsomes. The extents of inhibition of nuclear enzyme activities by the antibodies against corresponding microsomal enzymes were almost the same as those of the microsomal activities. It was concluded that a microsomal type electron transport system exists in rat liver nuclei, and that nuclear drug-oxidizing activities are inducible by PB or β-NF as their microsomal counterparts are.

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Sulphatides (ceramide galactose-3-sulphate) were isolated from human erythrocyte membranes. The amount obtained was 3.3mg from 6.7kg of wet cells, or 1.5×10^-9 mol per g dry cells. The polar part was shown to be galactose-3-sulphate by chromatographic analysis, infrared spectrometry, and mass spectrometry after solvolytic desulphation. The ceramide part consisted of three major molecular species, sphingosine-palmitic acid, sphingosine-2-hydroxy-palmitic acid, and phytosphingosine-2-hydroxypalmitic acid, as shown by thin-layer chromatography, mass spectrometry of galactosylceramides after desulphation, and gas chromatography of components after hydrolysis. The composition differed from other human erythrocyte sphingolipids.
Although the amount of sulphatides is very low for erythrocyte, the ratio of sulphatide concentration and Na⁺-K⁺-ATPase activity [EC 3. 6. 1. 3] is similar to the situation found for several animal tissues with an increased level of Na⁺ transport. This finding is discussed in relation to a recent model of sulphatide function in a transport unit for Na⁺ and K⁺ (cofactor site model).

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The molecular weight of yeast aldehyde dehydrogenase determined by sucrose density gradient centrifugation was 207, 000±13, 000. The enzyme activity was proportional to the enzyme concentration in the range of 2×10^-11 M to 1×10^-7 M.
Cross-linking patterns obtained with yeast aldehyde dehydrogenase after treatment with a series of diimidoesters of increasing chain lengths with different reaction times resulted in the appearance of tetramers as the largest cross-linked product of the enzyme subunits. The molecular weights of its monomer, dimer, trimer, and tetramer were, 57, 000, 114, 000, 171, 000, and 228, 000, respectively, as estimated from their mobilities on SDS-electrophoresis. In tetramers monomers are probably assembled in a heterologous square arrangement.

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Tryptic digestion of colicin E2, a bacteriocin of Escherichia coli, was carried out to obtain an active fragment. Native colicin E2^*, a complex of protein A or E2* (molecular weight 50, 000) possessing DNase activity and protein B or immunity protein (MW 10, 000), was digested with a low concentration of trypsin and two fragments, TI and T2, were obtained. The TI fragment was a polypeptide having a probable molecular weight of 35, 000, while T2 fragment was further dissociated by urea treatment into two components, T2A (MW 16, 000) and protein B. These tryptic fragmentation patterns suggest a close structural similarity of colicin E2 to colicin E3.
Tryptic fragment T2A retained almost all the DNase activity of protein A, which was neutralized by protein B. At high concentrations, protein A or T2A caused rapid single strand scission of closed-circular ColEl DNA followed by endonucleolytic cleavage.
Protein A showed full activities of native colicin E2 as regards killing sensitive cells and leakage of intracellular methyl-β-D-thiogalactoside (TMG) in λ-lysogenic cells, but none of TI, T2, and T2A showed these activities in vivo.

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The ATPase activity of chicken gizzard myosin was studied by varying the KCl concentration in the reaction medium. The following was thus found: (a) A sharp depression of the activity occurred when the KCl concentration was reduced to less than 0.3M, showing the minimum activity around 0.15M KCl. (b) The activity depression was removed by addition of urea or by papain-digestion, but not by addition of p -chloromercuribenzoate. (c) In the KCl concentration where the activity depression occurred, the ATPase reaction proceeded in two distinct phases; the activity was relatively high in the early phase of the reaction and declined into the later phase where the steady state reaction took place. (d) In the KCl concentrations higher than that particular concentration or in the presence of urea, the ATPase reaction proceeded in one phase. (e) The temperature dependence of the ATPase activity in the early phase was of an ordinary magnitude being approximately equal to that of the ATPase activity in 0.6M KCl. In contrast, the temperature dependence of the activity in the later phase was unusually small.
Gizzard myosin in various concentrations of KCl was also examined by measuring the turbidity and the light-scattering intensity, and by observation under an electron microscope. The following was thus found: (a) In the KCl concentration where the activity depression occurred, there was a stagnation in the turbidity decrease as the KCl concentration was gradually increased and also the formation of “ thick filaments, ” each of which was approximately 0.6-0.9μm in length and 20-30nm in diameter with no central “bare zone.” (b) Addition of ATP induced dissociation of the thick filaments, and the dissociation occurred during the early phase of the ATPase reaction. (c) Moreover, the temperature dependence of the ATP-induced dissociation rate was approximately equal to that of the ATPase activity in the early phase.

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Iron-free aposuperoxide dismutase was prepared by two methods from a native enzyme from Pseudomonas ovalis. The first method was the incubation of the native enzyme with sodium carbonate buffer (pH 11.0) containing dithiothreitol and EDTA under anaerobic conditions with subsequent dialysis to return the pH to 7.8. The second method was the incubation of the native enzyme in 3-(N-morpholino)-propanesulfonic acid-NaOH buffer (pH 7.0) containing urea, dithiothreitol, and EDTA under anaerobic conditions with subsequent dialysis to remove the urea. The reconstituted enzyme was obtained by incubation of the apoenzyme with the corresponding buffer described above, with addition of ferrous ammonium sulfate under anaerobic conditions.
The apoenzyme had neither any significant iron content nor enzymatic activity. The reconstituted enzyme had the same amount of iron and the same enzymatic activity as the native one. The apoenzyme had no visible absorption, but the reconstituted enzyme had the same visible absorption as the native enzyme. The four sulfhydryl groups of the apoenzyme showed high reactivity with 5, 5'-dithiobis-(2-nitrobenzoic acid), while those of the native and reconstituted enzymes had little reactivity. The fluorescence intensity of the apoenzyme was about three times that of the native and reconstituted enzymes. Circular dichroism spectra of the apo- and reconstituted enzymes in the 200-250nm region were similar in shape to that of the native enzyme. In the 250-340nm region, the circular dichroism spectrum of the apoenzyme was different from that of the native enzyme; this difference was greatly reduced in the reconstituted enzyme.
The electron paramagnetic resonance spectrum of the reconstituted enzyme showed the same signal prameters as the native enzyme. Thermal inactivation kinetics of the reconstituted enzyme were compared with those of the native enzyme; the reconstituted enzyme was slightly less stable than the native enzyme.

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The nature of basic differential equations governing the concentration of substrate and product species in a reaction of maltose catalyzed by sacchariying α-amylase from B. subtilis was investigated by analyzing their behavior near zero time. Near zero time, the differential equations could be approximated by simultaneous linear differential equations with respect to time. A characteristic equation was formulated from the coefficient matrix, and the eigen values, which are functions of initial maltose concentration and kinetic constants, were calculated. The eigen values were found to be negative real numbers except for one eigen value. This one eigen value had a negative real value at low maltose concentrations and a positive one at high concentrations.
Threshold concentration was defined as the initial maltose concentration at which the eigen value had zero value. The concentration was estimated to be 38.2mM. Above the threshold concentration, the concentrations of oligomers having chains longer than maltose increased explosively at the initial stage of reaction and the concentrations at the steady state increased sharply with increase of substrate concentration, showing a typical threshold phenomenon. This type of threshold phenomenon may not necessarily be characteristic of this enzyme. Enzymes which have activities for both degradation and synthesis may also show a threshold phenomenon.

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Pyridinoline is a crosslink compound isolated from bovine Achilles tendon collagen. It is a 3-hydroxypyridinium derivative with three amino and three carboxyl groups (Fujimoto, D., Akiba, K., & Nakamura, N. (1977) Biochem. Biophys. Res. Commun. 76, 1124-1129). The contents of pyridinoline in collagens from various sources were determined. The pyridinoline content of bovine Achilles tendon was 0.16 residue per 1, 000 residues and that of rat Achilles tendon collagen was 0.017 residue per 1, 000 residues. Besides Achilles tendon collagens, pyridinoline was found in collagens from costal cartilage, rib and femoral bone of rat. It was not found in collagens from the tail tendon and skin of rat. A crosslinked, triple-chained peptide containing pyridinoline was isolated from bovine Achilles tendon collagen after digestion with pronase. Its amino acid composition suggests that the peptide may be involved in an intermolecular crosslink among a carboxyterminal sequence, a sequence near the aminoterminus and a sequence in the helical region.

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Actin was obtained in high purity and with a high yield from acetone powder of fresh chicken gizzard muscle without pre-extraction of myosin.
1. The gizzard actin migrated as a single band in SDS-gel electrophoresis, showing a molecular weight of 42, 000±1, 000. The amino acid composition of gizzard actin was almost identical with those of uterine and skeletal actins. Gizzard actin contained approximately 6 cysteinyl residues per mol of actin whereas skeletal actin contained 5 cysteinyl residues per mol.
2. Two forms of F-actin, 83S and 36S, were obtained depending on whether or not calcium ions were present in the medium used for extraction and depolymerization. 83S F-actin was similar to skeletal F-actin in viscosity, sedimentation coefficient and the degree of activation of myosin-Mg²⁺-ATPase activity. 36S F-actin solution showed extremely low viscosity, steady ATP splitting, and slightly activated myosin ATPase.
3. When dialyzed against a buffer without added calcium, gizzard G-actin, which was otherwise capable of polymerizing to 83S F-actin, was transformed to 36S-forming G-actin with concomitant loss of a considerable amount of calcium.

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5-Fluorotryptophan (5FT) inhibited the activity of anthranilate synthetase (AS) of Brevibacterium flavum as strongly as did tryptophan. The concentration of 5FT required for complete inhibition of AS was similar to that for growth inhibition. Higher concentrations of 5FT repressed the synthesis of the tryptophan enzymes with a non-coordinate relationship between AS or anthranilate-phosphoribosylpyrophosphate phosphoribosyltransferase (PRT) and indole-3-glycerol phosphate synthetase (InGPS) or tryptophan synthetase (TS)-B. In a mutant resistant to low concentrations of 5FT, strain 251, which produced anthranilate and had an AS less sensitive to feedback inhibition by tryptophan, all the tryptophan enzymes showed normal levels. On the other hand, in a mutant resistant to higher concentrations of 5FT, which was derived from strain 251 and produced L-tryptophan, the synthesis of all the tryptophan enzymes except PRT had been derepressed. In mutants resistant to high concentrations of 5FT, strains 1288 and 399, which were derived directly from the wild-type strain and produced L-tryptophan, the syntheses of all the tryptophan enzymes and of all except TS had been derepressed, respectively, in addition to desensitization of AS to feedback inhibition. From these results, it was concluded that the syntheses of the five enzymes in the tryptophan pathway are regulated independently of each other in B. flavum and that derepression of PRAI and of InGPS, as well as desensitization of AS to feedback inhibition, is essential for tryptophan overproduction. Among L-tryptophan-producing mutants, the amount of L-tryptophan produced depended on the extent of desensitization of AS rather than the specific activities of the derepressed enzymes.

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Lipid micelles consisting of a glyceride mixture (triolein, diolein, and monoolein) and lecithin bound adrenaline-14C more strongly than did micelles consisting of the glyceride mixture only. Lipid micelles consisting of the glyceride mixture and phosphatidic acid also bound adrenaline-¹⁴C effectivily, whereas lipid micelles consisting of the glyceride mixture and diglyceride, obtained from lecithin, did not bind the hormone strongly. Both phenoxybenzamine (an α-blocker) and propranolol (α β-blocker) strongly inhibited the association between adrenaline-¹⁴C and lipid micelles consisting of the glyceride mixture and lecithin. Propranolol, inhibited adrenaline-induced lipolysis in both fat cells and fat globules, whereas, phenoxybenzamine, did not affect adrenaline-induced lipolysis. Both agents reduced adrenaline-induced adenylcyclase activation in fat cell ghosts. Phospholipid was also found to be related with adrenalinemediated adenylcyclase activation.

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A species of lysozyme (SE lysozyme) was purified from culture filtrate of Streptomyces erythraeus. The enzyme has a molecular weight of 18, 500 as determined by ultracentrifugation. Its isoelectrie point is 9.5, and it shows optimal activity at pH 4.0 with an optimal ionic strength of 0.1. Investigation of the substrate specificity showed SE lysozyme to be an N-acetyl-muramidase. The simplest product in the digest of cell walls of Micrococcus lysodeikticus was identified as a disaccharide, [GlcNAcβ(1→4) MurNAc]. While S. aureus as well as M. lysodeikticus was lysed by this lysozyme, chitin and its derivatives were not.

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An improved negative staining method has been used to visualize monomeric myosin molecules. Each lobe (subfragment 1) of the molecule looked like an elongated pear, and the widths of the thick and thin portions were about 95 and 55Å, respectively. The length of each lobe was about 210Å. It appeared capable of moving azimuthally and altitudinally, utilizing its juncture with the rod portion as the base. The rod portion of the molecule was about 1400Å long and 30Å wide. It also appeared to possess a considerably flexible region at a point about 680Å from the tail-end.

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