Fibronectin was isolated from porcine plasma by affinity chromatography with gelatin-linked Sepharose 4 B. Porcine fibronectin had a chemical composition similar to those of human and other fibronectins and reacted with antiserum raised against human fibronectin. It showed hemagglutination activity with trypsin-treated rabbit erythrocytes, though the activity was far less than that of human fibronectin. Porcine plasma fibronectin consisted of two subunit chains of about 230, 000-daltons linked by disulfide bond (s). Limited proteolysis of this protein with porcine liver cathepsin B yielded five major fragments which were investigated by affinity chromatography with gelatin- and heparin-linked Sepharose 4 B. One fragment (Mr=50, 000) was bound to gelatin but not to heparin, while the remaining four were bound to heparin but not to gelatin, suggesting that plasma fibronectin takes a discrete domain structure with respect to interaction with these two macromolecules. The three larger heparin-binding fragments, Mr=175, 000, 150, 000, and 130, 000 were eluted with different concentrations of a mixture of NaCl and urea from the heparincolumn, suggesting that they have different interactions with heparin, the 130, 000-dalton fragment being the one with the strongest interaction. After reduction with 2-mercaptoethanol, the 175, 000-dalton fragment was converted to the 150, 000-dalton region fragment, which, together with the unchanged 150, 000-dalton fragment, appeared to be equivalent in amount to the 130, 000-dalton fragment. This finding suggests that the 150, 000- and 130, 000-dalton fragments may have originated from different subunit chains. Since the 175, 000-dalton fragment was not produced by cathepsin B digestion of fibronectin which had been treated with plasmin, it was concluded that the 175, 000-dalton fragment contained interchain disulfide bond (s) which had linked the native subunit chains. These results suggest that porcine plasma fibronectin has non-identical subunit chains composed of domains which differ in interaction with heparin and in susceptibility to cathepsin B.

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Rat liver nuclei were isolated in aqueous solutions of low ionic strength or anhydrous glycerol. The presence of ribonuclease H (RNase H) [EC 3. 1. 4. 34] activity in the cytoplasm is due to extraction of the nuclear enzyme by buffer and inorganic salts. Two forms of RNase H were separated from rat liver nuclei by affinity chromatography using a DNA-cellulose column. When the RNases H in the wash solution of nuclei with 0.3M sucrose and in nuclear solution extracted with 0.15M NaCl were fractionated on a single-stranded DNA-cellulose column, two peaks corresponding to Mn²⁺- and Mg²⁺-dependent RNases H were eluted at 0.1M and 0.2M NaCl, respectively, and a peak having both RNase H activities was recovered in the wash-through fraction from the column. Among the enzymes from these two fractions in the nuclei, the activity of the Mg²⁺-dependent RNase H which binds to DNA-cellulose increased several-fold within 24 h of a single injection of thioacetamide. The activities of Mg²⁺-dependent RNase H extracted with highersalt solution from the nuclei and recovered in the flow-through fraction from the DNA-cellulose column and the Mn²⁺-dependent RNase H activities were relatively unaffected by an injection of thioacetamide.

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Cholestanol (5α-cholestan-3β-ol) in human serum was determined by gas-liquid chromatography after removal by epoxidation of cholesterol which is present in amounts about 400 times those of cholestanol and disturbs the accurate determination of minor sterols in serum. Epicholestanol (5α-cholestan-3α-ol) was used as an internal standard. By this method the cholestanol content in sera of 7 patients with cerebrotendinous xanthomatosis was determined. The cholestanol levels in those sera were significantly higher than those of normal subjects and the present method proved to be useful in a biochemical diagnosis of cerebrotendinous xanthomatosis.

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The phosphorylation of endogenous membrane proteins by an endogenous protein kinase was studied in isolated plasma membranes from AH-66 hepatoma ascites cells using [γ-³²P] ATP as a precursor. The phosphorylation occurred very rapidly in the presence of 10mM Mg²⁺ and reached a maximal level at 2min. Ca²⁺ strongly inhibited the phosphorylation reaction and antagonized the activation produced by Mg²⁺. Neither cyclic AMP nor cyclic GMP had a significant effect on the phosphorylation. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography showed that only membrane proteins ranging in molecular weight from 125, 000 to 200, 000 were heavily phosphorylated and those of less than 60, 000 molecular weight were not phosphorylated at all. The protein kinase activity was readily extractable from the plasma membranes with 1mM EDTA at pH 8.5. Among exogenous substrates, the extracted protein kinase catalyzed the phosphorylation of histone, protamine and phosvitin rather than casein. When the extracted protein kinase was subjected to chromatography on DEAE-Sepharose, a single major peak of cyclic AMP-independent protein kinase was eluted at a position quite different from those of the cytosolic protein kinases.

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L-Galactonolactone oxidase was found to be inactivated by various sulfhydryl reagents: the order of inactivation rate was HgCl₂>p-chloromercuribenzoate>4, 4'-dipyridyldisulfide>2, 2'-dipyridyldisulfide>5, 5'-dithio-bis-(2-nitrobenzoate)>N-ethylmaleimide>iodoacetamide. The inactivation by 4, 4'-dipyridyldisulfide was studied in detail, and it was found that the maximum degree of inactivation attained with increasing reagent concentration was 93%, and that the kinetics of inactivation was first order with respect to the reagent concentration. The pH dependence of the second-order rate constant of the inactivation revealed that a sulfhydryl group with a pK_a of approximately 9.8 was involved in the inactivation process. The value of pK_a, is high compared with that of low-molecular weight thiols, indicating that the ionization of the sulfhydryl group is affected by the electric field of a negatively charged group located in its vicinity. The values of K_m and V_max for the L-galactonolactone oxidase reaction did not change greatly around pH 9.8. This finding is consistent with the view that the sulfhydryl group does not participate in the catalytic process. The velocity of inactivation in the presence of substrate was considerably greater than that observed in its absence. It appears that the sulfhydryl group becomes more reactive during the catalytic cycle of the enzyme. It was also noted that the absorption spectrum of the fiavin prosthetic group (the oxidized form) was modified by adding p-chloromercuribenzoate.

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The preferential solvent interaction with bovine serum albumin in aqueous solutions of polyhydric alcohols (ethylene glycol, glycerol, xylitol, sorbitol, mannitol, and inositol) was investigated by a densimetric method with the application of multicomponent theory. This protein was preferentially hydrated in all solvent systems examined: the extent depended on the number and the steric configuration of the hydroxyl groups of alcohols. The absolute interactions of these alcohols with the protein were estimated by assuming that the amount of hydration of protein at every solvent composition used is identical with that in pure water. The preferential hydration of the protein in 30% aqueous solutions of glycerol and sorbitol was found to decrease as the temperature was increased, indicating that the increase in chemical potential of protein on transferring it from water to both aqueous solvents is generated by a large positive enthalpy change, sufficient to compensate for the positive entropy change in the transfer process. On the basis of these results, the mechanism of stabilization of protein structure by these alcohols was discussed from the viewpoint of the solvation of protein.

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In order to clarify the mechanism of polyol-induced stabilization of protein, the thermodynamic parameters (ΔG°, ΔH°, and ΔS°) of thermal denaturation of chymotrypsinogen have been measured in aqueous solutions of some polyols (ethylene glycol, erythritol, adonitol, sorbitol, mannitol, and inositol) by a differential spectrophotometric method. On increasing the alcohol concentration and the number of hydroxymethyl groups of the alcohols, ΔG° increased as a result of a large decrease in ΔS° compensating for a decrease in ΔH°. This result means that the stabilization of this protein by polyols is due to the entropy effect, and that the free energy change of transfer of the denatured protein from water to aqueous media containing these alcohols must be larger than that of the native protein. This strongly supports the previous proposal that the driving force of protein stabilization induced by polyols is a solvent medium effect or a solvent ordering effect. The decreases in ΔH° and ΔS° with polyols are expected to be more due to the effects of polyols on peptidewater interactions than to exposed nonpolar groups of denatured protein.

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Thiophosphorylated HMM was prepared by incubating chicken gizzard HMM with a partially purified preparation of gizzard myosin light-chain kinase and ATP-γ-S. The ATPase reactions catalyzed by gizzard un-thiophosphorylated HMM (or gizzard un-thiophosphorylated myosin) and by acto-gizzard un-thiophosphorylated HMM (or acto-gizzard un-thiophosphorylated myosin) were compared with those catalyzed by gizzard thiophosphorylated HMM (or gizzard thiophosphorylated myosin) and by acto-gizzard thiophosphorylated HMM (or acto-gizzard thiophosphorylated myosin). The following results were obtained.
1. The second-order rate constants for the ATP-induced fluorescence enhancement and for the P_i burst of gizzard HMM (or gizzard myosin), and those for ATP-induced dissociation of acto-gizzard HMM (or acto-gizzard myosin) were practically unaffected by thiophosphorylation of the light-chain subunit of HMM (or myosin) either in 5mM KCI or in 0.5M KCl.
2. The ATPase activity (Δv₀) of acto-gizzard HMM, the apparent first-order rate constant (v_recomb) for recombination of gizzard HMM^ADP_P with F-actin, which occurred after hydrolysis of ATP, and the extent (1-α) of binding of F-actin with gizzard HMM during the ATPase reaction were measured in 5mM KCl. The actinactivated ATPase reaction, either with gizzard un-thiophosphorylated HMM or with gizzard thiophosphorylated HMM, was found to fit the following equation:
Δv₀=(1-α)Δv₀+α•v_recomb,
where Δv₀ was obtained by extrapolating Δv_o to an infinite concentration of F-actin. 3. The first-order rate constant (Κ_recomb) for recombination of HMM^ADP_P with F-actin at the infinite concentration of F-actin, was increased approximately 3 times by thiophosphorylation of the light-chain subunit. Δv₀ was increased approximately 10 times and the apparent dissociation constant of acto-gizzard HMM in the presence of ATP was decreased to approximately one tenth by thiophosphorylation of the light-chain subunit. As a result, Κ_recomb became much smaller than Δv₀ in the KCl concentration range from 5 to 90mM. In other words, ATP hydrolysis via direct decomposition of acto-HMM^ADP_P was the main pathway in a wide KCl concentration range.
4. The extent (α) of AMPPNP-induced dissociation of acto-gizzard HMM was practically unaffected by thiophosphorylation of the light-chain subunit, whereas, as described above, the extent (α) of dissociation of acto-gizzard HMM during the ATPase reaction was markedly decreased by thiophosphorylation of the light-chain subunit. On addition of F-actin to HMM^ADP_P which was produced by preincubating gizzard thiophosphorylated HMM with ATP, the amount of Pi liberation linearly increased with reaction time, showing no lag phase in the early stage. On the basis of these results, we suggest that in the ATPase reaction catalyzed by acto-HMM, the reaction equilibrium, acto-HMMATP^→_←acto-HMM^ADP_P, was shifted by thiophosphorylation of the light-chain subunit, and that the refractory state did not occur on the reaction of gizzard HMM with ATP.

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Inorganic pyrophosphatase [EC 3. 6. 1. 1] from Streptococcus faecalis ATCC 8043 was purified to homogeneity as judged by slab gel electrophoresis in the presence and absence of sodium dodecyl sulfate. The purification consisted of the following steps: streptomycin sulfate precipitation, (NH₄)₂SO₄ treatment in a Sepharose CL-4 B column, DEAE-Sepharose CL-6 B chromatography, gel filtration on Ultrogel AcA 34, and preparative slab gel electrophoresis.
Gel filtration on Ultrogel AcA 34 was used to determine the molecular weight and slab gel electrophoresis in the presence of sodium dodecyl sulfate to study the subunit molecular weight. The enzyme appeared to be composed of four subunits with molecular weights of approximately 32, 500. The molecular weight of the native enzyme was about 128, 000.
The enzyme catalyzed the hydrolysis of inorganic pyrophosphate, and no activity was found with a variety of other phosphate esters. The cation Mg²⁺ was required for maximum activity; Co²⁺ and Ca²⁺ supported 24% and 5.6% of the activity observed with Mg²⁺, respectively. 2, 4, 6-Trinitrobenzene sulfonic acid inhibited and reductants containing SH-groups activated the enzyme suggesting that lysine and cysteine have essential roles in the enzyme.

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After Tris (hydroxymethyl) aminomethane [Tris buffer]-treatment cross-linking of the chloroplast thylakoid peptides of 11, 13, 18, 43, 55, and 87 kdaltons was observed on SDS-polyacrylamide gel electrophoresis. Tris-induced disulfide formation was suggested by the decrease of thiol groups of the chloroplast thylakoids in Tris medium at pHs above 8. In addition to the finding that Tris coordinates to Cu²⁺ in the forms of Tris-Cu²⁺ and Tris₂-Cu²⁺ whose successive stability constants are 5.78×10³M^-1 and 4.46×10⁶M^-2, respectively, it was observed that Tris-Cu²⁺ complexes catalyze cysteine oxidation. Since release of copper from the chloroplast thylakoids is enhanced by increasing the concentration of Tris, oxidation of thiols by Tris-Cu²⁺ complexes formed in chloroplast thylakoids is a possible mechanism for the cross-linking of chloroplast thylakoid peptides.

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The ATPase activity of purified coupling factor 1 (CF₁) of spinach chloroplasts [EC 3. 6. 1. 3] was reversibly enhanced in some aqueous organic solvents, notably methanol, ethanol, and acetone. Pretreatment of CF₁ with 20% (v/v) methanol did not affect the subsequent activity. The activity depended entirely on the final concentration of methanol in the reaction mixture. In the presence of 20%. methanol, the K_m of Ca²⁺-ATPase for ATP was lowered from 0.4mM to 0.2mM. Not only Ca²⁺, but also Cd²⁺, Mg²⁺, Mn²⁺, and Zn²⁺ supported the ATPase activity at rates of higher than 7 μmol•mg protein^-1•min^-1• Co²⁺, Ni²⁺, and Pb²⁺ supported the activity at rates of 0.5-1.0 μmol•mg protein^-1•min^-1. The activities supported by the following cations, if any, were less than 0.2 μmol•mg protein^-1•min^-1; Ba²⁺ Cu²⁺ Fe²⁺ Hg²⁺, Sn²⁺, and Sr²⁺. The optimum concentration of methanol for Ca²⁺-ATPase and Mg²⁺-ATPase activities was about 30% (v/v). The optimum pH values for Ca²⁺-ATPase and Mg²⁺-ATPase activities were about 8.0 and 8.8, respectively.
The enhancing effect of organic solvents appears to be associated with their relative lipophilic character as defined by the octanol-water partition coefficient.
The Ca²⁺-ATPase activities of the trypsin-activated and the heat-activated CF₁ were inhibited and their Mg²⁺-ATPase activities were enhanced by the presence of methanol in the reaction mixture.

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RNA polymerase II was purified from mouse Ehrlich ascites tumor cells, and was used to transcribe chromatin from Friend leukemic cells at various stages of induction. The transcription was entirely dependent on exogenous RNA polymerase, and was inhibited by actinomycin D, or a low dose of α-amanitin.
Globin-specific sequences of the transcripts were determined by hybridization of ³H-labeled transcripts with plasmid DNA immobilized on filters. When chromatin from uninduced cells was used, less than 0.01% of the total radioactivity was retained on the globin DNA-filters, whereas about 0.05% was retained when chromatin was made from induced cells. This value was about one-ninth of the content of cytoplasmic globin mRNA which was pulse-labeled in induced cells.
The transcription of whole chromatin or globin genes in chromatin was inhibited almost completely by a rifampicin derivative, but in the case of chromatin, both sense and anti-sense strands of globin genes were transcribed. From these results, it is suggested that isolated chromatin retains some native conformation but is transcribed aberrantly.

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1. RNase St was inactivated by iodoacetate. The inactivation was most rapid at pH 5.0-7.0. Competitive inhibitors protected RNase St from inactivation by iodoacetate. The protective effect of 2'-GMP was most effective among nucleotides tested.
2. RNase St was inactivated with the concomitant incorporation of one molar equivalent of carboxymethyl group. The carboxymethyl group incorporated into RNase St was liberated by treatment with 0.2 N NaOH or 1M hydroxylamine. Thus the incorporation of a carboxymethyl group into a carboxyl group was demonstrated.
3. ¹⁴C-labeled CM-RNase St was digested successively with alkaline protease and aminopeptidase M. The ¹⁴C-labeled amino acid was identified as the carboxymethyl ester of glutamic acid by means of column chromatography.
4. By digestion of reduced carboxymethylated CM-RNase St with trypsin, a peptide containing a ¹⁴C-carboxymethyl group was isolated by Dowex AG-50 W column chromatography. α-Chymotryptic digestion of the radioactive tryptic peptide, Glu₄₈-Lys₆₅, produced a tetrapeptide containing a ¹⁴C-carboxymethyl group, that is, Tyr₅₉-His₆₀-Glu₆₁-Tyr₆₂. Therefore, it was concluded that GLu₆₁ in RNase St was the site of carboxymethylation.
5. When RNase St was inactivated by iodoacetamide at pH 8.0, about 2 histidine residues were modified. The molar ratio of the products of carboxyamidomethylation were 52.3%., 21.7%, 21.0%, and 4.8%, for 3-CAM-His, 1, 3-di-CAM-His, 1-CAM-His, and di-CM-Lys, respectively.
6. CD spectra of CM-RNase St and CAM-RNase St were practically the same as that of the native RNase St indicating the maintenance of the native conformation during modification.
7. The binding constants of CM-RNase St and CAM-RNase St with 2'-GMP were about 1/150 and 1/38 of that of the native enzyme, respectively.

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Mesoheme bound to heme oxygenase protein was easily degraded to mesobiliverdin by incubation with NADPH-cytochrome c reductase and NADPH. The features of mesoheme degradation were very similar to those of protoheme degradation catalyzed by the heme oxygenase system; an intermediate compound having its absorption maximum at 660nm appeared in the course of mesoheme degradation and this compound is presumably equivalent to the 688 nm compound which appears in the course of protoheme degradation.
Hydroxymesoheme was chemically prepared and a complex of hydroxymesoheme and heme oxygenase was prepared. The complex was fairly stable in air, but when the complex was incubated with the NADPH-cytochrome c reductase system, the hydroxymesoheme bound to heme oxygenase was readily converted to mesobiliverdin through the 660nm compound as an intermediate. It is evident that hydroxyheme is a real intermediate of heme degradation in the heme oxygenase reaction and that the 688nm compound (or the 660 nm compound in the mesoheme system) is located between hydroxyheme and the biliverdin-iron chelate. The ferrous state of heme-iron may also be necessary for the onset of further oxidation of hydroxyheme.

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The features of the charge distribution in the vicinity of the ATP-binding site of heavy meromyosin (HMM) were investigated by the technique of dynamic fluorescence quenching. Instead of ATP, 1, N⁶-ethenoadenosine triphosphate (ε-ATP), a fluorescent derivative of ATP, was attached to the ATP-binding site in the presence of an ATP-regenerating system. The I^- ion and acrylamide were used as negativeand zero-charged quenchers. In addition to these quenchers, we used the Tl⁺ ion, which has recently been found to be a highly efficient quencher with positive charge, and to be generally applicable to fluorescence-labeled proteins. The Stern-Volmer quenching constants of Tl⁺ and I^- for ε-ATP bound to HMM both decreased with increasing ionic strength of the solvent. This result means that there is an electrostatic attractive force between the fluorophore and both Tl⁺ and I^-. On the other hand, the Stern-Volmer quenching constant of acrylamide was not significantly affected by a change in ionic strength. This result confirms that no significant change in protein conformation in the vicinity of the ε-ATP-binding site of HMM occurs with change in ionic strength. In order to interpret these results, we propose a model in which a positive charge is located on one side of the ε-adenine ring and a negative charge is located on the opposite side. The negative charge is attributed to the phosphate group in ε-ATP and the positive one is probably attributable to a lysyl residue in the polypeptide chain of HMM.

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1. Although the mean ornithine decarboxylase (ODC) activity induced by feeding in alloxan-diabetic rats was 60% of that in normal rats, it was markedly suppressed in pancreatectomized rats depending on the degree of pancreatectomy.
2. There was a correlation between plasma glucagon concentration and hepatic ODC activity under various dietary conditions.
3. When glucagon or insulin was given to pancreatectomized rats, dietary induction of ODC was restored to a level found in normal rats. The effects of glucagon and insulin were not additive, suggesting that these hormones act with a similar mechanism. Feeding 75% casein diet resulted in a considerable induction of ODC even in pancreatectomized rats. The enzyme was only slightly induced, however, when pancreatic hormones were given to starved rats.
4. Findings from amino acid analysis indicated that the influx of some amino acids into liver was stimulated by administration of glucagon.
5. These results suggest that increased influx of amino acids into liver is a critical factor for dietary induction of ODC and that both glucagon and insulin play a role in stimulating amino acid influx.

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Mutants of Shigella sonnei (S. sonnei) deficient in DNA polymerase I were isolated after mutagenesis with nitrosoguanidine. The isolation of the mutants was facilitated by the use of a strain harboring plasmid pBR 313 which required DNA polymerase I for its multiplication. The mutants isolated could not maintain the plasmid and became sensitive to methyl methanesulfonate (MMS) and to ultraviolet light (UV) irradiation. Assays performed on crude extracts established that the mutants were deficient in an enzyme with DNA polymerase activity. All of these properties are the same as those of E. coli polA. Several MMS-resistant revertants isolated from one of the S. sonnei polA mutants regained 3-120% of the DNA polymerase activity found in the extracts of the wild-type parent strain. Most though not all of the revertants could support the multiplication of plasmid pBR 313.

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Anhydroglucitol in human plasma was studied by high pressure liquid chromatography, which was employed to simplify sampling procedures for the polyol assay and to avoid enzymatic treatment of the plasma for removal of glucose. Polyols in the column effluent were assayed by a flow fluorometric method, which involved two continuous successive reactions: periodate oxidation and the Hantzsch reaction. Plasma, 1ml each, from 55 diabetic and 18 control subjects was deproteinized, concentrated, and subjected to the liquid chromatography. The results of this analysis indicated that the plasma anhydroglucitol level showed an inverse correlation with the plasma glucose level, while the correlation of glycerol and glucose levels was obscure.

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Skeletal muscle troponin C (TN-C) labeled with N-(p-(2-benzimidazolyl)phenyl)-maleimide (BIPM) shows about 5% fluorescence increase and 82% fluorescence increase upon Ca²⁺ binding and Mg²⁺ binding to the high affinity Ca²⁺-binding sites (sites III and IV) of TN-C, respectively. TN-C labeled with N-(1-anilinonaphthyl-4) maleimide (ANM) shows about 26% fluorescence decrease and 22% fluorescence increase upon Ca²⁺ binding and Mg²⁺ binding to the high affinity Ca²⁺-binding sites, respectively. These findings indicate that environmental change around Cys-98, where the maleimide reagents bind, induced by Ca²⁺ binding to the high affinity Ca²⁺-binding sites is very different from that induced by Mg²⁺ binding to the same sites. These dye-protein conjugates do not show fluorescence intensity change upon Ca²⁺ binding to the low affinity Ca²⁺-binding sites (sites I and II). Dansylaziridine (DANZ)-labeled TN-C shows more than 100% fluorescence increase upon Ca²⁺ binding to the low affinity Ca²⁺-binding sites of TN-C. Hence, we can observe the kinetic processes which TN-C undergoes upon Ca²⁺ binding to or removal from the high affinity Cal-binding sites by following the fluorescence intensity change of ANM-labeled TN-C and those upon Ca²⁺ binding to or removal from the low affinity Ca²⁺-binding sites by following the fluorescence intensity change of DANZ-labeled TN-C, respectively. The kinetic processes of the fluorescence intensity change associated with the Ca²⁺ binding and removal reactions with the high affinity Ca²⁺-binding sites (rate constants, 3.7-157 s^-1) are slower than the kinetic processes associated with the low affinity Ca²⁺-binding sites (rate constants, equal to or higher than 230 s^-1) in the absence of MgCl₂. In the presence of 2mM MgCl₂, a new slow phase (rate constant, 10-16 s^-1) appears in the kinetic processes associated with the low affinity Ca²⁺-binding sites, in addition to the rapid phase which is already observed in the absence of MgCl₂. Kinetic properties associated with the high affinity Ca²⁺-binding sites are not essentially altered by addition of 2mM MgCl₂ to the system, but ANM-labeled TN-C shows lower rate constants (0.67-26 s^-1).

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The reactive lysyl residues located in the subfragment-1 (S-1)/subfragment-2 (S-2) link region of cardiac myosin heavy chains (HCs) were preferentially labeled with a fluorescent reagent, N-methyl-2-anilino-6-naphthalenesulfonyl chloride(Mns-Cl). The effect of Mns-labeling of these residues on the ability of actomyosin to superprecipitate and on the actin-activated ATPase of myosin was investigated using two types of labeled myosins; one was myosin labeled in the absence of divalent metal ions, where one or two reactive lysyl residues were labeled (termed E-1-Mns-M or E-2-Mns-M, respectively) and the other was myosin labeled in the presence of Ca²⁺, where both reactive residues were intact but another lysyl residue was labeled (termed C-1-Mns-M).
Both the rate and extent of superprecipitation of actomyosin reconstituted from actin and two types of labeled myosins were reduced. Although the Mg²⁺-ATPase activity of E-1-Mns-M or E-2-Mns-M was practically unchanged, its actin-activated ATPase activities at both low and high ionic strengths were reduced. This reduction was saturated at an incorporation of about two moles of Mns groups per mol of myosin. Neither partial release nor partial labeling of light chain-2, which occurred during the labeling of the reactive lysyl residues, was responsible for the reduction in the actin-activated ATPase activity of myosin. The apparent dissociation constant for actin, K_m, obtained from the double-reciprocal plot of actin-activated ATPase activity of E-1-Mns-M or E-2-Mns-M at low ionic strength was increased 4- or 6-fold over that of unlabeled myosin, respectively. On the other hand, changes in the actin-activated ATPase activity and in the K_m of myosin were hardly observed when Ca²⁺ protected the reactive residues from the labeling, and another residue was labeled.
These results suggest that in the presence of ATP the actin binding site “senses” changes in the S-1/S-2 link region of the cardiac myosin HC including the reactive lysyl residue.

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Rat liver 40 S ribosomal subunits were treated with a bifunctional imidoester, dimethyl 3, 3'-dithiobispropionimidate (DTP), and the neighboring protein pairs were identified. The cross-linked proteins were analyzed by acrylamide/SDS diagonal gel electrophoresis (Sommer & Traut (1974) Proc. Natl. Acad. Sci. U. S. 71, 3946-3950). The cross-linked components that fell off the diagonal upon adding 2-mercaptoethanol in the second dimension were labeled with ¹²⁵I in the acrylamide gel and identified by two-dimensional acrylamide/urea gel electrophoresis, followed by radioautography.
Considering these results and the molecular weights, we propose the following ten pairs, according to our numbering system (Terao & Ogata (1975) Biochim. Biophys. Acta 402, 219-229): S 3-S 5 (S 3/S 3 a-S 4), S 3-S 14 (S 3/S 3 a-S 14), S 3-S 17 (S 3/S 3 a-S 16), S 5-S 22 (S 4-S 23/S 24), S 10-S 12 (S 8-S 11), S 9-S 16 (S 9-S 18), S 9-S 22 (S 9-S 23/S 24), S 6-S 23 (S 5-S 25), S 17-S 21 (S 16-S 19), and S 16-S 26 (S 18-S 27). The designations according to the proposed uniform nomenclature (McConkey et al. (1979) Mol. Gen. Genet. 169, 1-6) are given in parentheses.

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Creatine kinase has been isolated in a good yield from porcine skeletal muscle. The enzyme was purified about 8-fold from crude extract of porcine skeletal muscle in a yield of about 44%. The purified enzyme was homogeneous, as judged by sedimentation velocity (s⁰_{20, W}=5.31 S), sedimentation equilibrium, and polyacrylamide gel electrophoresis. The molecular weight of the enzyme was determined to be 83, 200 by high-speed meniscus-depletion sedimentation equilibrium analysis. The molecular weight of its subunit was estimated to be 43, 000-44, 000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The amino acid composition of the enzyme was determined.

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A methanol dehydrogenase [EC 1. 1. 99. 8] was purified and crystallized from methanol-grown Methylomonas J (formerly Pseudomonas sp. J), an obligate methylotroph. Its molecular weight was estimated to be 135, 000 by gel chromatography. The sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis revealed two bands and their molecular weights were approximately 60, 000 and 10, 000. The enzyme was relatively stable at pH 6 and 10, and was unstable at pH 8. The enzyme activity was lost at pH 4.0; however, the prosthetic group was not liberated from the enzyme. Its isoelectric point was pH 9.3. The visible-ultraviolet absorption, fluorescence, CD, and ESR spectra were measured. The amino acid composition was analyzed after separation of the two components. Primary alcohols and formaldehyde served as substrates. Phenazine methosulfate (PMS) was an effective electron acceptor of the enzyme in the presence of either NH₄Cl or methylamine. The enzyme was inhibited partially by metal chelators and completely by Mn²⁺ and Co²⁺.

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Two kinds of c-type cytochromes, cytochrome c-551 (I), and cytochrome c-551 (II), were highly purified and crystallized from cell-free extract of methanol-grown Methylomonas J (formerly Pseudomonas sp. J) and their physicochemical and biochemical properties were studied. Cytochrome c-551 (I) had an absorption peak at 409 nm in the oxidized form, and peaks at 417, 523, and 551 nm, and a shoulder at 532 nm in the reduced form. The millimolar extinction coefficient of the α-peak of the reduced form was 25.3. The isoelectric point was at pH 5.3 and its standard redox potential was 0.29 V at pH 7.0. The molecular weight was estimated to be 16, 000. Cytochrome c-551 (II) had absorption maxima at 409 nm in the oxidized form, and at 416, 521, and 551 nm in the reduced form. The millimolar extinction coefficient of the α-peak of the reduced form was 22.4. The isoelectric point was at pH 4.3 and its standard redox potential was 0.24 V at pH 7.0. The molecular weight was estimated to be 12, 500. The two cytochromes were reduced by methanol dehydrogenase [EC 1. 1. 99. 8] of this bacterium, and formaldehyde was detected as an oxidation product. Ammonium chloride was not essential for reduction of the cytochromes. No significant reduction of the cytochromes was observed by methylamine dehydrogenase isolated from methylamine-grown cells or by 2, 6-dichlorophenol-indophenol (DCPIP)-dependent aldehyde dehydrogenase of the methanol-grown cells. The reduced forms of the cytochromes were oxidized by blue copper protein of the methanol-grown cells.

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Two forms of urokinase [EC 3. 4. 99. 26] with molecular weights of 51, 600 and 34, 500 were purified from human urine. The specific activities of the high molecular weight urokinase (HMW-UK) and low molecular weight urokinase (LMW-UK) were 157, 400 and 246, 700 International Units (IU/mg), respectively. Purified HMW-UK was 97% active and LMW-UK was 88% active, as judged by using p-nitrophenyl-p'-guanidinobenzoate. LMW-UK had five multiple isoelectric subforms, compared with HMW-UK which had only one. Not only HMW-UK but also LMW-UK was composed of two polypeptide chains linked by disulfide bond (s). The molecular weight of the heavy chain of both forms was the same (34, 000 daltons), while the molecular weight of the light chain of HMW-UK was 17, 600 and that of LMW-UK was approximately 1, 200-3, 400. Enzyme kinetic studies revealed that the kinetic constants, K_m and κ_cat, of both forms toward the synthetic substrates, acetyl-Gly-Lys-methylester (AGLMe) and glutaryl-Gly-Arg-4-methylcoumarin-7-amide (GGA-MCA), were almost the same, but the dissociation constant of HMW-UK toward Glu-plasminogen was 2.4-2.6 times less than that of LMWUK. HMW-UK incubated at 37°C was converted into LMW-UK in an autocatalytic digestion manner leading to no loss of the total activity. These results show that HMW-UK with a higher affinity toward Glu-plasminogen is converted into LMW-UK with a lower affinity, a greater portion of the light chain of HMWUK splitting off.

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The structure of the calcium-activated neutral proteinase (CANP) from rabbit skeletal muscle was examined. The purified CANP was homogeneous as judged by disc gel electrophoresis, while it showed two bands (M. W.=80, 000 (80 K) and 31, 000 (30 K)) on SDS-gel electrophoresis. After denaturation of CANP, each subunit was separated and could be renatured alone or in combination with the other subunit. The results indicated that all the machinary necessary for the proteolytic activity is present in the 80 K subunit, but for the full activity the existence of the 30 K subunit is required.

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Whale heparin was separated by affinity chromatography on an antithrombin III-Sepharose column into two distinct fractions. The high-affinity fraction accounted for most of the anticoagulant activity of the unfractionated material, while the low-affinity fraction was relatively inactive. The yields of the two fractions were substantially equivalent. No significant difference was observed between these fractions in terms of electrophoretic mobilities on cellulose acetate membrane and analytical data except for the contents of N-acetylglucosamine and N-sulfoglucosamine. The highly active form contained more N-acetylglucosamine and less N-sulfoglucosamine than the relatively inactive form.
The two fractions were separately subjected to sequential digestion with purified heparinase and heparitinase, and the oligosaccharide fractions were isolated from the digests by DEAE-cellulose column chromatography, followed by preparative paper chromatography. The purified compounds were then characterized by routine chemical and physical methods. Compound 1, Δ_{4, 5}hexosyluronic acidl→4 N-acetylglucosamine, was exclusively obtained from the highly active form, whereas compound 3 a, Δ_{4, 5}hexosyluronic acidl→4 N-acetylglucosamine 6-sulfate, and compound 3 b Δ_{4, 5}hexosyluronic acidl→4 N-sulfoglucosamine, were the only ones obtained from the relatively inactive form. The yields of other oligosaccharide fractions from both forms were comparable. The present data suggest that an N-acetylglucosamine-containing oligosaccharide structure in whale heparin is essential for binding to antithrombin III.

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Droplet counter-current chromatography (DCC), a novel liquid-liquid partition technique, is a useful method for the separation of glycolipids. After a series of investigations on suitable experimental conditions for DCC, mouse brain gangliosides were separated into GM₁, GD_1a, GT_1b with the solvent system of chloroformmethanol-0.5% CaCl₂- n-propanol (50:60:40:6) by the ascending method with 500 standard columns. GM₁ and cerebroside sulfuric ester (CSE) from whale brain glycolipid fraction were separated by DCC into two peaks and three peaks, respectively, with chloroform-methanol-water-n-propanol (50:60:40:2) and 500 standard columns. The occurrence of two peaks of GM₁ was due to the differences of sphingenine and fatty acid compositions. In the former peak of GM₁, the majority of long chain base and fatty acid were 18:1 sphingenine and stearic acid respectively, while the latter contained 18:1 (32%), 20:1 (68%) sphingenine and 16:0 (14.5%), 18:0 (61.4%), and 20:0 (21.0%) fatty acids. The three peaks of CSE were due to the difference of carbon chain length and to the presence or absence of a hydroxyl group in fatty acids. The first polar fraction (CSE-I) had hydroxy 23:0 (34.0%) and 24.1 (53.4%) fatty acids and the second fraction (CSEII) contained hydroxy 24:0 (70.5%) and 25:1 (12.8%) fatty acids, while the third fraction (CSE-III) had normal 23:0 (11.9%), 24:1 (56.4%), hydroxy 25:0 (11.4%) and 26:1 (9.7%) fatty acids. Preparative DCC was also applied to the glycolipid fraction from Tay-Sachs' brain to give 242mg of practically pure GM₂ from 700mg of crude brain extract without any preliminary purification.

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Modification of E-64 focused on the terminal agmatine for practical use led to some potent analogs which were successfully obtained by a stereoselective synthesis using D-tartaric acid as a starting material. Ep-475 (id. E-64-c), in which the agmatine was replaced by 3-methyl-butylamine, was found to react with the essential SH of papain in accord with the decrease of activity. [³H] Ep-475 was irreversibly incorporated into papain in an equimolar ratio. None of the analogs showed any effect on thiol enzymes other than proteolytic ones.

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The troponin T₂ (m. w. 13, 000) subfragment showed a Ca²⁺ sensitizing action on the superprecipitation and ATPase activities of actomyosin in the presence of tropomyosin, troponin I, and troponin C. On the other hand, troponin T₂β (m. w. 11, 000), the properties of which were similar to those of troponin T₂α except for the lower affinity for tropomyosin, did not show the Ca²⁺ sensitizing action.

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The N-terminal and C-terminal residues as well as amino acid compositions were determined for the chymotryptic subfragments from rabbit skeletal troponin T. The positions of the subfragments in the sequence of troponin T were N-terminal acetylserine-tyrosine-158 for troponin T₁ (m. w. 18, 700), leucine-159-C-terminal lysine-259 for troponin T₂α (m. w. 11, 900), and leucine-159-phenylalanine-242 for troponin T₂β (m. w. 10, 200), respectively.

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The content of epidermal growth factor (EGF) as a high molecular weight complex (HMW-EGF) in the submandibular glands of mice was measured simply by a single radial immunodiffusion method. In female mice, the amount of HMW-EGF was increased 10-fold by tri-iodo-L-thyronine (T₃) and 60-fold by 5α-dihydrotestosterone (5α-DHT). In mice with testicular feminization (Tfm), which are genetically deficient in androgen receptor, T₃ but not 5α-DHT increased the HMW-EGF from a non-detectable level to 5.4±0.94 μg/mg protein. It was concluded that EGF is also synthesized under the control of thyroid hormone in vivo, and that androgen was not involved in this induction of EGF by thyroid hormone.

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β-Galactosidase was partially restored by protease inhibitors, leupeptin, chymostatin and E-64 in cultured fibroblasts from three patients with β-galactosidaseneuraminidase deficiency. Pepstatin did not activate this enzyme. Neuraminidase was not affected by any of these compounds in the culture medium. It was concluded that the activating effect was produced by a specific inhibition of thiol proteases.

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Ca²⁺-activated neutral protease (CANP) usually requires mM Ca²⁺ for activation. The sensitivity of CANP to Ca²⁺ is greatly enhanced by passing it through a casein-Sepharose column in the presence of Ca²⁺ ions. This conversion is ascribed to autolysis of CANP. The converted enzyme required 40 μM Ca²⁺ for 50%. activation. Various properties of the converted enzyme were very similar to those of CANP-I, recently found in canine heart muscle. Names of “m-CANP” and “μ-CANP” are proposed for CANPs which require mM and μM order Ca²⁺ for activation, respectively.

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Heterogeneous expression of myosin light chain 1 was observed in a human slowtwitch muscle fiber on two dimensional gel electrophoresis. Three spots were found around the L1 region and one of them corresponded to fast type L1. The nature of the heterogeneity is discussed.

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A new thiol-protease inhibitor, E-64-c (N-[N-(L-3-carboxyoxirane-2-carbonyl)-L-leucyl]-isoamylamine), specifically suppressed the disappearance of the Z-line induced by calcium in rat soleus muscle. The result suggests that calcium-activated neutral protease (CANP) may be involved in such calcium-mediated degeneration of myofilaments.

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Crystalline actin tubes can be formed in the presence of Pr (III) and examples can be selected which are opened along their length. The structural unit cell seen in electron micrographs were computer-averaged to obtain the projected size and shape of the actin monomer. There are two monomers per unit cell, each related by a two-fold axis perpendicular to the plane of the projection. Each 3.3×5.5 nm monomer was markedly asymmetric. Skeletal muscle actin strongly resembles Acanthamoeba actin (Aebi et al. (1980) Nature 288, 296) although the projected dimensions may vary slightly.

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The enzyme reactions from NNM-A to NNM-E and from NNM-E to NNM-B were established in a cell-free system containing an extract from the nanaomycin producer Streptomyces rosa var. notoensis. The enzyme which catalyses the former epoxide-forming reaction (NNM-A→NNM-E) requires NADH (or NADPH) and O₂ suggesting that it is the monooxygenase-type; thus, NNM-A monooxygenase (4a, 10a-epoxidizing) is proposed as its name. The other enzyme which catalyses the reductive epoxide-opening reaction (NNM-E→NNM-B) requires NADH or NADPH and is tentatively named NNM-B synthetase. Such a reductive epoxideopening reaction is a novel enzyme reaction.

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The amino (N)-terminal sequence of the larger subunit (α) of the MoFe-protein from Clostridium pasteurianum was determined up to 179 amino acid residues by analyses of BrCN and tryptic peptides of the original subunit. Apparent similarities exist among the sequence of the clostridial α-subunit, that of the smaller subunit (β) of the Azotobacter vinelandii MoFe-protein, and those predicted from the nucleotide sequences of nif D genes in Klebsiella pneumoniae and Anabaena 7120. In comparing the sequences of C. pasteurianum and K. pneumoniae, 45% of residues are identical of a total of 184 sites. Therefore, the larger subunit of the clostridial MoFeprotein must correspond to the nif D gene product of K. pneumoniae.

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