Human colostral macrophages stimulated by opsonized zymosan or phorbol myristate acetate (PMA) released superoxide anions (O₂^-) and hydrogen peroxide (H₂O₂) with activities comparable to those of monocytes and about one-fourth of those of polymorphonuclear leukocytes (PMNL) of blood. The O₂^--forming oxidase in the macrophages stimulated by PMA was dependent on NADPH as an electron donor with an apparent K_m value for NADPH of 27.6±4.0 μM, which is comparable to those obtained for the stimulated monocytes and PMNL of blood. The V_max was 1.86±0.33 nmol O₂/min/10⁶ cells, which is essentially the same as that of monocytes and about half of that of PMNL. p-Chloromercuribenzoate or cetyltrimethylam-monium bromide completely inhibited oxidases of all three types of phagocytes. A b-type cytochrome was identified in the macrophages but the concentrations in the macrophages and monocytes were less than half of that in PMNL. These results suggest that the differences in the O₂^--forming activities of the three types of phagocytes are quantitative rather than qualitative. The macrophages and monocytes showed very low activities of myeloperoxidase [EC 1. 11. 1. 7] in contrast to PMNL. The activity of β-glucuronidase [EC 3. 2. 1. 31] in the macrophages was much higher than those of the monocytes and PMNL, but little difference was observed in the activities of lysozyme [EC 3. 2. 1. 17], catalase [EC 1. 11. 1. 6] and superoxide dismutase [EC 1. 15. 1. 1] among the three types of phagocytes examined. Electron micrographs of the macrophages showed little increase of vacuoles upon exposure to PMA, in contrast to the cases of monocytes and PMNL.

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Specific antibodies against I-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine (platelet activating factor, PAF) were raised in rabbits. The anti-PAF IgG significantly agglutinated the PAF antigen and inhibited the platelet aggregation induced by PAF. In the double immunodiffusion test, the anti-PAF IgG specifically produced a single precipitin line with only PAF. Neither the 1-acyl-linked nor the non-acetylated analogue exhibited antigenic properties, which implies that the precise chemical structure is essential for the antigenic expression. Cross-reactivity with various PAF analogues was also examined by the radioimmunoprecipitation technique.

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The dark interaction of furocoumarins with DNA has been studied by a fluorescence quenching technique. The intrinsic fluorescence of psoralen, 4, 5', 8-trimethylpsoralen (TMP) and 8-methoxypsoralen (8-MOP) was quenched to an appreciable extent upon their noncovalent binding to DNA molecule. The analysis of the binding data revealed that TMP binds to DNA with higher efficiency than 8-MOP and psoralen, their apparent Scatchard binding constants being 13.2×10⁵M^-1, 7.1×10⁵M^-1, and 12.2×10⁵M^-1, respectively. The interaction of furocoumarins with DNA was strongly dependent on the conformational stability of DNA in the particular interaction media. The perturbation of DNA structure by changing the ionic environment decreased its interaction with furocoumarins. However, the interaction was facilitated by the presence of an electron-donating moiety in the parent compound, psoralen.

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An acidic isozyme of a chymotrypsin-like esteroprotease from the mouse subman-dibular gland was purified and its properties were compared with those of the basic isozymes purified previously (Takuma, T., et al. (1983) Biochim. Biophys. Acta 755, 70-75), bovine pancreatic α-chymotrypsin, and other chymotrypsin-like enzymes of mice. The isoelectric point of the purified enzyme was pH 4.7, and the molecular weight was estimated to be 25, 000 by gel filtration on Sephadex G-100. The enzyme hydrolyzed benzoyl-L-tyrosine ethyl ester (Bz-Tyr-OEt) 7 times more slowly than basic isozymes did, but hydrolyzed casein as slowly as the basic isozymes did. The acidic isozyme was 40 times more sensitive to chymostatin than basic isozymes were, but 10 times less sensitive than α-chymotrypsin was. Moreover, acidic and basic isozymes were immunologically distinct. Chymotrypsin-like esteroproteases in the submandibular gland were antigenically unique among chymotrypsin-like enzymes in various tissues of mice.

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Two distinct dipeptidyl aminopeptidases, which were designated DPP-A and DPP-B, were purified from soluble fraction of monkey brain using Leu-enkephalin as the substrate. The enzymes were purified 187 and 136 fold, respectively. Both enzymes showed the optimum pH in neutral range. Their molecular weights were almost equal and were estimated to be about 100, 000. Their K_m values with Leuenkephalin as the substrate were 5.6×10^-5 and 1.1×10^-5M, respectively. Among synthesized substrates, the highest affinity of the enzymes was toward arginyl-arginine β-naphthylamide with the K_m values of 6.25×10^-5 and 6.41×10^-5M, respectively. Both enzyme activities were inhibited by the metal-chelators DFP and PCMB. Two hundred fifty μM arphamenine A inhibited DPP-A and -B with inhibition of 36.6% and 44.1%, respectively. Beta-endorphin, ACTH, and glucagon inhibited only DPP-B, while β-lipotropin and angiotensin II inhibited both DPP-A and -B when Leu-enkephalin was used as the substrate.

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Thermophilic malate dehydrogenase [L-malate:NAD⁺ oxidoreductase, EC 1. 1. 1. 37] was denatured at pH 2.0 with complete loss of enzyme activity but without dissociation to monomers, suggesting the presence of strong intersubunit contact. On the other hand, the enzyme was completely denatured and dissociated to monomers in the presence of 5M GdnHCl.
Inactivation and denaturation of the enzyme by acid and GdnHCI were rever-sible. Upon dilution of the denaturants, the inactivated enzyme regained enzyme activity and the native structure with high yield (80-90%).
Kinetic analyses of reactivation of the enzyme denatured by GdnHCl and by acid revealed that the reaction obeyed first-order kinetics. The rate constant and Arrhenius activation energy of the reactivation of the acid-inactivated enzyme were almost the same as those of the enzyme inactivated by GdnHCl. These results suggest that the rate-limiting steps in the reactivation processes of the enzyme denatured by GdnHCI and by acid are the same and that a conformational change of the inactive dimer to active dimer is the rate-limiting step in the reactivation reaction.

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The inactivation mechanism of pyridoxal phosphate-linked mitochondrial aspartate transaminase (pig heart) by gostatin (5-amino-2-carboxy-4-oxo-1, 4, 5, 6-tetrahydro-pyridine-3-acetic acid), a novel amino acid produced by Streptomyces sumanensis, was investigated. Gostatin is a time-dependent inhibitor of the enzyme giving an enzyme half-life of 1.8 min at 3.1 μM (25°C). The kinetic properties of the inhibitor suggest that it is a suicide substrate (mechanism-based inhibitor) of the enzyme, and the observed K₁ is 59 μM and K_cat is 0.11 s^-1 at 25°C. Incubation of the enzyme with a stoichiometric amount of the inhibitor (1 mol of inhibitor/1 mol of enzyme monomer) results in complete inactivation. Spectrophotometric titration and gel filtration experiments indicate the binding of 1 mol of gostatin with 1 mot of enzyme monomer. Gostatin serves as an efficient titrant for the enzyme.
Liberation of a compound having inhibitory activity against the apo-form enzyme from the enzyme-inhibitor complex under denaturing conditions suggests irreversible modification of the cofactor.

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Rat M₁- and L-type pyruvate kinases were inactivated by photo-oxidation mediated by methylene blue at pH 8.0 and 0°C according to first-order kinetics. The pH profiles of the inactivation rates of these isozymes showed that amino acid residues having a pK value of 7.0-7.5 were involved in the inactivation. Three histidine residues per subunit of M₁- or L-type enzyme were destroyed by the photo-oxidation with complete loss of the enzyme activities. However, two of the three photo-oxidized histidine residues in the L-type enzyme were more important in the inactivation reaction. The kinetics of the partially inactivated L-type enzyme suggests that complete inactivation is achieved via an intermediate form having low affinity for phosphoenolpyruvate (PEP). These observations revealed the involvement of essential histidine residues of two different kinds in the catalytic mechanism of the L-type enzyme. In the photo-oxidation of M₁ -type enzyme, no intermediate form was observed.
Addition of PEP or pyruvate to the reaction mixture markedly prevented the photo-oxidative inactivation of only the M₁-type enzyme in the presence of K⁺ and Mg²⁺; the addition of ADP or ATP was ineffective even in the presence of both metal ions. This protective effect of PEP was counteracted by further addition of ATP but not by ADP. However, photo-oxidative inactivation of the L-type enzyme was not prevented even by the addition of PEP in the presence of both metal ions, owing to the low affinity for PEP at 0°C, in spite of the presence of fructose 1, 6-bisphosphate (Fru-1, 6-P₂). These results indicate that the photo-oxidizable histidine residues in these isozymes participate in the PEP binding site, but their precise roles in the catalytic mechanism are not identical in the two isozymes.

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Sarcoplasmic reticulum membranes were subjected to limited proteolysis by trypsin under various conditions and the products were analyzed with SDS-polyacrylamide gel electrophoresis. Besides the fragments well characterized in the literature, i.e. A, B, A₁, and A₂ [Thorley-Lawson, D. A. & Green, N. M. (1977) Biochem. J., 167, 739] of Mr 55, 000, 45, 000, 30, 000, and 20, 000, respectively, two closely related fragments derived from fragment A₁, and another migrating a little faster than these (Mr 27, 000-28, 000), were produced at 35°C. Appearance of these smaller fragments was accompanied by the disappearance not only of A₁ but also of A₂. This subfragmentation was prevented at 0°C, so that a large amount of A₁ fragment was accumulated. Ca²⁺ and AMP-P (NH) P, alone or in combination, also affected the susceptibility of the ATPase protein to tryptic digestion, resulting in a marked stabilization of A₁ and A₂ fragments. These findings afforded us a basis of devising a procedure for efficient production of these fragments, which would serve as useful starting materials for protein chemical analyses of this enzyme. The observed changes in susceptibility to proteolytic digestion are consistent with our current knowledge on the conformational changes of this ATPase.

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Ca²⁺-Mg²⁺-ATPase of sarcoplasmic reticulum was subjected to tryptic digestion under various conditions and the cleavage patterns were compared. The first tryptic cleavage to yield the NH₂-terminal A-fragment (Mr_??_55, 000) and COOH-terminal B-fragment (Mr_??_45, 000) [Thorley-Lawson, D. A. & Green, N. M. (1977) Biochem. J. 167, 739-748] was little affected by adding ligands such as Ca²⁺ and AMP-P(NH)P. On the other hand, subsequent splitting of A-fragment into A₁ (Mr_??_30, 000) and A₂ (Mr_??_20, 000), and further cleavages giving rise to three smaller fragments of Mr_??_27, 000-28, 000 (A_1a, A_1b, and C) [Saito, K., et al. (1984) J. Biochem. 95, 1297] were profoundly affected by these ligands. A difference in cleavage sites was noted depending on Ca²⁺ ion concentration; thus, Alb and C were the major components remaining after digestion in the presence and absence of Ca²⁺, respectively. AMP-P(NH)P markedly stabilized both A₁ and A₂ fragments, but the effect was much more prominent when Ca²⁺ was simultaneously present on the transport site. These findings suggest that conformational changes of the ATPase molecule upon binding of Ca²⁺, AMP-P(NH)P, or both are accompanied by corresponding changes in the susceptibility to tryptic digestion. Fragments A₁ and A₂ were both quite stable and fragmentation did not proceed beyond A₁, when sarcoplasmic reticulum membranes were treated with trypsin at 0°C. Significant further fragmentation of A₁ was observed only above 20°C, suggesting a conformational transition of the ATPase protein around that temperature.

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NADPH-dependent trans-2-enoyl-CoA reductase was purified to homogeneity from Escherichia coli. The molecular weight of the native enzyme was estimated to be 80, 000 by gel filtration and that of the subunit was estimated to be 40, 000 by sodium dodecyl sulfate polyacrylamide gel electrophoresis.
The enzyme was most active toward trans-2-hexenoyl-CoA and trans-2-octenoyl-CoA but was less active toward longer chain substrates, whereas the K_m values decreased progressively with increasing carbon chain length of substrates.
The reductase appears to have a functional thiol group. The enzyme activity was rapidly decreased by p-hydroxymercuribenzoate and 5, 5'-dithiobis (2-nitrobenzoic acid), and slowly by N-ethylmaleimide. The enzyme was protected from inhibition by these SH-reagents by the addition of NADPH. The enzyme was also inhibited by saturated acyl-CoA esters.

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A simple and quantitative method for the modification of sialic acid carboxyl group in ganglioside is described.
Methyl iodide was added to the ganglioside in dimethylsulfoxide. The reaction was completed quickly at room temperature, giving the methyl ester with practically no by-products.
Reduction of the methyl ester was achieved by sodium borohydride. These modified gangliosides were chemically characterized. Reduced GM₁ has a strong antigenicity compared with the original GM₁, and it raised high titer antisera which did not crossreact with the original GM₁ nor with its methyl ester.
Peanut agglutinin, which binds strongly to asialo GM₁ but weakly to GM₁, bound to the methyl ester of GM₁ and reduced GM₁.
Cholera toxin, which is specific for GM₁, also reacted with the methyl ester of GM₁ and reduced GM₁ to a certain extent.

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Hyaluronic acid was purified from the horny layer of guinea pigs and its biochemical and physical properties were studied. The horny layer, obtained by applying n-hexadecane to guinea pig skin, was digested with pronase, and glycosaminoglycans in the digest were separated from UV-absorbing material by Sephadex G-75 chromatography (sample A, 17.5mg). On DEAE-Sephadex chromatography, the fraction obtained with 0.5M NaCl was found to contain 94% of the total uronic acid. This fraction, consisting mainly of hyaluronic acid, was dialyzed and lyophilized (sample B, 12.5mg).
Sample B, consisting of 26.1% uronic acid and 27.0% glucosamine on a dry weight basis, could be digested completely with Streptomyces hyaluronidase. Sample B had a low reduced viscosity which showed almost no concentration dependence. The intrinsic viscosity of sample B was 0.83 dl/g and its molecular weight, calculated from its viscosity, was 34, 000. Sample B was eluted from Sepharose CL-6B as a broad peak between the void volume and the total column volume. The enzyme levels of hyaluronidase, β-glucuronidase, and β-N-acetylglucosaminidase in the n-hexadecane treated guinea pig skin increased to 1.7 to 2.5 fold those of controls after 6 days of the experiment. These results suggested that hyaluronic acid in the horny layer of n-hexadecane treated guinea pig skin might be degraded by hyaluronic acid degrading enzymes in the hyperkeratinized tissue.

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A 26K fragment of troponin T, which was produced by endogenous proteases in rabbit skeletal muscle, was isolated by SE-Sephadex column chromatography. This fragment sensitized both superprecipitation and ATPase of actomyosin to calcium ions, to the same extent as troponin T. There was no difference in affinity for tropomyosin between this fragment and troponin T as examined by affinity chromatography. Amino acid analysis showed that this fragment consisted of residues Ala-46-Lys-259 of troponin T. The N-terminal 45 residues of troponin T, therefore, are not essential for the physiological action of troponin T. It was also observed that Ca²⁺-activated neutral protease digested troponin T into the 26K fragment in the native thin filament, while the protease digested troponin T in a different way in the reconstituted thin filament.

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We investigated the effect of tryptic digestion of S-1 (95K) into three segments (27K, 50K, and 20K) on the binding of F-actin with S-1, using an ultracentrifugal separation method and a light-scattering method. The tryptic digestion of S-1 decreased the affinity of S-1 for F-actin both in the absence of nucleotide and the presence of AMPPNP or ATP, suggesting that the peptide cutting impairs the structures participating in the binding of S-1 or S-1-nucleotide complex with F-actin. Although nucleotides markedly weakened the affinity of S-1 for F-actin, the ratios of affinity of digested S-1 for F-actin to that of intact S-1 were all about 1/10 both in the absence and presence of nucleotides. This may be understood if we accept the assumption that two kinds of structure participate in the binding of S-1 with F-actin; one is independent of nucleotides and the other is dependent on them, with only the former being affected by the tryptic digestion of S-1.

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Alkanoyl-N-methylglucamides, nonionic detergents, were utilized to solubilize membrane proteins of Escherichia coli and were used to reconstitute them into liposomes. First, critical micelle concentrations (CMC) of nonanoyl-N-methylglucamide and decanoyl-N-methylglucamide were determined to be 25mM and 7mM, respectively, by photometric assay. Then solubilization and reconstitution of the melibiose transport carrier were performed using these detergents at concentrations above the CMC. Melibiose counterflow activity was observed with the proteoliposomes reconstituted from the extracted proteins and phospholipids. The proton-trans-locating ATPase complex (F₁-F₀) was also solubilized with these detergents. These results indicate that nonanoyl- and decanoyl-N-methylglucamide are useful detergents for solubilization and reconstitution of membrane proteins.

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The stage-specific antigen, gp68 (Hirano, T., Yamada, H., & Miyazaki, T. (1983) Biochim. Biophys. Acta 742, 224-234), was purified from a phenol/water extract of aggregation-competent cells of Dictyostelium discoideum by preparative polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS). Anti-gp68 was produced against purified gp68 which was determined to be homogeneous by silver staining on analysis by SDS-polyacrylamide gel electrophoresis. The cross-reactivity of anti-gp68 against cellular antigens was estimated by immunoaffinity chromatography on anti-gp68 immunoglobulin G (IgG)/Sepharose. When the whole cell lysate was applied to this affinity column, three major proteins, with molecular weights of 80, 000, 68, 000, and 56, 000, were obtained in the absorbed fraction. When the butanol extract, which was enriched in contact site A, an adhesion mediating glycoprotein, was applied to the same column, two major proteins with molecular weights of 80, 000 (corresponding to contact site A) and 56, 000 were obtained in the absorbed fraction but, however, gp68 was negligible. Reversely, when the phenol/water extract was applied to anti-contact site A-IgG/Sepharose, only gp68 was obtained in the absorbed fraction. Moreover, contact site A was seen to compete with [³H] mannose-labeled gp68 in a competition radioimmunoassay using anti-gp68 serum. The effect of Pronase or exo-α-mannosidase digestion on the antigenic activity of gp68 was examined by radioimmunoassaying. The results indicated that the α-mannosyl residue of the non-reducing terminal in the carbohy-drate moiety of gp68 was a major immunodeterminant. However, the polypeptide chain did not participate in the antigenic reactivity against anti-gp68. Both anti-gp68 and anti-contact site A agglutinated heat killed-yeast cells. Also, both antisera inhibited EDTA-stable cell adhesion of aggregation-competent cells in the presence of Fab from goat anti-rabbit IgG. These results indicate that gp68 and contact site A have a common antigenic determinant against anti-gp68, and that the target antigen of anti-gp68 was somehow involved in cell adhesion.

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Truncated templates of a cloned actin gene (actin 5) of Dictyostelium discoideum were transcribed in a HeLa cell extract from a position indistinguishable from the in vivo initiation site. The efficiency of this accurate transcription, however, was very low compared with that of the adenovirus 2 major late gene in the same system. Transcription of the actin 6 and 8 genes, of which the 5'-flanking regions differ in their base sequences from the corresponding region of the actin 5 gene, was not good in either fidelity or efficiency. When the partially purified RNA polymerase II of D. discoideum was used instead of the HeLa cell extract, no accurate transcription of the actin 5 gene occurred. However, simultaneous addition of RNA polymerase II and HeLa cell extract into the cell-free system caused a shift of the in vitro initiation point of transcription of the actin 5 gene to a position about 20 nucleotides downstream from the presumed in vivo initiation site. The reason for the shift remains unknown, but it is supposed that some factor (s) other than RNA polymerase II are involved in the accurate initiation of transcription and the maintenance of transcription efficiency of the actin genes.

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A unique form of cytochrome P-450 (called P-450₂) with high affinity for 7-alkoxy-coumarins was purified from liver microsomes of phenobarbital-treated rabbits with an overall yield of about 0.6%. The purified preparation had a specific cytochrome P-450 content of 15-16 nmol per mg of protein and was homogeneous on sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis. The apparent molecular weight estimated for P-450₂ was 50, 500. In the oxidized state its absorption spectrum was characteristic of low-spin cytochrome P-450. The activity of purified P-450₂ to catalyze O-dealkylation of several 7-alkoxycoumarins in the reconstituted system was compared with those of P-450₁, the major phenobarbital-inducible form in rabbit liver microsomes, and rat P-448₂, purified from liver microsomes of β-naphthoflavone-treated rats. With 7-ethoxycoumarin as substrate, the pH optimum for P-450₂ was 6.5, whereas those for P-450₁ and rat P-448₂ were 7.5-8.0. P-4502 showed K_m values of 0.38 to 1.4 μM for the five alkoxycoumarins tested, whereas the corresponding K_m values determined for P-450₁ and rat P-448₂ were 40- to 700-fold higher. The K_m values of P-450₂ and P-450₁ were relatively unaffected by the length of the alkyl chain in the alkoxycoumarins, whereas those of rat P-448₂, decreased markedly as the length of the alkyl chain increased. For all the three forms of cytochrome P-450, the highest V_max values were observed with 7-ethoxycoumarin, and V_max values tended to decrease with increase in the alkyl chain length in the substrate. Only P-450₂ catalyzed 7-hydroxylation of coumarin, though at a low rate. Progressive and irreversible inactivation of P-450₂, but not P-450₁ and rat P-448₂, was observed during the catalysis of 7-methoxycoumarin O-demethylation, and this inactivation was considerably prevented by inclusion of cytochrome b₅ in the reconstituted system.

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The activities of template-engaged and “free” RNA polymerase I in isolated prostatic nuclei from rats treated with testosterone or cycloheximide were measured by the previous method with modifications, and the following results were obtained. (1) Castration resulted in a gradual decrease in “engaged” RNA polymerase I activity, finally to a 40%. level, while the “free” enzyme activity showed a transient increase at 12 h after castration. Administration of testosterone to castrated rats scarcely affected the “free” enzyme activity while the “engaged” enzyme activity increased with time up to 24 h, when it reached a value of about 2.6 fold the castrated control value. On the other hand, testosterone administration did not alter the total activity or content of RNA polymerase I solubilized from nuclei until 12 h after the hormone injection although a 2 to 3 fold increase was observed at 24 h after the hormone injection. (2) Cycloheximide caused a rapid decrease in the “engaged” enzyme activity in the case of normal rats, but did not affect it in the case of castrated rats. The activity of “free” enzyme in normal prostates was found to show a transient increase after cycloheximide treatment, in contrast to a gradual increase of the activity in the nuclei from castrated rat prostates. (3) Incubation of prostatic slices in the presence and absence of α-amanitin showed that “engaged” RNA polymerase I activity in the isolated nuclei was diminished in the case of normal rats but not in the case of castrated rats. The results obtained here, together with observations previously reported, suggest that there are short-lived protein(s) which are completely androgen-dependent and play a role in the activation of “dormant” RNA polymerase I but not in the transformation of the “free” form to the “engaged” form of the enzyme.

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1. DNase I in human urine was purified to an electrophoretically homogeneous state by column chromatographies on DEAE-lignocellulose, hydroxyapatite, DEAE-cellulose, Sephadex G-75 and elastin-celite.
2. The purified enzyme was immunologically identical with human pancreatic DNase I, but not with bovine pancreatic DNase I.
3. The molecular weight and isoelectric point of the enzyme were estimated to be 4.1×10⁴ and 3.6, respectively. The amino acid analysis revealed that I mol of the enzyme contained 8 mol of half-cystine. The N-terminal amino acid was identified as leucine by the dansyl chloride method.
4. The enzyme was active in the presence of Mg²⁺, Co²⁺, or Mn²⁺. The optimum pH was around 6.5. The enzyme was stable in the pH range from 5.0 to 9.0 and at temperatures lower than 45°C. The rate of hydrolysis of native DNA by the enzyme was twice as fast as that observed with heat-denatured DNA. This enzyme exhaustively degraded about 20% of the phosphodiester bonds in native DNA. The enzyme also degraded poly (dA) and poly (dT), but hardly degraded poly (dG) and poly (dC).

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The catalytic actions of the native type and the type modified by a protease attack of P. notatum phospholipase B were studied. The native and modified enzymes showed a significant difference in pH dependence of their catalytic activities. In the native enzyme, the optimum pH of phospholipase B activity (which catalyzes the complete deacylation of diacylglycerophospholipids) was pH 5.5 toward egg yolk phosphatidylcholine and its lysophospholipase activity (which catalyzes the deacylation of monoacylglycerophospholipids) showed a wide optimum pH range (pH 3.8-6.5). On the other hand, phospholipase B activity of the modified enzyme toward egg yolk phosphatidylcholine was strongly depressed between pH 3.0 and 7.0. The reduction in phospholipase B activity was due to the reduction of hydrolyzing activities for fatty acyl ester bonds at both positions 1 and 2 of diacylglycerophospholipids. However, toward 1, 2-dioctanoyl-sn-glycero-3-phosphocholine and egg yolk lysophosphatidylcholine, the catalytic activities of the modified enzyme were almost the same as those of the native enzyme below pH 4.0, although above pH 4.0 they were significantly lower.
The binding activity to micellar substrates was investigated by following the fluorescence spectral changes of the enzymes on adding the enzymatically nondegradable substrate, 1-O-alkyl-sn-glycero-3-phosphocholine. At the optimum pH of the native enzyme (pH 5.0), the binding constant of the modified enzyme (0.46×10⁵ M^-1) was almost one order of magnitude lower than that of the native one (3.8×10⁵ M^-1), showing that the affinity to the substrate of the modified enzyme was decreased.
In addition, at the same pH, 5.0, the negative ellipticities in the far-ultraviolet region of the circular dichroism spectra of the modified enzyme were increased compared with those of the native one. It was suggested that the conformational change in protein structure of phospholipase B resulted in the different pH dependence and low affinity to substrates in lipid-water interfaces.

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The binding of various combinations of chymotryptic troponin T subfragments, troponin I and troponin C to tropomyosin, troponin C and troponin I was examined semiquantitatively by using affinity chromatography. The interaction between troponin T₂ and troponin I intensified the interaction between troponin T₂ (or troponin T) and tropomyosin. When a mixture of troponin T₂ and troponin C was applied to a tropomyosin-Sepharose 4 B column, neither troponin T₂ nor troponin C was retained in the presence of Ca²⁺ ion, while only troponin T₂ was bound in the absence of Ca²⁺ ion. Such a Ca²⁺-dependent effect was not observed with troponin T. Troponin T₂ subfragments, except troponin T₂βIII, were retained by troponin C-Sepharose 4 B in the presence of troponin I, even in the solution containing 1.0M NaCl, in the presence and absence of Ca²⁺ ion. On the basis of these findings, the interactions among troponin components and tropomyosin are discussed.

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Connectin is an elastic protein of vertebrate striated muscle, and consists of doublet components, α and β (also called titins 1 and 2). In the present study, β-connectin isolated in the native state was investigated in order to characterize its molecular size and shape. The molecular weight was approximately 2.1×10⁶ (SDS gel electrophoresis) or 2.7×10⁶ (sedimentation equilibrium). The sedimentation coefficient (s⁰_{20, W}) was 17 S in 0.1 M phosphate buffer, pH 7.0. The intrinsic viscosity measured in an Ostwald-type viscometer was 1.8 dl/g. However, the viscosity was greatly dependent on the velocity gradient, and at a very low velocity gradient of 0.0007 s^-1, a solution of connectin (0.3mg/ml) showed a viscosity value of 17, 000 cp. Flow birefringence measurements suggested a length distribution ranging from 300 to 450 nm. Electron microscopic observations revealed that connectin is a long flexible filament and the peaks of frequency of length distribution were at 150, 300, 450, and 600 nm. It was tentatively assumed that the connectin molecule is 300-400 nm long and 34-38 nm wide. It is likely that β-connectin is derived from α-connectin, which has an apparent molecular weight of 2.8×10⁶.

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Human plasma fibrinogen rapidly incorporated stoichiometric amounts of [³²P]-phosphate when incubated with [³²P] ATP and calcium-activated, phospholipid-dependent protein kinase purified from pig spleen. Half-maximal fibrinogen kinase activity was attained at less than 0.1mM calcium acetate. The optimum concentration of phosphatidylserine was about 50 μg per ml. Diolein slightly potentiated the stimulatory effect of phosphatidylserine. The α-chain of fibrinogen, which is reported to contain endogenous phosphate (Blombäck, B., Blombäck, M., Edman, P., & Hessel, B. (1962) Nature 193, 883-884 and Doolittle, R. F., Watt, K. W. K., Cottrell, B. A., Strong, D. D., & Riley, M. (1979) Nature 280, 464-468) was phosphorylated by the protein kinase. The apparent K_m value for the phosphorylation reaction (0.3-0.6 μM fibrinogen) was comparable with the Km values reported for the hitherto most effective substrate proteins for protein kinase C. Up to 5 mol phosphate per mol fibrinogen could be incorporated, indicating at least three phosphorylatable sites per half molecule.

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The complete amino acid sequence of the B chain derived from Tachypleus tridentatus coagulogen was determined. It consisted of a total of 129 amino acid residues with a NH₂-terminal glycine and COOH-terminal phenylalanine. Sequence studies of the whole B chain and the fragments obtained from the digests with trypsin, α-chymotrypsin, thermolysin and Staphylococcal protease V8 showed the following sequence:
NH₂-_??_-F-S-I-F-S -H-H-P-_??_-F-R-E-C-G-K-Y-E-C-_??_-T-V-R-P-E-H-S-R-C-Y-N-F-P-P-F-T-H-F-K-L-E-C-P-V-S-T-R-D-C-E-P-V-F-G-Y-T-V-A-G-E-F-R-V-I-V-Q-A-P-R-A-G-F-R-Q-C-V-W-Q-H-K-C-R-F-G-S-N-S-C-G-Y-N-G-R-C-T-Q-Q-R-S-V-V-R-L-V-T-Y-N-L-E-K-D-G-F-L-C-E-S-F-R-T-C-C-G-C-P-C-R-S-F-OH
These structural studies of the B chain and the previously established amino acid sequences of the A chain and peptide C derived from T. tridentatus coagulogen, now make it possible to complete the whole sequence of coagulogen consisting of 175 amino acid residues with the molecular weight of 19, 723.

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Amylose was fractionated according to its molecular weight by high performance gel chromatography using columns of a TSK-GEL PW series. Elution from the columns was monitored with a low-angle laser light scattering photometer and a precision differential refractometer. The following results were obtained indicating that the procedure is highly efficient for characterizing an amylose preparation with respect to its molecular weight: 1) the weight-average molecular weight of lily amylose used as a test material was determined to be 786, 000±26, 000 (n=7); 2) the molecular weight distribution curve of the amylose was worked out from the chromatographic data; and 3) based on the concept of the universal calibration curve, the Mark-Houwink-Sakurada equation of the amylose was presumed to be [η]=2.27×10^-4M^0.62 (dl/g). The technique saves time and sample significantly compared with the conventional ones, and consequently enables the characterization of amylose in aqueous solvents without either the degradation or association peculiar to the amylose molecule.

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Rabbit factor H, a control protein of the alternative complement pathway, was isolated from rabbit serum by polyethylene glycol precipitation, DEAE-Sephacel chromatography, and gel chromatography on Sephadex G 200. The protein migrated as a single-chain polypeptide with a molecular weight of 160, 000 on sodium dodecyl sulfate-gel electrophoresis with Laemmli's buffer system, but hardly migrated into the gel with Fairbanks' buffer system. Physical and chemical properties of rabbit H were similar to those of human H, except that fragments produced by limited tryptic digestion from rabbit H had different molecular sizes from those produced from human H.
Significant species-specificity was observed in the functional activity of factor H; activation of the alternative complement pathway was inhibited more efficiently with homologous H than with heterologous H. In contrast, factor H inhibited the hemolysis of homologous erythrocytes less than that of heterologous erythrocytes.

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Mouse cells deficient in the enzyme thymidylate synthase [TS; EC 2. 1. 1. 45] were serially transformed with human DNA to yield primary and secondary transformants which produced human TS [Ayusawa, D., Shimizu, K., Koyama, H., Takeishi, K., & Seno, T. (1983) J. Biol. Chem. 258, 48-53]. Southern blot hybridization of their genomic DNA showed that six secondary transformants examined contained in common a 5.5 kb EcoRI fragment hybridized with a human Alu sequence. From the secondary transformant genomic library constructed with phage λ Charon 4 A, two recombinant phage clones carrying Alu sequences were isolated. Restriction endonuclease mapping revealed that the insert DNAs of the two phage clones overlapped and covered a region of 19 kb in total. Within this region at least six Alu sequences were located. A 2.0 kb DNA fragment, prepared from an EcoRl fragment subcloned in plasmid pBR322 and free of Alu sequences, hybridized to a single band on RNA blots of primary and secondary transformant poly (A)⁺ RNA, but not to RNA of mouse wild-type and recipient cell lines. The relative amount of the presumed human TS mRNA was linearly correlated with the relative activity of human TS in various types of mouse transformant cells. These results indicate that these two phage clones contain genomic DNA sequences encoding human TS.

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The leucine genes of alkalophilic Bacillus No. 221 were cloned into the HindIII sites of pBR322 and pGR71, and recombinant plasmids pHK101 and pHK111 were constructed. The cloned 7.2 kb DNA consisting of HindIII fragments of 4.0 kb and 3.2 kb could substitute for leucine genes (α-isopropylmalate (α-IPM) synthetase gene, β-IPM dehydrogenase gene, and α-IPM isomerase gene) of E. coli and B. subtilis, but not ilvB gene of B. subtilis. The 4.0 kb HindIII fragment could substitute for α-IPM synthetase gene of E. coli, and the 3.2 kb HindIII fragment could substitute for α-IPM isomerase gene of E. coli. Both 4.0 kb and 3.2 kb fragments are necessary to substitute for β-IPM dehydrogenase gene. The expression of β-IPM dehydrogenase gene of alkalophilic Bacillus No. 221 was repressed by the addition of leucine in the culture medium of B. subtilis carrying the plasmid pHK111. These results indicated that the 7.2 kb fragment contains the leucine gene cluster and its regulatory region.

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Three human decidual prolactin (PRL) cDNA clones (pdPL-1:344 base pairs, pdPL-2:584 base pairs, pdPL-3:807 base pairs without poly (A) tract) were prepared and sequenced. The insert DNA of the largest clone pdPL-3 contained the coding region corresponding to 217 amino acid residues including 18 amino acid residues of the signal peptide, and 157 nucleotides of the 3'-untranslated region. A comparison of the pdPL-3 cDNA sequence with that of human pituitary PRL cDNA revealed 4 silent nucleotide differences. Two of the base changes occurred in the third position of the amino acid codons, and the other two occurred in the 3'-untranslated region. Therefore, the amino acid sequence of decidual PRL deduced from the nucleotide sequence of pdPL-3 was identical with that of pituitary PRL.
Minor changes were also observed among the three PRL mRNAs: a silent change in the third codon in the translated region, and another change in the 3'-untranslated region. Southern blot hybridization of human cellular DNA with the pdPL-3 probe indicates that PRL gene may occur once per haploid genome. Therefore, these minor changes may reflect microheterogeneity of PRL gene.
The existence of multiple poly (A) -adjacent sequences was shown in the three species of human decidual PRL mRNA and in human pituitary PRL mRNA. This variation may be due to heterogeneity in the processing at the 3' terminus of mRNA or the termination sites of the transcription.

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Lipids were extracted from the kidney of house musk shrew (Suncus murinus), which belongs to the order Insectivora. Acidic glycosphingolipids were purified from lipid extracts by mild alkaline methanolysis followed by column chromatographies on DEAE-Sephadex and silica beads (Iatrobeads). Purified glycolipids were analyzed by thin-layer chromatography, mild acid hydrolysis, gas liquid chromatography of the methyl glycosides after methanolysis and gas chromatography-mass spectrometry of the partially methylated alditol acetates. The kidney of suncus was unique in that it contained ganglioside GM2 (NeuAc type, 28.7 nmol and NeuGc type, 15.8 nmol/g tissue) as the major ganglioside. GM4 (NeuAc) (2.6 nmol/g), and GM3 (NeuAc type, 11.5 nmol and NeuGc type, 8.7 nmol/g) were also present. The content (298.9 nmol/g) of galactosyl sulfatide (GalCer-I³-sulfate) was higher than the values reported previously for other animal species. The total amount of acidic groups in glycolipids of suncus kidney was compared with the values for the kidney of 4 placental mammals to obtain an allometric correlation:
log Y=0.266+0.780 log X
where X designates body weight, kg and Y, total acidic groups, μmol. A highly significant correlation (r=0.999) was obtained among values from 5 representative placental mammals which live in mesic environments, suggesting that acidic glycosphingolipids are essential for the kidney function.

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The amino acid sequence of ferredoxin-NADP⁺ oxidoreductase [EC 1. 18. 1. 2, FNR] from Spirulina sp., a blue-green alga, was determined. Spirulina ferredoxin-NADP⁺ oxidoreductase was composed of 294 amino acid residues and the molecular weight of the holoenzyme was 34, 135. An apparent homology of the amino(N)-terminal region was found between ferredoxin-NADP⁺ reductases from Spirulina and spinach. We also found some sequence similarities in human erythrocyte glutathione reductase and p-hydroxybenzoate hydroxylase from Pseudomonas fluorescens, both of which are NADPH-dependent FAD enzymes.

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A sensitive enzyme-immunochemical staining method was developed for detection of N-glycolylneuraminic acid (NeuGc)-containing glycosphingolipids (GSLs) on silica gel thin-layer chromatography. The procedure consists of immune reaction among NeuGc-containing GSLs, affinity-purified chicken anti-NeuGc-LacCer and horseradish peroxidase-conjugated rabbit anti-chicken IgG, and the peroxidase reaction using 4-chloro-l-naphthol as a chromogenic substrate. Quantitative determination was achieved by direct densitometric scanning of the enzyme-immunostained spots on the chromatogram. As little as 0.5 pmol of NeuGc-LacCer, NeuGcnLcOse₄Cer, and NeuGc-nLcOse₆Cer (0.64-1.0 ng) could be detected with a good signal-to-noise ratio. A semi-linear detector response was observed up to 50 pmol of each GSL. This procedure can be applied easily to other glycolipid antigen systems.

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Restriction fragments composed of only hop stunt viroid (HSV) cDNA were prepared from recombinant clone pHS-P4P, which carries four tandemly repeated HSV cDNAs, and inoculated into cucumber cotyledons. The results showed that doublestranded cDNAs consisting of 1 to 3 units of HSV sequences were infectious. In cucumber plants inoculated with these double-stranded cDNAs, infectious RNA molecules indistinguishable from authentic HSV were propagated.

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Mild acid treatment of in vitro translated cytochrome P-450 (SCC) (pre-P-450 (SCC)) peptide cleaved the peptide into two fragments. Comparison of the sizes and the NH₂ terminal amino acids of the fragments with those of the corresponding fragments from mature P-450 (SCC) suggested that the prepiece of pre-P-450 (SCC) was present at the NH₂-terminal end of the peptide. This conclusion was confirmed by radio-sequencing of the NH₂-terminal portion of pre-P-450 (SCC).

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The molecular (Mr=120, 000; s_{20, w}=5S) and catalytic properties (K_m (ATP)=3 μM; K_m (nicked DNA)=0.2 μM; K_m (Mg²⁺)=3mM) of DNA ligase from mouse Ehrlich ascites tumor cells are similar to those of the enzymes from calf thymus and rodent liver. The activity level of DNA ligase from the tumor cells is about 10-fold higher than that from mouse liver. Immunochemical titration of DNA ligase with antibodies against the calf thymus enzyme showed that the higher level of DNA ligase activity in the tumor cells is due to an increase in enzyme quantity and not to elevation of the catalytic efficiency of the enzyme molecule. These results suggest that there is little apparent difference between the qualities of DNA ligases from the tumor cells and normal tissues of rodents and calf.

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