Previous studies in our laboratories (Miyata, T., et al. (1982) Proc. Natl. Acad. Sci. U. S. 79, 6132-6136) showed that the structural defect in a hereditarily abnormal plasminogen, plasminogen Tochigi, is due to replacement of Ala by Thr at position 600 from the NH₂-terminal end. In the present studies, two abnormal plasminogens, plasminogens Tochigi II and Nagoya, obtained from other family members were analyzed to identify the structural impairment in these molecules. Amino acid sequence analysis of one of the tryptic peptides isolated, respectively, from plasminogens Tochigi II and Nagoya indicated that in both cases, Ala-600 (equivalent to Ala-55 of the chymotrypsin numbering system) had been replaced by Thr. No other substitutions at the active site and substrate-binding site residues, namely, His-57, Asp-102, Ser-195, and Asp-189, were found in the plasmin light chain variants, indicating that all these residues are intact. Moreover, the NH₂-terminal heptapeptide sequences of the plasmin light chain variants isolated from plasminogens Tochigi II and Nagoya were identical to the sequence determined for the normal control. These results indicate that the absence of proteolytic activity of both abnormal molecules is due to the same amino acid substitution as that of previously reported plasminogen Tochigi.

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To elucidate the mechanism of biliary occurrence of γ-glutamyl transferase [EC 2. 3. 2. 2] and alkaline phosphatase [EC 3. 1. 3. 1], the effect of bile acids on the biliary level of these enzymes was studied in vivo and in vitro. Following intravenous administration of taurocholate, the activities of both enzymes in rat bile increased markedly with a concomitant increase in the excretion of the bile acid. The biliary levels of these enzymes increased to reach a maximum at 10-20min after administration of the bile acid and decreased thereafter.
Right-side-out oriented rat liver canalicular membrane vesicles which localize γ-glutamyltransferase, aminopeptidase M and alkaline phosphatase on their outer surface (Inoue, M., Kinne, R., Tran, T., Biempica, L., & Arias, I. M. (1983) J. Biol. Chem. 258, 5183-5188) were prepared. Upon incubation of the vesicles with either intact or heat-treated bile samples, the membranous enzymes were released from the vesicles in a time-dependent manner. Incubation of these vesicles with physiological concentrations of taurocholate also solubilized these enzymes from the membranes. Affinity chromatographic analysis on concanavalin A-Sepharose revealed that the transferase thus solubilized retained the hydrophobic domain responsible for anchoring the enzyme to membrane/lipid bilayers. These results indicate that bile acid (s) excreted into the bile canalicular lumen solubilized these enzymes from the apical membrane surface of the biliary tract cells by their detergent action.

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When pig liver phosphorylase kinase was assayed at various concentrations of Mg²⁺, about 2-fold stimulation was observed around 2-3mM Mg²⁺ (Mg²⁺/ATP ratio, 20-30) compared with the activity at 0.3mM Mg²⁺ (Mg²⁺/ATP ratio, 3). This stimulation was specific for Mg²⁺ among the divalent cations tested and the process was reversible. K_m values for ATP and phosphorylase b were decreased 3.6- and 9.5-fold, respectively, at 3mM Mg²⁺ compared with those obtained at 0.3mM Mg²⁺ These results indicate that the activity of liver phosphorylase kinase is influenced by free Mg²⁺

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DNA sequences coding for the immunogenic capsid protein VPI and/or VP 3 of poliovirus strain LSc-2 ab (Sabin 1) were prepared by digesting the cloned complementary DNA with restriction endonuclease PstI. The DNA fragments were inserted into the unique Pstl site of Escherichia coli plasmid vectors pBR 322, pKT 280 and/or pKT 287 that lay in the region expressed under control of the penicillinase promoter system. In the expression vectors, poliovirus sequences were designed to be read in phase and therefore to be expressed as fusion proteins with the bacterial peptides. In addition, the Escherichia coli tryptophan operon promoter-operator system was inserted upstream of the penicillinase system to obtain stronger expression of the poliovirus sequences. Escherichia coli transformed with these plasmids appeared to produce the antigenic polypeptides, which were detected by immunoprecipitation with antibodies to capsid proteins VPl and/or VP 3 followed by SDS-polyacrylamide gel electrophoresis.

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A crystalline acylphosphatase has been obtained from porcine skeletal muscle. The purification procedure consists of extraction with water, phosphocellulose column chromatography, CM-cellulose column chromatography, and crystallization. The enzyme was homogeneous by polyacrylamide gel electrophoresis. A high yield (39%) of the pure enzyme was attained by the use of buffers containing 10mM 2-mercaptoethanol to prevent dimerization of the enzyme in the purification process. Activity assay in the presence of bovine serum albumin showed a high specific activity of the enzyme (about 7, 000 μmol/min/mg at 25°C with benzoyl phosphate as substrate). The molecular weight was determined to be 11, 100 by sedimentation equilibrium. The amino acid composition of the enzyme was determined. The amino-terminus of the enzyme was blocked and the carboxyl-terminal residue was tyrosine.

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Fluorescence titration and fluorescence stopped-flow studies were performed on carp muscle parvalbumin components 1, 2, 3, and 5 (the latter three components were modified with a SH-directed fluorescent reagent, dansyl-L-cysteine). Apparent binding constants (Kapp) of Ca²⁺ to these components decrease in the order of component 2 (K_app=2.8±0.9×10⁸ M^-1)>component 1 (K_app=1.25±0.25×10⁸ M^-1)>component 3=component 5 (K_app=4.0±0.5×10⁷ M^-1) in 30mM KCl, 50mM Na-cacodylate-HCl, pH 7.0 at 20°C. The rate constant of the conformational change of parvalbumin induced by Ca²⁺ binding or removal decreases in the order of component 2>component 1>component 5^>₌component 3; that is, component 2 undergoes the fastest conformational change and component 3 the slowest in response to the rapid free Ca²⁺ concentration ([Ca²⁺]) change in the protein solution. The fluorescence titration curves and [Ca²⁺]-dependences of the rate constants are analyzed by a simple two-state model, (partially unfolded state)κ₁_??_κ₂ (folded state). It is shown that the equilibrium constant K=κ₁/κ₂ depends on the second power of [Ca²⁺], the rate constant κ₁ on the first power of [Ca²⁺] and κ₂ on the inverse first power of [Ca²⁺], respectively

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To investigate the role of carboxyl groups of glucoamylases [EC 3. 2. 1. 3] from a Rhizopus sp. (Gluc₁ and Gluc₂), the modification of Gluc₁ and Gluc₂, with a water-soluble carbodiimide, 1-cyclohexyl-3-(2-morpholinyl-(4)-ethyl) carbodiimide metho-p-toluenesul-fonate (CMC), was studied. The inactivation of Gluc₁ proceeded with the incorporation of about 3 CMC moieties. In the presence of maltose, the modification of about 2.2 carboxyl groups of Gluc₁, proceeded with a slight loss of enzymatic activity. In the re-modification of Gluc₁ modified in the presence of maltose, Gluc₁ was inactivated by further modification of about 1.3 carboxyl groups. Therefore, one carboxyl group, which was protected by maltose, was thought to be a crucial one. The inactivation of Gluc₂ proceeded similarly to that of Gluc₁, but the number of CMC moieties incorporated was about one less than in the case of Gluc₁. Thus, it was suggested that one of the reactive carboxyl groups of Gluc₁, was located in the N-terminal part of Gluc₁, which is deficient in Gluc₂. From the results of kinetic studies on CMC-modified Gluc₁, it was suggested that the hydrolysis mechanism of malto-oligomers differs somewhat from that of PNPG.

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F-Actin and myosin subfragment-1 (S-1) were covalently crosslinked in the absence and presence of nucleotides, adenyl-5'-yl imidodiphosphate (AMPPNP), ATP, and ADP, at various KCl concentrations (0-0.5M), using a zero-length crosslinker, 1-ethyl-3-[3-(dimethylamino) propyl] carbodiimide (EDC), The rate of production of the covalent acto-S-1 complex was almost proportional to the amount of the acto-S-1 (-nucleotide) complex existing in the reaction medium. However, the Mg-ATPase activity of the covalent acto-S-1 complex thus produced was affected by the presence of nucleotides. When S-1 was crosslinked to F-actin in the absence of nucleotide at 0-0.5M KCl, the Mg-ATPase activity of S-1 was enhanced from 0.1-0.3 to 12-15 s^-1. The Mg-ATPase activity of covalent complex produced in the presence of AMPPNP at 0M KCl was 13 s^-1 which was equal to that produced in the absence of nucleotide. The activity decreased with increasing KCl concentration of the crosslinking medium, reaching 6 s^-1 at 0.5M KCl. Covalent acto-S-1 complex was also produced when acto-S-1-ADP-P was formed during ATP hydrolysis. The Mg-ATPase activity of the covalent acto-S-1 complex crosslinked during ATP hydrolysis at 0 M KCl was about 5 s^-1 which was about half of that obtained in the absence of nucleotide, and it decreased to about 2 s^-1 at 0.15 M KCl of crosslinking medium. In contrast, the acto-S-1-ADP complex gave a covalent complex which was indistinguishable in its extent and Mg-ATPase activity from that obtained in the absence of nucleotide. These results suggest that the structures of acto-S-1-AMPPNP and acto-S-1-ADP-P complexes are different from those of acto-S-1 and acto-S-1-ADP complexes.

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Active site Ser-195-fluorine-labeled derivatives of α-chymotrypsin were prepared from a series of N-(trifluoromethylphenyl)-fluorosulfonylphenyl carboxamides whose synthesis is described. The six new ¹⁹F spin labels varied in the position of the -CF₃ substituent (o-, m-, and p-) and the fluorosulfonyl substituent (m- or p-). The chemical shifts of these covalently bound analogs of “tosyl-chymotrypsin” were each uniquely sensitive to their environment in the catalytic center as evidenced by differences in resonance line position for each label. Upon titrating these derivatives with the reversible competitive inhibitor, indole, a downfield shift was observed (with all but one label), which could be fit in each case to an apparent dissociation constant for indole binding. Indole binding to the p-sulfonyl derivatives was essentially unaltered from that for the native enzyme, while the m-sulfonyl derivatives required some additional free energy of binding to saturate the enzyme. The results are consistent with a partial embedding of the phenylsulfonyl moiety in the aromatic specificity pocket.

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A rearranged human γ₁, immunoglobulin gene was introduced into fertilized mouse eggs. The phage Ch 4 A-VCE-γ₁, was constructed by ligating an EcoRI and BglII fragment of pBR 322-CESSV (CE-1) containing the VDJ region with an EcoRI and BamHI fragment of Ch 4 A-HIgγ₁-10 containing the γ₁, constant region. About 200 copies of Ch 4 A-VCE-γ₁, genes were introduced into fertilized mouse eggs. Of 489 eggs injected with these genes, 319 survived and were transferred to oviducts of foster mothers. Thirtyeight mice were born and were screened for the presence of human γ₁, immunoglobulin genes by Southern blot hybridization. Five of these 38 mice had integrated human γ₁, immunoglobulin genes. None of the human γ₁, copies in each mouse had undergone deletions or rearrangements as judged by the Southern blotting patterns for several restriction enzymes. Human γ₁, gene was present in several different tissues. All the mice tested so far transmit the human γ₁, gene to a fraction of their offspring in an autosomal dominant manner.
Spleen cells from transgenic mice were analyzed for immunoglobulin production by reverse plaque assay or immunofluorescence staining of cytoplasmic immunoglobulin, but synthesis and secretion of human γ₁, chains could not be detected. No human γ₁, immunoglobulin mRNA was detected in the liver and spleen of a transgenic mouse. The presence of the human γ₁, immunoglobulin gene appeared to have no effect on the expression of endogenous mouse immunoglobulin genes.

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DNA polymerase β's from mouse myeloma, chick embryo, and cherry salmon testis were all composed of a single polypeptide of about 40K daltons as judged by a sodium dodecyl sulfate-polyacrylamide gel electrophoresis of extensively purified enzyme preparations. Although the enzyme from bullfrog ovary was not fully purified, its molecular weight was estimated to be the same as that of the chick enzyme by immunological detection after electrophoresis. All the enzymes tested cross-reacted immunologically with the antibody against chick DNA polymerase β, indicating that they have a common molecular structure, at least in part.
Two-dimensional maps of radioiodinated tryptic peptides directly showed the presence of highly conserved amino acid sequences among mouse, chick, and cherry salmon enzymes. This conserved structure is thought to be essential for the enzyme activity, which is very similar among all these vertebrates.

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Spinach ferredoxin was found to lose its iron-sulfur center during trinitrophenylation. The loss of the iron-sulfur center resulted in disappearance of its activities as electron mediators in the NADP⁺ photoreduction and NADPH-cytochrome c reduction systems, whereas it did not affect the activity in the cytochrome c photoreduction system. Based on the experimental evidence that ovalbumin, originally inactive in electron transport, became active in the cytochrome c photoreduction system after trinitrophenylation, the retained activity of the modified ferredoxin was concluded to be due not to the original function of the iron-sulfur center, but to artificial electron transport mediated by TNP groups introduced into the protein molecule. Trinitrophenylated cytochrome c was also active as a mediator, but lost its ability as a terminal acceptor in the system.

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The binding of Ricinus communis lectins to HeLa cells, Sarcoma 180 ascites tumor cells and human erythrocytes was studied in detail. Scatchard plots of binding of ¹²⁵I-lectins to these cells gave biphasic lines except for HeLa cells at 0°C. The association constants of lectins for the three cell types at 37°C were lower than those at 0°C. The numbers of total binding sites were estimated to be 7 to 16×10⁷ per HeLa cell, 3 to 4×10⁷ per Sarcoma 180 ascites tumor cell and 0.4 to 1×10⁶ per erythrocyte. A fraction, 16 to 27% of the total amount of cell-bound lectin at 37°C, appeared to be bound irreversibly as judged by non-removal on washing with 0.1M lactose, whereas no lectin was irreversibly bound at 0°C. In the case of erythrocytes, no lectin became irreversibly bound even at 37°C.
The toxicity of lectins on HeLa cells and Sarcoma 180 ascites tumor cells was investigated. The toxicity of ricin D was 50 times for Sarcoma 180 ascites tumor cells and 140 times for HeLa cells as much as that for castor bean hemagglutinin. As to the sensitivities of both cell types to these lectins, it became apparent that Sarcoma 180 ascites tumor cells were more susceptible than HeLa cells.

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Chemically modified spinach plastocyanin, in which negatively charged carboxyl residues are replaced with positively charged amino residues, has been prepared. Four distinct species of chemically modified plastocyanin, having I to 4 mol of modified carboxyl residue per mot of plastocyanin, could be separated by ionexchange chromatography on DEAE-Sephacel. The rate of electron transfer from reduced cytochrome ƒ to oxidized singly substituted plastocyanin was only 30% of that of the native unmodified plastocyanin, and the reaction rate decreased further with increasing number of modified carboxyl residues. These results indicate the importance of electrostatic interactions between the negative charges on plastocyanin and the positive charges on cytochrome ƒ in this reaction. Since the overall net charge of cytochrome ƒ is negative at neutral pH, the positive charges on cytochrome ƒ involved in the reaction should be localized ones. On the other hand, the rates of electron transfer from reduced singly and doubly substituted plastocyanin to photooxidized P 700 in the P 700-chlorophyll α protein complex were similar to that of native plastocyanin, which suggests that these carboxyl residues have only a minor role in the electron transfer to P 700. Although divalent cation is essential for the electron transfer from native plastocyanin to P 700 at neutral pH, the triply substituted plastocyanin could donate electrons to P 700 even without MgCl₂, and the rate of this reaction reached the maximum at a low concentration of MgCl₂ (<2.5mM). The modification of four carboxyl residues per plastocyanin molecule activated this reaction to the maximum level without MgCl₂. These results suggest the presence of different reaction site domains, at least partially, on the plastocyanin for the electron transfer to P 700 and for the electron transfer from cytochrome <. The results are discussed in relation to the electron input to and output from plastocyanin in vivo.

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Root pulps from bovine unerupted wisdom teeth produce a potent collagenase inhibitor together with latent collagenase when cultured in Eagle's minimal essential medium (Biochem. Int. 5, 763, 1982). The inhibitor was purified more than 700-fold from the explant medium using Con A-Sepharose, Ultrogel AcA 44 and DE-52 cellulose columns. It showed a single band (MW=36, 000) on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, but showed multiple bands on basic (pH 8.3) polyacrylamide gel electrophoresis and electrofocusing. The inhibitor is a sialo-glycoprotein containing approx. 20% carbohydrate by weight and its composition suggests that it contains complex-type oligosaccharides. The electrophoretic heterogeneity of the inhibitor was proved to be due to the attachment of different numbers of sialic acid residues. All the SH groups were demonstrated to exist as six disulfide linkages which might be involved in the inhibitory activity. The bovine pulp inhibitor does not combine with collagen. The addition of the inhibitor to activated collagenase resulted in dose-dependent inhibition of the enzyme activity, but the interaction between the inhibitor and activated collagenase is not tight enough for the complex to remain intact during gel filtration column chromatography. A rabbit antiserum was prepared against the inhibitor, and immunoglobulin purified from the antiserum can completely abolish the inhibitory activity of the inhibitor.

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A bone Gla-containing protein (osteocalcin) of cat has been isolated and the complete primary structure has been determined to be YLAPGLGAOAPYPDPLXPKRXICXLNPDCDELADHIGFQDAYRRFYGTV. The protein consists of 49 amino acid residues (Mr. 5, 641) containing three Gla residues and a single disulfide bond. Although the C-terminal 30 residues (20th to 49th) of the sequences of cow, monkey, and human osteocalcins are identical, three amino acid substitutions occur at positions 22, 40, and 48 in the case of cat. These substitutions can be explained by single point mutations.

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Chromoprotein of a light-driven chloride pump, halorhodopsin (HR), was isolated from Halobacterium halobium L-33, which contains HR and “slowly cycling rhodopsin-like pigment” (SR) but lacks bacteriorhodopsin (BR). The isolation was run in the presence of more than 2M NaCl, which was required to preserve this halophilic retinal protein. Cell envelope vesicles were washed with Tween-20 to remove 80% of the proteins. The residual membranes were solubilized with 0.5% C₁₂E₉, which had little effect on the photochemical activities of HR and SR. HR was purified by passing it through a hydroxyapatite and then a phenyl-Sepharose column in 2M NaCl and 0.5% C₁₂E₉. The absorption maximum of HR was 578 nm and the ratio of absorbance at 280 nm to 580 nm was 1.52. The apparent molecular weight of HR was 20, 000 on polyacrylamide gel electrophoresis in the presence of SDS. The characteristic, bilobed CD spectrum of HR in the visible region suggested that HR exists as an oligomer in both its membrane-bound and isolated forms.

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A number of L-amino acids and derivatives were tested as substrates for the purified Pseudomonas L-phenylalanine oxidase. The reaction products of these amino acids were analyzed by high performance liquid chromatography and the kinetic properties of the reactions were partially characterized.
In addition to L-phenylalanine, L-tyrosine, DL-o-tyrosine, DL-m-tyrosine, p-fluoro-DL-phenylalanine and β-2-thienyl-DL-alanine served as substrates for both oxidation and oxygenation catalyzed by the enzyme. On the other hand, L-methionine and L-norleucine were enzymically converted to the corresponding α-keto acids with the consumption of oxygen and with the formation of ammonia and hydrogen peroxide in stoichiometric amounts. Kinetic studies showed that the K_m values for oxidation and oxygenation of L-phenylalanine by the enzyme were 2.04mM and 1.96mM for oxygen, and 13.3 μM and 11.1 μM for L-phenylalanine, respectively. ω-Phenyl fatty acids such as phenylacetic acid, 3-phenylpropionic acid and 4-phenylbutyric acid were competitive inhibitors of the enzyme towards L-phenylalanine. Both oxidation and oxygenation of L-phenylalanine by the enzyme were also inhibited by phenylacetic acid competitively.

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In a primary culture of rat hepatocytes, [5, 6, 8, 11, 12, 14, 15-³H] prostaglandin E₂ showed maximal incorporation of radioactivity (5 to 8% of total added) into the whole cells at 15 to 30min (37°C) and the incorporation then decreased gradually. Treatment of the cells at acidic pH (at 2.5 for 10min, 4°C) followed by thin layer chromatographic analysis revealed that most of the radioactivity incorporated was associated with the cell surface and consisted of intact prostaglandin E₂ and its metabolite (s). In the culture medium after 20min incubation, the concentration of prostaglandin E₂ had decreased by approximately 50% and its metabolite (s) appeared. However, incubation of [1-¹⁴C] prostaglandin E₂ under the same culture conditions led to intracellular incorporation of radioactivity; more than 30% of radioactivity accumulated during a 2 to 3 h incubation period, but it was not in the form of either intact prostaglandin E₂ or the metabolite corresponding to that of [5, 6, 8, 11, 12, 14, 15-³H] prostaglandin E₂.
These results indicate that prostaglandin E₂ was associated with the surface of cultured hepatocytes and degraded into at least two metabolites. The fragment containing ¹⁴C-labeled terminal COOH was incorporated into the intracellular fraction and the residual fragment consisting mostly of the ³H-labeled portion was released from the cell surface into culture medium.

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The mode of acetylcholinesterase release from mammalian erythrocyte membranes by the action of phosphatidylinositol (PI)-specific phospholipase C of Bacillus thuringiensis was studied. As regards intact erythrocytes, a larger amount of acetylcholinesterase was released from sheep or bovine erythrocytes than from horse erythrocytes. From horse erythrocyte ghosts, acetylcholinesterase was more easily released than from intact cells.
Bovine erythrocyte acetylcholinesterase released by PI-specific phospholipase C was purified by column chromatography on DEAE-cellulose, affinity gel and Sepharose 6 B, to a homogeneous state, as indicated by polyacrylamide gel electrophoresis, with a recovery of 39%. Also, bovine erythrocyte acetylcholinesterase was solubilized by Triton X-100 and partially purified. The properties of these acetylcholinesterase preparations obtained by the action of PI-specific phospholipase C and/or Triton X-100 were studied in detail. On elution from the Sepharose 6 B column, Triton X-100-solubilized acetylcholinesterase was eluted at the void volume while the enzyme obtained by further treatment with PI-specific phospholipase C was eluted in the region corresponding to M. W. 250, 000. Furthermore, the heat stability of acetylcholinesterase purified after solubilization with PI-specific phospholipase C was higher than that of the Triton X-100-solubilized acetylcholinesterase. The close association and direct interaction of PI with acetylcholinesterase in the erythrocyte membrane was suggested by the above results.

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A copper-containing dissimilatory nitrite reductase [nitric-oxide: ferricytochrome c oxidoreductase, EC 1. 7. 2. 1] was purified from a denitrifier, Alcaligenes sp. NCIB 11015, by ion-exchange chromatography on CM-cellulose, gel filtration on Sephadex G-150, and adsorption on hydroxyapatite. The preparation was homogeneous by SDS-polyacrylamide gel electrophoretic criteria, and its enzymatic activity increased considerably by freezing (at -20°C) and thawing. The enzyme consists of two subunits with a molecular weight of 37, 000, and the isoelectric point and redox potential are 8.4 and +260 mV (pH 7.2), respectively. The EPR spectrum and copper analysis clearly indicated that the enzyme contains two type I copper atoms per molecule but no other types of copper. This is the first blue copper protein that exhibits catalytic activity despite possessing only type I copper.

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The prothrombin-converting activity of Factor Xa was enhanced by thrombin-stimulated Factor V-deficient platelets and supplementary extraneous Factor Va, and also by thrombin-stimulated normal human platelets. Both extraneous Factor Va and intra-platelet Factor Va were equally inactivated by a γ-carboxyglutamic acid-containing plasma protease, activated protein C. However, a relatively larger amount of activated protein C was required for efficient inactivation of platelet-associated Factor Va as compared with the amount of activated protein C needed for inactivation of phospholipid vesicle-associated Factor Va. Protein S, another γ-carboxyglutamic acid-containing plasma protein, increased the rate of the inactivation of platelet-associated Factor Va about 25-fold. This stimulating effect was observed only slightly with the thrombin-modified protein S. Thus, it was concluded that protein S is essential for the process of inactivation of platelet-associated Factor Va by activated protein C.

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Previously, we reported two DNA polymerases (DPols), Pol I and Pol II, in the unicellular green alga, Chlorella (Aoshima, J., Nishimura, T., & Iwamura, T. (1982) Cell Struc. Funct. 7, 71-86). Changes in their activities during the cell cycle either in the normal and drug-inhibited courses indicated that Pol I and Pol II functioned to replicate nuclear and chloroplast DNAs, respectively. In the present work, we have examined their enzymic properties to characterize and distinguish them further. A number of inhibitors commonly used for such studies were also tested to determine their effects and the results were analyzed by use of the simple and useful “Tamiya plot.” We have also analyzed the data obtained in inhibitor studies on various eukaryotic DPols in the literature using the Tamiya plot, and the results will be presented elsewhere (Iwamura, T. & Aoshima, J. (1984) J. Biochem. in press). Comparisons of the algal DPols with mammalian enzymes as regards enzymic properties and inhibition modes have led us to conclude that: [1] the algal two DPols are significantly different from each other, despite having many similarities to each other; [2] they are related in properties to any of the three mammalian DPols-α, -β, and -γ; Pol I (n-DPol) was a little more like α than Pol II (ch-DPoI), which in turn more akin to γ. This feature was quantitized by using vectors in a three-dimensional α-β-γ-space. Another peculiar feature derived from the Tamiya plot of the inhibitions by araCTP and aphidicolin (both being competitive with cCTP) has led us to propose a specific allosteric role of cCTP in the reaction mechanism besides its role as one of the substrates.

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The binding of ADP to mitochondrial soluble F₁-ATPase was studied by a membrane filtration method. One mol of F₁, which contained about 1 mol of tightly bound adenine nucleotide, bound 4 mol of [α-³²P] ADP at a saturating concentration of [α-³²P] ADP. Two mol of the bound [α-³²P] ADP was readily exchanged with medium nonradioactive ADP while the remaining 2 mol of the bound [α-³²P] ADP was hardly exchanged (tightly bound [α-³²P] ADP). F₁ catalyzed the synthesis of [α-³²P] ATP from the medium [α-³²P] ADP and P₁. However, when exchangeably bound [α-³²P] ADP was replaced by nonradioactive ADP, no [α-³²P] ATP formation was observed. Furthermore, tightly bound [α-³²P] ADP was not released from F₁ during ATP hydrolysis catalyzed by the enzyme. These results indicate that both ATP synthesis and hydrolysis catalyzed by F₁ occur at the exchangeable binding site and not at the tight binding site on the enzyme.

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F₁-ATPase isolated from bovine heart mitochondria catalyzes the synthesis of enzyme-bound ATP from externally added ADP and P₁ in the presence of dimethylsulfoxide (DMSO) (Sakamoto, J. & Tonomura, Y. (1983) J. Biochem. 93, 1601-1614). When the concentration of DMSO in the reaction medium was decreased from 40% to 10% (w/v), the maximal amount of ATP formed decreased from 0.50 to 0.14 mol/mol F₁ and the P₁ concentration required for the half-maximal amount of ATP formed increased from 0.7 to 11mM. On the other hand, the ADP concentration required for the half-maximal value and the rate of ATP formation were unaffected by the decrease in the DMSO concentration. These results suggest that DMSO increases the affinity of F₁ for P₁ and shifts the equilibrium from the enzyme-ADP-P₁ complex to the enzyme-ATP complex during the ATP synthesis.

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A low-molecular-weight acidic protein was isolated from human whole saliva by DE 32 column chromatography and designated as SAP-1. The amino acid sequence was determined by conventional methods to be _??_PGGIYDADLNDEWVQRA_??_HFAISEYNKATEDEYYRRP_??_QVLRAREQTFGGVNYFFDV_??_VGRTICTKSQPNLDTCAFH_??_QPELQKKQLCSFEIYEVPW_??_DRMSLVDSRCQE_??_. The protein consisted of 113 residues and the calculated molecular weight was 12, 552. Computer analysis revealed the presence of 54% sequence homology between SAP-1 and γ-trace, a basic microprotein present in cerebrospinal fluid and in urine of patients with renal failure.

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The interaction of the muscle elastic protein connectin with myosin and actin filaments was investigated by turbidimetry, viscosity, flow birefringence measurements, and electron microscopic observations. In KCl concentrations lower than 0.15M at pH 7.0 at 25°C, both myosin and actin filaments were aggregated by connectin. Myosin filaments were entangled with each other in the presence of connectin. Actin filaments were assembled into bundles under the influence of connectin just as under that of α-actinin. The physiological significance of the interactions of connectin with myosin and actin filaments is discussed in relation to the localization of connectin in myofibrils.
The Mg²⁺-activated ATPase activity of actomyosin was appreciably enhanced by connectin in the presence of KCl concentrations lower than 0.1M. The extent of activation by connectin was smaller than by α-actinin. The enhancement of the ATPase activity may be due to acceleration of the onset of superprecipitation of actomyosin.

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N-Acetylneuraminate lyase [N-acetylneuraminic acid aldolase EC 4. 1. 3. 3] from Escherichia coli was purified by protamine sulfate treatment, fractionation with ammonium sulfate, column chromatography on DEAE-Sephacel, gel filtration on Ultrogel AcA 44, and preparative polyacrylamide gel electrophoresis. The purified enzyme preparation was homogeneous on analytical polyacrylamide gel electrophoresis, and was free from contaminating enzymes including NADH oxidase and NADH dehydrogenase.
The enzyme catalyzed the cleavage of N-acetylneuraminic acid to N-acetyl-mannosamine and pyruvate in a reversible reaction. Both cleavage and synthesis of N-acetylneuraminic acid had the same pH optimum around 7.7. The enzyme was stable between pH 6.0 to 9.0, and was thermostable up to 60°C. The thermal stability increased up to 75°C in the presence of pyruvate.
No metal ion was required for the enzyme activity, but heavy metal ions such as Ag⁺ and Hg²⁺ were potent inhibitors. Oxidizing agents such as N-bromosuc-cinimide, iodine, and hydrogen peroxide, and SH-inhibitors such as p-chloromer-curibenzoic acid and mercuric chloride were also potent inhibitors.
The Km values for N-acetylneuraminic acid and N-glycolylneuraminic acid were 3.6mM and 4.3mM, respectively. Pyruvate inhibited the cleavage reaction competitively; K₁ was calculated to be 1.0mM. In the condensation reaction, N-acetyiglucosamine, N-acetylgalactosamine, glucosamine, and galactosamine could not replace N-acetylmannosamine as substrate, and phosphoenolpyruvate, lactate, β-hydroxypyruvate, and other pyruvate derivatives could not replace pyruvate as substrate.
The molecular weight of the native enzyme was estimated to be 98, 000 by gel filtration methods. After denaturation in sodium dodecyl sulfate or in 6M guanidine-HCl, the molecular weight was reduced to 33, 000, indicating the existence of 3 identical subunits.
The enzyme could be used for the enzymatic determination of sialic acid; reaction conditions were devised for determining the bound form of sialic acid by coupling neuraminidase from Arthrobacter ureafaciens, lactate dehydrogenase, and NADH.

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A new tuberculin-active substance, designated TAS-1 D 3, has been purified from the extract of Mycobacterium bovis BCG by precipitation at pH 4.2, ethanol fractionation, and column chromatography involving CM-cellulose, QAE-Sephadex A-25, Sephadex G-100, and Sephadex G-75. TAS-1 D 3 was homogeneous in polyacrylamide gel electrophoresis and positive in both Coomassie brilliant blue and periodic acid-Shiff staining, suggesting that TAS-1 D 3 is a glycoprotein. The molecular weight of TAS-ID3 was estimated to be 26, 000 by gel filtration. In amino acid analysis, TAS-1 D 3 was distinctive in having proline as a dominant amino acid, and in that it lacked basic amino acids, sulfur-containing amino acids and aromatic amino acids. Moreover, TAS-1 D 3 was almost devoid of absorption at around 280 nm. In guinea pigs sensitized with BCG vaccine, the tuberculin activity of TAS-1 D 3 was about forty times more potent than that of purified protein derivative (PPD).

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Proton nuclear magnetic resonance studies of various mammalian metal lothioneins are described. Metallothioneins-1 and 2, isolated from rat and rabbit, and also metallothionein-2, from humans, were investigated. Proton NMR spectra showed that mammalian metallothioneins containing Cd have similar tertiary structures regardless of their sources. On the other hand, metallothioneins containing both Cd and Zn have a slightly different conformation from those containing Cd alone. Almost all methyl resonances of Cd-metallothioneins were assigned to those of Met, Ile, Leu, Val, Thr, and Ala residues by NMR double resonance techniques, pH titration, and the spectral comparison of these metallothioneins. α-Proton resonances of Ala residues were classified into two groups, one of which arose around 4.1 ppm, the other around 4.4 ppm. Moreover, the number of α-proton resonances around 4.1 ppm and 4.4 ppm agreed with the number of Ala residues in the COOH-terminal and NH₂-terminal domains of the metallothionein, respectively. This finding suggests that the chemical shift values of a-protons of Ala residues are reflective of the domain structure of the metallothioneins.

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Aspartase [L-aspartate ammonia-lyase, EC 4. 3. 1. 1] of Pseudomonas fluorescens was highly purified to homogeneity and crystallized. The purified enzyme sedimented as a monodisperse entity upon ultracentrifugation with a s⁰_{20, w} value of 8.6 S. Upon polyacrylamide gel electrophoresis (PAGE), the enzyme migrated as a single band. The molecular weight of the native enzyme was 173, 000±3, 000, as determined by sedimentation equilibrium analysis, and that of the enzyme subunit was determined to be 50, 000±1, 500 by sodium dodecyl sulfate (SDS)-PAGE. Cross-linking experiments using dimethyl suberimidate followed by SDS-PAGE indicated that the native enzyme was composed of four subunits with identical molecular weight. The amino acid composition of the enzyme was determined.

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The covalently bound flavoproteins in rat liver mitochondria were prelabeled by injecting [¹⁴C] riboflavin into a rat, then liver mitochondria were obtained and further labeled with [³H] pargyline, a suicide inhibitor of monoamine oxidase. When the mitochondria were subjected to osmotic lysis, two covalently bound flavoproteins having molecular weights of 110, 000 and 94, 000 were found in the supernatant. These proteins were identified as sarcosine dehydrogenases. Upon treatment of the membranous fraction with 1% Triton X-100, succinate dehydrogenase with a molecular weight of 70, 000 was found in the soluble fraction, while two well-separated proteins doubly-labeled with ¹⁴C and ³H were found in the insoluble fraction. Their molecular weights were 61, 000 and 57, 000. By isoelectric focusing, two ³H peaks were observed with pI values of 8.3 and 8.4. The former corresponded to the 61, 000-dalton protein, and the latter, to the 57, 000 one. From the data obtained by using selective inhibitors, deprenyl and clorgyline, the [³H] pargylinebinding proteins with molecular weights of 61, 000 and 57, 000 were assinged to proteins of monoamine oxidases of type A and type B, respectively.

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The degradation of protoheme in the heme oxygenase reaction involves three oxidation steps: from protoheme to hydroxyheme, from hydroxyheme to a 688-nm substance, a protein-bound intermediate, and from the 688-nm substance to a biliverdin-iron complex. The 688-nm substance has a ferrous iron and it readily binds carbon monoxide to form a CO-complex, called the 638-nm substance (Yoshida, T., Noguchi, M., & Kikuchi, G. (1980) J. Biochem. 88, 557-563).
The ferric 688-nm substance was prepared from the 638-nm substance by the addition of potassium ferricyanide together with aspiration to eliminate CO. The ferric 688-nm substance did not show any distinct absorption maximum in the red region of the absorption spectrum. The ferric 688-nm substance was readily reduced on the addition of the NADPH-cytochrome P-450 reductase system, but the ferric 688-nm substance could also be reduced spontaneously though at a very low rate. The ferrous 688-nm substance free from excess reducing agents was prepared by passing the 638-nm substance through a column of Sephadex G-25. The ferrous 688-nm substance was degraded to a biliverdin-iron complex much more rapidly in the presence of the NADPH-cytochrome P-450 reductase system than in its absence, indicating that a reducing equivalent is essential for the initiation of heme degradation even when starting from the ferrous 688-nm substance.
Cyanide was found to bind to the ferrous 688-nm substance to form a stable compound; the cyanide compound formed could revert to neither the ferrous 688-nm substance nor the 638-nm substance. The heme of the 688-nm substance was purified as a pyridine-complex by reversed-phase high-performance liquid chromatography and its absorption spectrum was determined.

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Trinitrobenzene selectively dinitrophenylates SH₁, a specific thiol in the myosin heavy chain which contains 1 mol of this cysteinyl residue. When the SH₁-DNP-myosin thus obtained was irradiated with a mercury lamp, a cross-linked product was formed with a molecular weight of about 220 K daltons. It was shown that this product was composed of both heavy and light chains by fluorescence labeling of the heavy chain at SH₂, another specific thiol, and immune reaction using an anti-light chain antibody, respectively.

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A simple and rapid method to remove myosin light chain phosphatase from gizzard myosin using hydroxyapatite chromatography was developed. Stable phosphorylated myosin without light chain phosphatase and kinase was also prepared by the same procedure. A convenient method using the urea gel system to detect minor contamination by phosphatase of myosin is also described.

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The amino acid sequence of soluble NADH-cytochrome b₅ reductase purified from normal human erythrocytes was determined as one approach to understand the hereditary disease of a deficiency of this enzyme. The protein is hydrophilic as a whole, but two regions, from Phe-36 to Ile-71 and from Met-231 to Phe-275, were found to be highly hydrophobic. The sequence of the latter region is particularly unique, and rich in proline (20%). The sequence of the amino-terminal region was very similar to the partial sequences of the corresponding regions of the enzymes from pig and steer liver microsomes.

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