The amino acid sequence of hen egg yolk-riboflavin binding protein (yolk-RBP) was determined by conventional methods. The sequence was identical with that of hen egg white-riboflavin binding protein except that their carboxyltermini were different, that of yolk-RBP lacked 11 or 13 amino acid residues, while hen plasma-RBP had the same C-terminal sequence as white-RBP. This indicated that the C-terminal 11 or 13 amino acid residues in plasma-RBP might be cleaved off during the incorporation from the blood into the oocyte or in the yolk fluid.
Yolk-RBP had the same characteristics as white-RBP, such as N-terminal pyroglutamic acid, polymorphism in the amino acid sequence (Lys/Asn) at the fourteenth residue from the N-terminal end, carbohydrate chains attached to both Asn (36) and Asn (147) residues, and phosphate groups bound to some serine residues in the sequence of Ser (185) to Ser (197) as a cluster.
These results led us to the conclusion that yolk- and white-RBPs are bio-syn-thesized from the same gene in the different organs (liver and oviduct).
The carbohydrate composition of yolk-RBP was identical to that of plasma-RBP but different from that of white-RBP showing that the processing of the carbohydrate chains in the liver was different from that in the oviduct.

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Variants of T. utilis tRNA^TYr containing deletions or substitutions of nucleotides in the D-loop region have been prepared by several enzymatic reaction steps in vitro. Although these variants lack the “conserved” nucleotides Gm¹⁸-G¹⁹ in their D-loop, their tyrosine-accepting capacities are indistinguishable from that of the native tRNA^Tyr. Thermal denaturation studies with tRNA^TYr variants lacking the Gm¹⁸-G¹⁹ sequence have revealed a biphasic nature of the melting profile, suggesting the loss of tertiary interactions between Gm¹⁸-G¹⁹ and somewhere in the molecule (probably in the TΨC-loop region). These results indicate that nucleotide sequences around Gm¹⁸-G¹⁹ (i.e. D¹⁶-D-Gm-G¹⁹ or Gm¹⁸-G-D-D-²¹) themselves are not essential sites for the recognition of tRNA^TYr by T. utilis tyrosyl-tRNA synthetase and that tRNA^TYr variants with an apparently “relaxed” conformation still have full aminoacylation capacities at around 30°C.

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Human urine urokinase [EC 3. 4. 21. 31] was found to be inactivated by dithiothreitol (DTT) much more severely than by 2-mercaptoethanol at the same concentration on the basis of -SH groups. Removal of DTT by dialysis restored the activities of esterase toward acetyl-glycyl-L-lysine methyl ester, plasminogen activation, and amidase toward 7-(glutaryl-glycyl-L-arginine-amido)-4-methyl coumarin. But the restoration of amidase activity was much less than that of esterase activity. The addition of DTT mediated the conversion of high molecular weight urokinase to low molecular weight urokinase, releasing several peptides. This suggests that the urokinase consists of several polypeptides linked by disulfide bonds. The molecular weight of urokinase produced with DTT was smaller than that of low molecular weight urokinase obtained by autodigestion of high molecular weight urokinase. The autodigestion was also accompanied by liberation of some peptides. But, those peptides released on autodigestion of high molecular weight urokinase were different from those appearing in the presence of DTT.

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Gel filtration of rat liver cytosol on Bio-Gel A-5m resolved the phosphatase activities into four peaks, all of which showed activity with either phosphatidase bound to microsomal membrane (PAmb) or phosphatidate dispersed in sonicated microsomal lipid (PAaq) as the substrate. A major part of the PAmb phosphatase activity (52 %) was eluted in a peak with an apparent molecular weight (Mr) of 500, 000 where the PAaq phosphatase activity was very low. A major PAaq phosphatase activity peak (48 %) was obtained in the void volume (V_o), where the PAaq phosphatase activity was higher than the PAmb phosphatase activity. The addition of 0.075 % Tween 20 to the elution buffer gave only the 500 kilodalton (kDa) peak. When the activity in the V_o peak obtained in the absence of Tween 20 was rechromatographed in the presence of the detergent, a part of the activity was dissociated into 500 kDa molecules having a preference for PAmb. These results suggest that the enzymes obtained in the V_o peak are formed by the association of the 500 kDa molecules with macromolecules and that the substrate preference of phosphatidate phosphatase is modified by the change in the physical state of the enzyme.
The microsomal phosphatidate phosphatase activities were also separated on Bio-Gel A-5m after solubilizing by sonication. Most of both the PAmb and PAaq phosphatase activities were coeluted in the V_o peak, in which the latter activity was higher than the former. When the gel filtration was performed in the presence of Tween 20, a major activity peak with a preference for PAmb was obtained at the elution volume corresponding to apparent Mr 500, 000, indicating a potential relationship between the cytosolic and microsomal activities.

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Double-headed protease inhibitors I, IIa, and IIc (AB I, AB IIa, and AB IIc) have been purified from azuki beans “Takara” (Vigna angularis) by conventional chromatographic methods and their amino acid sequences have been determined. AB I, AB IIa, and AB IIc had molecular weights of 9, 166, 8, 661, and 8, 756 daltons, consisting of 82, 78, 79 amino acid residues, respectively. The molecular weights of these inhibitors, determined by gel filtration at pH 8.0, were 18, 000 for AB I and 17, 000 for both AB IIa and AB IIc, indicating that the inhibitors are dimers. The inhibitors had isoelectric points of 4.7 (AB I), 6.8 (AB IIa), and 6.2 (AB IIc). AB I stoichiometrically inhibited both trypsin and chymotrypsin at a molar ratio of 1 : 1. On the other hand, AB IIa and AB IIc both inhibited trypsin at a molar ratio of about 1 : 2 and also inhibited chymotrypsin, though only weakly.
Sequence comparison with other double-headed inhibitors indicated the reactive sites of AB IIa and AB IIc for trypsin to be Lys²⁶-Ser²⁷ and Arg⁵³-Ser⁵⁴, and those of AB I for trypsin and chymotrypsin to be Lys²⁶-Ser²⁷ and Tyr⁵³-Ser⁵⁴, respectively. The differences between AB IIa and AB IIc were that AB IIa lacked the C-terminal aspartic acid residue, and that Glu¹⁰ and Arg⁶⁰ in AB IIa were replaced by Gln¹⁰ and His⁶⁰ in AB IIc. A comparison between AB IIc and AB I revealed 25 variant amino acids among the 78 residues of AB IIa; further, AB IIa lacked 4 amino acid residues in the C-terminal region of AB I.

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To obtain information about the adenine recognition site in myosin ATPase, ribose-modified fluorescent analogs of ATP, 3'-O-anthraniloyl and 3'-O-(N-methylan-thraniloyl) derivatives, were directly cross-linked to myosin subfragment-1 (S-1) ATPase by irradiation with visible light in the presence of FMN as a photosensitizer. The cross-linking of the fluorescent nucleotides was inhibited by addition of ATP or ADP. Tryptic digestion of the cross-linked S-1 revealed that fluorescence of the analog was associated predominantly with the 50 K fragment and its precursor, the 75 K one, and slightly with the 20 K fragment. However, fluorescence was scarcely associated with the 26 K fragment. The results were confirmed by crosslinking experiments using trypsin-split S-1, which mainly consists of the 50 K, 26 K, and 20 K fragments. These findings suggest that the adenine recognition site of the myosin ATPase is located predominantly on the 50 K domain.

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An aldehyde reductase catalyzing the NADPH-dependent reduction of D-erythrose 4-phosphate to D-erythritol 4-phosphate was purified from beef liver. It was proved to be homogeneous by polyacrylamide gel electrophoresis, sodium dodecyl sulfate polyacrylamide gel electrophoresis and ultracentrifugation analysis. The enzyme was proved to be a monomeric enzyme and its molecular weight was about 40, 000. The enzyme was able to reduce not only tetroses but also trioses, aromatic aldehydes, D-glucuronate and succinic semialdehyde. Apparent K_m-values for aromatic aldehydes were lower than those for tetroses, trioses, D-glucuronate and succinic semialdehyde. Barbiturates and valproate were potent inhibitors of the enzyme and their apparent K₁-values were in the range of 80-180 μM. Quercitrin was the most potent inhibitor and its K₁-value was about 7 μM. From the viewpoint of substrate specificity and inhibitor sensitivity, it seems that the enzyme belongs to the high-K_m type aldehyde reductases.

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A new low temperature EPR signal at g=1.92 was found in photoreduced membrane fractions prepared from blue-green algae, Nostoc muscorum, Tolypothrix tenuis, Phorrnidiaun persicinum, and Anabaena variabilis. The signal also appeared when the sample was chemically reduced in the dark. Judging from the spectrum, the signal was probably due to the reduced form of a membrane-bound iron-sulfur cluster, tentatively designated as Cluster D. The midpoint potential of Cluster D was determined to be -0.27 volts. Possible roles of the cluster in electron transfer systems are also discussed.

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The effects of prostaglandins (PGs) on the induction of alkaline phosphatase (ALP) were investigated in osteoblastic clone MC3T3-E1 cells cultured in serum-free medium. Prostaglandin E₂ (PGE₂) stimulated ALP activity in the cells in a dosedependent fashion with a maximal effect which was about twice that in the control cells at concentrations of 100-500 ng/ml. Actinomycin D and cycloheximide inhibited the stimulative effect of PGE₂ on ALP activity in the cells. PGE₂-induced and native ALPs in the cells were of the same type as that in adult mouse calvaria, being heat-labile, L-homoarginine- and levamisole-sensitive, and L-phenylalanine-insensitive. Isobutyl methylxanthine (IBMX), a cAMP phosphodiesterase inhibitor, stimulated the inductive effect of PGE₂ on ALP activity at 0.1mm, at which concentration IBMX alone had little effect on the activity. PGE₂ also increased the intracellular cAMP content in a dose-dependent fashion with a maximal effect at 100 ng/ml. PGE₁, PGF_1α, and PGF_2α. (primary PGs like PGE₂) increased the activity. Our present results suggest that PGs stimulate the differentiation of osteoblasts and are involved in bone formation in vivo, as well as in bone resorption.

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EDTA/KCl- or pyrophosphate-treated rough microsomes of rat liver clearly showed the co-translational cleavage of pre-human placental lactogen and translocation of the product into membrane vesicles. The signal peptidase fraction was isolated by chromatography on Sephacryl S-300 of deoxycholate-treated membranes and reconstituted into liposomes by dialysis or by the Biobeads SM-2 method. Assay of the signal peptidase activity was performed with pre-human placental lactogen synthesized by the reticulocyte lysate system programmed with human placental lactogen mRNA. The signal peptidase reconstituted into liposomes showed stable activity over the temperature range of 0 to 45°C in contrast, the detergent-solubilized signal peptidase of dog pancreatic membranes was completely inactivated at the unusually low temperature of 37°C. It was shown that this inactivation was due to the presence of detergent. Signal peptidase from rat liver was insensitive to a variety of protease inhibitors, like the enzyme from dog pancreas, but differed from the latter in being inhibited by chymostatin and TPCK.

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A rapid and sensitive homogeneous enzyme immunoassay (homogeneous EIA) was developed for determination of serum proteins such as α-fetoprotein (AFP). There are two assay systems, one is a competitive system including horseradish peroxidase (HRP)-labeled antigen, antibody and substrate, and the other is a non-competitive system including HRP-labeled antibody and substrate. When the aggregate was formed through the binding of HRP-labeled AFP and anti-AFP antibody or through the binding of HRP-labeled anti-AFP antibody and AFP, HRP of the aggregates, as compared with HRP of free conjugates, exhibited marked activity in the presence of 35mM H₂O₂. The extent of stimulation of HRP activity depended on the amount of AFP. This new assay method is very simple and sensitive, and can be used for the determination of any kind of protein, hormone, or drug.

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β-D-Glucosidase was purified from seeds of Japanese cycad by dialysis, chromatography on CM-Sepharose CL-6B, gel filtration on Biogel P-200, and chromatofocusing. By chromatofocusing, β-D-glucosidase was separated into four components whose isoelectric points were in a very narrow range (7.43-7.68). All these components were glycoproteins. The main component (pI=7.59) was homogeneous on gel isoelectric focusing, and was crystallized from ammonium sulfate solution. The molecular weight of the crystalline preparation was determined to be 137, 000 by gel filtration, and 67, 000 by sodium dodecylsulfate polyacrylamide gel electrophoresis, indicating the main component was composed of two subunits with the same molecular weight. The amino acid composition and sugar content of the main component were also determined.
All four components hydrolyzed not only o-nitrophenyl β-D-glucopyranoside but also o-nitrophenyl β-D-galactopyranoside, o-nitrophenyl β-D-fucopyranoside, and o-nitrophenyl β-D-xylopyranoside. Hydrolysis rates of each substrate by the four components were quite similar. Mixed substrate experiments using crystalline preparation proved that a single active site was responsible for the hydrolysis of these substrates.

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Thirteen monoclonal antibodies designated as MFC-1 to MFC-13 were obtained from hybridoma cells cloned after the fusion of mouse myeloma cells with spleen cells of mice immunized with purified human protein C. Studies were made to determine where the antibodies bound to the molecule of protein C and whether they affected the biological actions of protein C. By using the immunoblotting technique, six of these antibodies were shown to bind to the light chain of protein C, and five to the heavy chain of protein C and also activated protein C. The remaining two antibodies bound to neither the light chain nor the heavy chain, though both antibodies bound to the intact protein C. Antibodies specific for the light chain did not bind to the γ-carboxyglutamic acid-domain. Two of the antibodies specific for the heavy chain (MFC-13 and -1) inhibited the amidolytic activity of activated protein C. The MFC-13 also inhibited the activity of bovine activated protein C, but not that of human Factor IXa, Factor Xa, or thrombin. In addition to these two antibodies, another one for the heavy chain (MFC-10) and two antibodies for the light chain (MFC-9 and -11) inhibited the inactivation of Factor Va by human activated protein C. One of the antibodies which inhibited the enzymeactivity (MFC-1) blocked the inhibition of activated protein C by protein C inhibitor. Another one for the heavy chain (MFC-5) inhibited the activation of protein C by thrombin regardless of the presence or absence of thrombomodulin. Based on these results, we have established the positions of some monoclonal antibodybinding sites on the protein C molecule.

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A possible role of bovine platelets in the surface-mediated activation of Factor XII and prekallikrein was studied, using the contact system reconstituted with the purified proteins from bovine plasma. The washed platelets before and after aggregation by ADP, thrombin or collagen did not show any ability to trigger or accelerate the activation of Factor XII and prekallikrein. On the contrary, these aggregates showed a potent inhibitory activity on the activation of those zymogens triggered by kaolin, amylose sulfate and sulfatide. The inhibitory substances from the supernatant of the thrombin-induced aggregates were separated into two major fractions, a low affinity fraction and a high affinity fraction, on a heparin-Sepharose column.
The high affinity protein was identified as platelet factor 4, based on the amino acid composition. From the low affinity fraction, a β-thromboglobulin (β-TG)-like substance and three kinds of unknown proteins, named LA1, LA2, and LA3, were isolated by gel-filtration on a column of Sephadex G-100 or Sephadex G-75 followed by chromatography on a column of Mono S. The molecular weights of LA1, LA2, and LA3 were estimated to be 35, 000, 26, 000, and 11, 000, respectively, on SDS-PAGE. LA2 was identified as a carbohydrate-less LA1, as judged from the amino acid composition and carbohydrate content. The inhibitory activities of these five cationic proteins on the activation of Factor XII and prekallikrein mediated with amylose sulfate, sulfatide and kaolin were different from each other. In the case of kaolin-mediated activation, LA3 was the most potent inhibitor, while platelet factor 4 and β-TG-like substance did not show any significant inhibitory activity. Moreover, the inhibitory activities of all the cationic proteins were not correlated with their anti-heparin activities. Since these proteins were rapidly liberated from platelets by the action of the stimulants, the present results demonstrate a negative role of platelets in the surface-mediated activation of Factor XII and prekallikrein.

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A cDNA library was prepared from the poly(A) mRNA isolated from human peripheral blood lymphocytes which were induced by combined treatment with phytohemagglutinin and a phorbol ester. Recombinant plasmids containing human interferon-γ (HuIFN-γ) cDNAs were identified by the oligonucleotide-hybridization method. Nucleotide sequence analysis showed that the nucleotide and amino-acid sequences of HuIFN-γ cDNA in plasmid pIFNγ-G4 differed from the published data at amino acid position 9 (CAA for glutamine versus AAA for lysine). The cDNA in plasmid pIFNγ-G4 was expressed under control of the simian virus 40 early promoter in monkey COS cells and a biologically active HuIFN-γ was secreted from the cells. The cDNA was also inserted into an expression vector carrying an E. coli tryptophan promoter and was expressed in E. coli. The results suggest that the conversion from lysine to glutamine at amino acid position 9 might not affect the specific activity of HuIFN-γ.

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The sequence of the 5'-terminal 106 nucleotides of cucumber mosaic virus (strain Y) RNA 4, the mRNA coding for viral coat protein, has been determined. The first AUG was located at 77 nucleotides from the 5'-terminus and was confirmed to be an initiation codon by analysis of the N-terminal amino acid sequence of the protein. The nucleotide sequence (positions 77-106) beyond the AUG codon predicted the sequence of ten amino acids corresponding to the N-terminal region of the protein, which exactly matched the determined amino acid sequence containing an acetyl methionine as the N-terminal amino acid. The distance of the initiation codon AUG from the cap structure was 76 nucleotides and the longest among the mRNAs for coat protein of plant viruses so far reported (9-36 nucleotides). This noncoding region is rich in U residues (40%) and the number of G residues (21 nucleotides) is the largest among these mRNAs (usually 1 or 2 residues). A possible secondary structure is postulated for the region, which might be implicated in efficient translation of the RNA 4 in vivo.

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Streptomyces Metallo-Proteinase Inhibitor (S-MPI) consists of 102 amino acid residues, including one methionine and two disulfide bridges. The complete amino acid sequence of S-MPI, including two disulfide bridges, was determined by sequencing of tryptic and chymotryptic peptides of two fragments obtained by cyanogen bromide cleavage followed by reduction and S-pyridylethylation of the protein. Incubation of the inhibitor with thermolysin slowly cleaved one peptide bond, Cys(64)-Val(65), which might be a reactive site of S-MPI.

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Hydrogenase [hydrogen: ferricytochrome c₃ oxidoreductase, EC 1. 12. 2. 1] solubilized and purified from the particulate fraction of Desulfovibrio vulgaris Miyazaki F (IAM 12604) contains 8 iron and 8 labile sulfide ions in one molecule which is composed of two unequal subunits (M_r: 60, 000+29, 000). It does not contain nickel atoms. The EPR (electron paramagnetic resonance) spectrum has an isotropic signal at g=2.017 which is independent of the temperature. The peak-to-peak width of the signal is about 20 G. The signal intensity is nearly equivalent to 1 unpaired electron per molecule. No other signals can be detected in the field range between 2, 240 and 4, 240 G (which corresponds to g-values between 2.91 and 1.54). Ferricyanide has only a little effect on the shape and intensity of the EPR signal. The hydrogenase reduced under H₂ is EPR silent. The Mössbauer spectrum has no hyperfine splitting at 4 K. The isomer shift and quadrupole splitting at 77 K are 0.38 and 0.87mm/s, respectively. Based on these magnetic measurements, the structure of the active center of hydrogenase was suggested to be [4Fe-4S]³⁺+[4Fe-4S]²⁺.

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S-Adenosylhomocysteine hydrolase [EC 3. 3. 1. 1] was purified to electrophoretic homogeneity from mastocytoma P-815 cells. The purified enzyme had a molecular weight of 190, 000, as estimated by Sephadex G-200 chromatography, and a monomer molecular weight of 45, 000, as determined by polyacrylamide gel electrophoresis in the presence of SDS. The K_m value for adenosine was 0.29 μM and the V_max value 4.5 μmol S-adenosylhomocysteine•min^-1•mg^-1 in the synthetic reaction, while the K_m value for S-adenosylhomocysteine was 0.77 μM and the V_max 0.48 μmol adenosine•min^-1•mg^-1 in the hydrolytic reaction. The purified enzyme also had one binding site for adenosine (K_D=2.61×10^-7M) and one for cAMP (K_D=1.6×10^-7M). Using rabbit antiserum raised against the purified enzyme, it was shown that the enzyme activity and enzyme synthesis fluctuated during the cell cycle of mastocytoma cells, reaching the maximum levels as the cells changed from the G_l/S phase to the G₂ phase.

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Bile acid profiles in serum, urine and bile of Nagase analbuminemia rats (NAR) and Sprague-Dawley rats (SDR) were examined. Serum bile acid levels in NAR (2.02+0.51μg/ml, n=15, M±S.E.) were markedly decreased as compared with those in SDR (20.86±3.72μg/ml, n=10). The unbound fraction of bile acids in serum examined by equilibrium dialysis was about ten times higher in NAR than in SDR. In the profiles of urinary and biliary bile acids in NAR and SDR, as big differences as seen for serum bile acids were not observed. Low bile acid levels in serum of NAR may reflect low bile acid binding capacity of NAR serum because the absence of albumin was thought to be one of the major causes of low bile acid levels in serum of NAR.

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Proton translocation coupled to the reduction of nitrite was studied in anaerobically grown Escherichia coli. Extrusion of protons occurred by adding nitrite to an anaerobic suspension of wild-type cells. This extrusion was sensitive to a proton conductor, 3, 5, -di-tert-butyl-4-hydroxybenzylidenemalononitrile (SF6847) or carbonylcyanide-p-trifluoromethoxyphenylhydrazone. Dicyclohexylcarbodiimide, an inhibitor of H^--ATPase, prevented the proton extrusion linked to nitrite reduction, whereas this reagent had no effect on respiratory nitrate reduction to nitrite. Proton extrusion was undetectable when nitrite was added to a suspension of mutant cells defective in H^--ATPase. These results indicate that the proton extrusion associated with nitrite reduction to ammonia is not by redox pumps but by H⁺-ATPase. From the results obtained by the measurement of proton extrusion in nitrite reductasedeficient mutants, NADH-nitrite reductase system is suggested to involve the proton extrusion in whole cells of E. coli.

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The inhibition of the binding of ¹²⁵I-labeled Clostridium botulinum type C neurotoxin to synaptosomes by unlabeled toxin indicated that there were two kinds of receptors on the synaptosomal membrane. The dissociation constants (K_d) were calculated as 79 pM and 35 nM from the concentration of unlabeled toxin that induced half-displacement of bound ¹²⁵I-toxin. These values agree satisfactorily with the values obtained from direct binding experiments (Agui, T, Syuto, B., Oguma, K., Iida, H., & Kubo, S. (1983) J. Biochem. 94, 521-527).
The inhibition of the binding of ¹²⁵I-toxin to synaptosomes and N-acetylneuraminyl(α2-3)galactosyl(β1-3)N-acetylgalactosaminyl(β1-4) [N-acetylneuraminyl(α2-8)N-acetylneuraminyl(α2-3)]galactosyl(β1-4)glucosyl(β1-1)ceramide (GT_1b) by unlabeled heavy chain indicated that heavy chain facilitates the binding of toxin to synaptosomes and GT_1b. The synaptosomal and heavy chain complex K_d values were estimated as 12 nM and 24 μM.
Monoclonal antibodies C-9 and CA-12 recognized the binding sites to GT_1b and synaptosomes, respectively. Antigenic determinants against the two antibodies are presumably partially overlapping, and the overlapping area seems to be essential to the reaction between toxin and C-9 antibody.

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Three-dimensional images of the actin-tropomyosin-myosin subfragment-1 (S1) complex were reconstituted from both minimal- and high-dose electron micrographs by using a conventional reconstruction technique. Higher resolution (1/15 Å^-1) than those of the previous reconstructions was attained.
A multi-domain structure similar to that of the actin-S1 complex described in the previous paper (l) was observed and a new diagram of the multi-domain structure of the actin-tropomyosin-S1 complex is presented.
The shape of S1 molecules in the rigor complex was clearly resolved. In a view perpendicular to the filament axis, S1 had an axially bent profile; only the tail portion, which was thin but was not small in diameter, was steeply inclined. These features were more prominent in the model from minimal-dose images than that from high-dose images.

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To assign the actin molecule in the three-dimensional image of the actin-tropomyosin-myosin subfragment-1 (actin-TM-S1) complex, the three-dimensional image of the actin-tropomyosin complex was correlated to that of actin-TM-S1. To assess the similarity of two structures in a quantitative manner, we used a normalized cross-correlation function (“similarity function”). The calculation of similarity indicated that domain A and domain B defined in (1, 2) correspond to actin-tropo-myosin. This assignment indicates that one S1 molecule strongly interacts with only one actin molecule, but at least two regions of S1 contribute to the binding. Comparison of the reconstituted models of thin filaments with those of decorated thin filaments suggested a change in the shape of the actin molecule.

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Spleen cells from a CBF₁ (BALB/c X C57BL/6) mouse immunized with rat tyrosine 3-monooxygenase were fused with NS-1 mouse myeloma cells. From 188 hybrid cells, 2 stable clones secreting anti-tyrosine 3-monooxygenase antibody were obtained. Antibody from one clone was coupled to CNBr-activated Sepharose 4 B and the monoclonal antibody-Sepharose was shown to be very useful for isolating rat tyrosine 3-monooxygenase from crude preparations.
Analyses by monoclonal antibody chromatography followed by SDS-polyacrylamide gel electrophoresis and by gel filtration revealed that tyrosine 3-monooxygenases from nerve cell bodies, nerve terminals, and adrenal medullae were indistinguishable with respect to their molecular structures. However, there were serious differences in the catalytic properties between the enzymes from the brain tissues and adrenal medullae, although there appeared to be no significant difference between the enzymes from nerve cell bodies and nerve terminals. The possibility that the activity of the enzyme may be strongly suppressed especially at the physiological pH in brain tissues is also discussed.

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Kinetic parameters for the trypsin-catalyzed hydrolysis of the oxygen and sulfur “inverse substrates, ” p-amidinophenyl and p-amidinothiophenyl acetates and trimethylacetates, have been compared. The results suggest that both series of compounds are hydrolyzed via an identical pathway. Appreciable differences, however, were observed in the efficiency of the acylation process in both series, possibly reflecting the spatial requirements of the enzyme's active site toward these substrates. As reported previously, acceleration in deacylation by a positively charged molecule is a characteristic feature of trypsin-catalyzed hydrolysis of “inverse substrates.” In the present investigation, it was shown that p-amidinothiophenol is ineffective as an activator, whereas its oxygen counterpart behaves as a potent activator toward oxygen and sulfur substrates. It is assumed that some ionic interaction between the enzyme and the ligand molecule could prevent the rate enhancement.

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Spectral changes of hemoproteins in the near ultraviolet region on binding to a ligand and on oxidation-reduction of the heme-iron were studied by computercontrolled spectrophotometry.
1. Near ultraviolet difference spectra between the low spin and high spin forms of ferric hemoproteins were classified into three groups: Those showing two absorption peaks having maxima at around 285 and 295 nm, those showing a peak at around 275 nm, and those showing a peak at around 300 nm. No corresponding absorption peak was observed with model heme complexes of low molecular weight.
2. The intensity of the peak in cyanide difference spectra of catalase and horseradish peroxidase in the near ultraviolet region was dependent on the concentration of added cyanide and paralleled the intensity of the spectral changes in the Soret region. The spectral changes in both the near ultraviolet and Soret regions developed within 6 ms after the addition of cyanide.
3. Difference spectra between the reduced and oxidized forms of cytochrome c, cytochrome oxidase-cyanide complex, hemoglobin, and lactoperoxidase-cyanide complex showed a characteristic peak at around 285-290 nm.
4. Various difference spectra of hemoglobin in the near ultraviolet region were also measured.
5. The observed positions, shapes, combinations, and relative intensities of the peaks were compared with those of solvent perturbation difference spectra and pH difference spectra of proteins and aromatic amino acids and also with the diacetylchitobiose-induced difference spectrum of lysozyme. The kinds of aromatic amino acid residues possibly responsible for the observed difference peaks were discussed on the basis of the results of the comparison.
6. Based on the results obtained, the common occurrence of a heme-linked functional response of the hemoprotein conformation was suggested.

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The mechanism of the ATPase [EC 3. 6. 1. 3] reaction of porcine platelet myosin and the binding properties of platelet myosin with rabbit skeletal muscle F-actin were investigated.
The kinetic properties of the platelet myosin ATPase reaction, that is, the rate, the extent of fluorescence enhancement of myosin, the size of the initial P₁ burst of myosin, and the amount of nucleotides bound to myosin during the ATPase reaction, were very similar to those found for other myosins. Strong binding of platelet myosin with rabbit skeletal muscle F-actin, as found for smooth muscle myosin, was suggested by the following results. (i) The rate of the ATP-induced dissociation of hybrid actomyosin, reconstituted from platelet myosin and skeletal muscle F-actin, was very slow. (ii) The amount of ATP necessary for complete dissociation of hybrid actomyosin was 2 mol/mol of myosin, although skeletal muscle actomyosin is known to dissociate completely upon addition of 1 mol ATP per mol of myosin. (iii) Unlike skeletal muscle myosin, the EDTA(K⁺)-ATPase activity of platelet myosin was inhibited by skeletal muscle F-actin. These observations indicate that ATP hydrolysis by vertebrate nonmuscle myosin follows the same mechanism as with other myosins and that the binding properties of nonmuscle myosin with F-actin are similar to those of smooth muscle myosin but not to those of skeletal muscle myosin.

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ECMA 2 and ECMA 3 antigens defined by two monoclonal antibodies are preferentially expressed in early embryonic cells of the mouse. The antigens were isolated from F9 embryonal carcinoma cells by detergent solubilization followed by indirect immunoprecipitation. Both antigens were glycoproteins, which, upon extensive pronase digestion, released the high-molecular-weight glycan (embryoglycan). The immunoprecipitation reactions were inhibited by the glycan, indicating that the two antigens were carried by it. Furthermore, binding of anti-ECMA 2 antibody to the glycan was directly demonstrated by a modified Farr's assay. The antigenic determinant of ECMA 2 antigen was found to involve an a-galactosyl residue, since a-galactosidase from coffee bean, but not other glycosidases, abolished the antigenic activity. Serological experiments indicated that ECMA 2 antigen is different from other a-galactosyl antigens, namely blood group B and P₁ antigens and an antigen defined by antibodies in the sera of patients with ovarian germ cell tumors.

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When various lectins were mixed with radioactively labeled embryoglycan (polylactosamine-type glycoprotein-bound carbohydrates from early embryonic cells) isolated from F9 embryonal carcinoma cells and the resulting complex was precipitated with ammonium sulfate, the glycan was found to react with the following lectins: Helix pomatia agglutinin (HPA), soybean agglutinin (SBA), Sophora japonica agglutinin (SJA), and Ricinus communis agglutinin-1 (RCA-1). Furthermore, affinity chromatography on lectin-agarose revealed that receptors for Griffonia simplicifolia agglutinin-I (GS-I) were also carried by the glycan. Together with the previous finding that the glycan carries receptors for Dolichos biflorus agglutinin (DBA) and peanut agglutinin (PNA), the present result established that the glycan has receptors for a variety of lectins recognizing N-acetylgalactosamine and/or galactose in teratocarcinoma cells. Intact molecules carrying GS-1 receptors and SJA receptors were isolated from F9 cells and teratocarcinoma OTT6050 and were shown to be high-molecular weight glycoproteins similar to DBA receptors isolated from the same sources.

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A staphylocoagulase-binding region in human prothrombin was studied by utilizing several fragments prepared from prothrombin by limited proteolysis. Bovine prothrombin, prethrombin 1, prethrombin 2, and human diisopropylphosphorylated a-thrombin strongly inhibited formation of the complex (“staphylothrombin”) between human prothrombin and staphylocoagulase, but bovine prothrombin fragment 1 and fragment 2 had no effect on the complex formation, indicating that the binding region of human prothrombin for staphylocoagulase is located in the prethrombin 2 molecule. To identify further the staphylocoagulase-binding region, human a-thrombin was cleaved into the NH₂-terminal large fragment (Mr=26, 000) and the COOH-terminal fragment (Mr=16, 000) by porcine pancreatic elastase. Of these fragments, the COOH-terminal fragment, which includes Asn-200 to the COOH-terminal end of the a-thrombin molecule, partially inhibited the complex formation between staphylocoagulase and human prothrombin. In contrast, the NH₂-terminal large fragment did not show any inhibitory effect on the staphylothrombin formation. These results suggest that the staphylocoagulase interacts with human prothrombin through the COOH-terminal region of a-thrombin B chain. Other plasma proteins, factor X, factor IX, protein C, protein S, protein Z, all of which are structurally similar to prothrombin, did not inhibit the staphylothrombin formation at all, indicating that a specific interaction site with staphylocoagulase is contained only in the prothrombin molecule.

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A fragment of the cloned gene for the mouse Cε chain, coding for the first, second, third, and fourth domains, has been coupled to the SV40 promotor region (pSV2-mCε). About 50 copies of pSV2-mCε or its PvuII-EcoRI fragments were introduced into Cos-7 cells. Expression of PvuII-EcoRI fragments of pSV2-mCε was observed in about 50% of the Cos-7 cells by indirect fluorescence staining. However, no expression of circular pSV2-mCε was observed. About 200 copies of linearized pSV2-mCε with EcoRI were introduced into fertilized mouse eggs. Two of 78 mice born from these eggs had integrated mouse Cε genes. Mouse Cε gene was shown to be integrated in a tandem array and as intact structures without undergoing gross deletions or rearrangements, judged from the Southern blotting patterns from several restriction enzymes. The first transgenic mouse was mated to a normal male to examine whether mouse Cε gene were stably transmitted to progeny. Among 5 mice to which the Cε gene had been transmitted, one deleted 5 copies of this gene and another deleted one junction fragment, thus demonstrating relatively unstable transmission. No Cε mRNA was detected in the liver, kidney, brain, lung, skeletal muscle, heart, testis, or spleen of a transgenic mouse.

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Intracellular transport of a newly synthesized asialoglycoprotein receptor was studied biochemically using a monospecific antibody for the receptor. Pulse-labeling by intravenous injection of [³H]leucine and pulse-chasing after 10min by cycloheximide injection resulted in the maximal labeling of the receptor in the rough microsomes at 15min, in the smooth microsomes and the heavy Golgi subfraction (GF₃) at 25min and in the intermediate plus light Golgi subfraction (GF₁₊₂) at 30min. By 60min, the labeling in GF₁₊₂ had decreased and leveled off. In the plasma membrane fraction, the labeled receptor first appeared at 20min, increased rapidly and also reached a constant level at 40-60min. Intracellular movement of the newly synthesized receptor in the GF₁₊₂ and plasma membrane fractions was also investigated by purifying the receptor protein from the GF₁₊₂ and plasma membrane fractions by affinity chromatography. It was revealed that the specific radioactivities of the receptor in the two fractions become equilibrated after 60-120min.
The receptor of the various membrane fractions was also pulse-labeled in vivo for 20min simultaneously with [³H]glucosamine and [¹⁴C]leucine, and pulse-chased for the following 40min. After pulse-labeling for 20min, the ratio of the radioactivity of [³H]glucosamine or [³H]sialic acid to [¹⁴C]leucine of the receptor from the rough and smooth microsomes, and GF₃, GF₂, and GF₁ increased in that order. That of the receptor from the plasma membrane fraction was infinitely higher, because, while a significant amount of ³H-radioactivity was incorporated into the receptor in the Golgi apparatus, only a negligible amount of ¹⁴C-radioactivity was incorporated into the same receptor in the plasma membrane due to the delay in the arrival of [¹⁴C]leucine labeled receptor to the plasma membrane. After chasing for 40min, however, the same radioactivity ratios of the GF₁ and plasma membrane fractions approached each other. All these results strongly suggest that the distribution of the newly synthesized receptor becomes rapidly equilibrated between the trans-Golgi components and plasma membranes probably by repeated recycling of the receptor protein between the two membranes.

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The fate of palmitoyl-lysophosphatidylcholine (lysoPC) incorporated into the membrane of intact human erythrocytes from a medium was investigated under nonhemolytic conditions at 37°C by means of ¹⁴C-labeled tracers.
The lysoPC was first incorporated into the outer half of the membrane lipid bilayer and then gradually translocated into the inner half during the incubation. At the same time it was metabolically converted into phosphatidylcholine (PC) and free fatty acid (FFA) plus glycerophosphorylcholine by the actions of acyltrans-ferase and lysophospholipase, respectively. The half times of the conversion were about 14 h, while the value of 0.5 h was obtained when the half time was measured with the hemolysate of the lysoPC-loaded erythrocytes.
Chymotrypsin treatment of unsealed ghosts caused a definite decrease in lysophospholipase activity, while similar treatment of resealed ghosts did not. This together with other evidence already reported in the literature suggests that both lysophospholipase and acyltransferase may be located in the inner surface of the membrane.
The above findings strongly suggest that the most of the lysoPC loaded to the membrane is gradually translocated from the outer to the inner half of the bilayer and soon converted to either PC or FFA.

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A novel modified nucleoside located in the first position of the anticodon of yeast tRNA^Val_2a was isolated and its chemical structure was characterized as 5-carbamoylmethyluridine by means of ultraviolet absorption spectrum, mass spectrum, and nuclear magnetic resonance spectrum.

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Autoactivation of the proenzyme form of a subunit of the first component (C1r) was performed in the presence and absence of diisopropyl fluorophosphate (DFP). The time-course of autoactivation of zymogen C1r followed a sigmoidal curve and was accelerated by addition of the enzyme C_??_r and by increasing the concentration of C1r, suggesting that autoactivation of C1r consists of two intermolecular reactions, i.e. zymogen(C1r)- and enzyme(C1r)-catalyzed reactions. In the presence of 10mM DFP, the enzyme-catalyzed autoactivation of C1r was completely inhibited, while the zymogen-catalyzed autoactivation still proceeded depending upon C1r concentration. These results suggested that the zymogen-catalyzed autoactivation of C1r is a DFP-insensitive second-order reaction and is mediated by an active site generated in a single chain C_??_r through a conformational change (Kasahara et al. (1982) FEBS lett. 141, 128-131).
Based on these results, a possible reaction process of autoactivation of C1r was proposed, as follows:
_??_
where C_??_r represents a conformational isomer which catalyzes the autoactivation of C1r, and the rate constants, k₂ and k₃, are of second-order. Utilizing a computer, we simulated the autoactivation of C1r and found the above scheme to be a reasonable model of C1r autoactivation.
Evidence which supports the formation of a conformational isomer of C1r, C_??_r, as an intermediate in its autoactivation was also obtained by a surface radio-labeling method.

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Limited proteolysis of C3b by C3b inactivator (factor I) consists of a two-step reaction; rapid cleavage of C3b to yield a nicked C3b derivative, iC3b, and followed by slow cleavage of iC3b to yield two antigenically distinct fragments, C3c and C3d, g. Using a fluorescence-labeled C3b as a substrate for I, we have investigated in detail the optimal conditions for the sequential cleavages of C3b by I.
The pH optimum for the first cleavage was markedly affected by the ionic strength of buffers. The cleavage was maximum at pH 6.0 under physiological ionic strength but at pH 8.5 under low ionic strength (such as 1.7 mS). The second cleavage was a slow reaction and occurred only under low ionic strength and within a narrow pH range around pH 6.0. One of the products of the second cleavage, C3d, g, was isolated and shown to be a single polypeptide chain of 41, 000 daltons with pI 5.0. C3d, g had leucocytosis-inducing activity, like C3d-k, which is a C3d fragment released by the action of plasma kallikrein. Trypsin digestion of C3d, g produced two fragments of 30, 000 and 10, 000 daltons and the 10, 000-dalton fragment retained the leucocytosis inducing activity.

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dATP at high concentrations was capable of replacing ATP required in the synthesis of Okazaki pieces in isolated HeLa cell nuclei. In addition, the levels of synthesis of high molecular weight DNA were observed to vary depending on the lot of dATP used. Analysis by HPLC revealed that dATP samples of a particular source contained ATP in the range of 0.25-0.43 mol%. With ATP-free dATP, almost no synthesis of high molecular weight DNA was observed, while with impure dATP, a small but significant amount of high molecular weight DNA was synthesized. While this observation confirmed our previous finding that dATP can replace ATP in the synthesis of Okazaki pieces but not in the joining of the pieces, it is also a warning to users of commercial dATP in biochemical and biological studies.

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Parathyroid hormone (PTH) increased the activity of spermidine/spermine N¹-acetyltransferase, a rate-limiting enzyme of polyamine biodegradation, in rabbit costal chondrocytes in culture. The enzyme activity increased in a dose-dependent manner after addition of PTH to the culture, reaching a maximum at 8 h. The increase in the enzyme activity was abolished by cycloheximide or actinomycin D. Dibutyryl cyclic AMP also induced the acetyltransferase to some extent. These results suggest that the induction of spermidine/spermine N¹-acetyltransferase by PTH may play some significant role in the expression of the differentiated pheno-type of chondrocytes.

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