As reported previously, the lysate of liquid paraffin-induced guinea pig peritoneal macrophages contains a hemolytic factor which is composed of two components:the soluble(S) and membrane-bound (M) components. To investigate the mech-anism wherby the factor hemolyses sheep erythrocytes, an attempt was made to identify the S and M components. The fractionation of the cytosol of macrophages by DEAE-cellulose chromatography an the failure of the lysate from L-ascorbate-depleted macrophages to lyse erythrocytes demonstrated that the S component was L-ascorbate. In addition, L-ascorbate was found to be replaced by NADPH, a substrate of the membrane-bound NADPH oxidase, showing that L-ascorbate acts as a donor of active oxygen. When L-ascorbate was combined with the phos-pholipids isolated from the membrane fraction by extraction with chloroform-methanol and thin layer chromatography, it became able to lyse erythrocytes. The results so far obtained indicate that the hemolysis by the macrophage lysete is de-pendent on the formation of peroxidized phospholipids in the membrane fraction with certain active oxygen species produced either from L-ascorbate or by the NADPH oxidase.

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Highly purified recombinant human tumor necrosis factor (TNF) (molecular mass determined as 17 kilodaltons (kDa) by sodium dodecyl sulfate-polyacrylamied gel electrophoresis and as 36 kDa by Sephadex G-100 gel chromatography) was labeled with ¹²⁵I to a specific activity of 5μCi/μg without appreciable loss of activity. The binding of ¹²⁵I-TNF to eighteen human and twelve animal cell lines was examined. The binding varied considerable among different cell lines. In most cell lines, the binding was inhibited up to >90% by the addition of a 100-fold excess of unlabeled TNF. Some human and mouse cell lines showed no significant binding above background levels, suggesting that these cell lines had no receptors for TNF. Among the TNF receptor-positive cell lines, there was no direct correlation between the level of specific TNF binding and the level of sensitivity to the cytotoxic or cytostatic effect of TNF. Some cell lines were sensitive to TNF, whereas others were not affected at all by TNF. The TNF receptor-negative cell lines were also resistant to TNF. Therefore, although the existence of TNF receptor seens to be necessary, it does not alone determine cellular sensitivity to TNF. Scatchard analysis of the binding data revealed that human HeLa S₃ and THP-1 had about 50, 000 and 10, 000 receptors/cell with a dissociation constant (K_D) of 0.3-0.5nM, respectively. Similarly, mouse L-929 and L-M cells had about 5, 000 receptors/cell with K_D of 3-5nM. ¹²⁵I-TNF bound to HeLa S₃ cells was rapidly internalized at 37°C, presumably by receptor-mediated endocytosis, and degraded to acid-soluble products. The turnover of TNF receptors on HeLa S₃ cells seemed to be rapid, since the level of specific binding quickly decreased after treatment with 100μg/ml of cycloheximide at 37°C with a half-life of about 1.5h. The crosslinking of the cell-bound ¹²⁵I-TNF with the use of disuccinimidyl suberated yielded a complex of 105 kDa for HeLa S₃ and THP-1 cells, and a complex of 100 kDa for U937 cells. The crosslinking was completely inhibited by the addition of a 100-fold excess of unlabeled TNF. Assuming that the complex was due to a one-to-one association of the dimeric form of TNF (34 kDa) with the receptor, we estimated the molecular size of the human TNF receptor to be 71 kDa for HeLa S₃ and THP-1, and 66 kDa for U937.

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The associatio and reduction reactions of ten different 4-carboxy-2, 6-dinitrophenyl (CDNP) horse heart cytochromes c, singly modified at lysines 8, 13, 27, 39, 60, 72, 73, 86, 87, and 99, with Saccharomyces cerevisiae cytochrome b₂ were studied to determine the region of cytochrome c interacting with cytochrome b₂. In the presence of higher ratios of free cytochrome c to cytochrome b₂, native cytochrome c, and the CDNP-lysine 39, 60, and 99 derivatives associated with cytochrome b₂ with a bingding stoichiometry close to 2:1, while CDNP-cytochromes c modified at lysines 8, 13, 27, 72, 73, 86, and 87 formed only 1:1 complexes. In the presence of lower ratios of free cytochrome c, modification of lysines 8, 27, 86, and 87 had more inhibitory effects on the association of cytochrome c with cytochrome b₂ than modification of lysines 13, 39, 60, 72, 73, and 99. This tendency was similar to that on removal of free cytochrome c, except in the case of CDNP-lysine 13 and 73 derivatives. The rate of reduction of cytochrome c by cytochrome b₂ was decreased by carboxydinitrophenylation of lysines 8, 13, 27, 72, 73, 86, and 87. In contrast, the rate of reduction of cytochrome c was not affected by modifications of lysines 39, 60, and 99. Since lysines 8, 13, 27, 72, 73, 86, and 87 are located on the front surface and lysines 39, 60, and 99 on the back side, and since different effects of modifying lysine redidues located onthe front surface may be interpreted in terms of effects on the complementary interaction of cytochrome c and cytochrome b₂, these results indicate that the region of cytochrome c interacting with cytochrome b₂ is located on the front surface of the cytochrome c molecule containing the exposed heme edge.

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The neutral sphingolipids and gangliosides were isolated from 62- and 63-day-old chicken livers and characterized. The total concentration of neutral sphingolipids was 59 nmol/g of liver, and that of gangliosides was 330nmol/g of liver. The major neutral sphingolipids were free ceramide, galactosylceramide, glucosylceramide, lactosylceramide, galabiolsylceramide, and Forssman glycolipid. Galactosylceramide was the most abundant and free ceramide was the second most abundant. The major gangliosides were sialosylgalactosylceramide (GM₄) and sialosyllactosyl-ceramide (GM₃), each of which contained only N-acetylneuraminic acid as a sialic acid. Sphingosine (d18:1) was a major long-chain base in all the sphingolipids. Considerable amounts of 2-hydroxy fatty acids were present in free ceramide, galactosylceramide, and GM₄.

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A single cysteine residue present in human plasma arproteinase inhibitor was labeled with a fluorescent sulfhydryl reagent, N-iodoacetyl-N'-(5-sulfo-l-naphthyl)ethyl-enediamine. The resulting fluorescent inhibitor retained nearly full inhibitory ac-tivity and formed complexes with bovine chymotrypsin, porcine pancreatic elastase, and bovine trypsin as revealed by sodium dodecyl sulfate-polyacrylamide gel electro-phoresis. Association rate constants for the interactions of the labeled inhibitor with the proteinases were determined to be 1.5(±0.4) × 10⁶, 3.3(± 0.3)×10⁵, and 1.4(±0.3)×10⁵M^-l forchymotrypsin, elastase, and trypsin, respectively. These values were found to be only slightly lower than those of the unlabeled inhibitor. Fluorescence emission spectra of the labeled inhibitor in the absence and presence of each proteinase were also examined, and little difference was observed between them.

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The correlations among the potentiating activity of various PS analogs on con-canavalin A (Con A)-induced rat mast cell degranulation, the hemolytic activity and the incorporation into the mast cell membrane were studied. The following results were obtained.
1. Lysophosphatidylserine (LysoPS) caused rat mast cell activation (degra-nulation) in the presence of Con A. The order of the activity was as follows: 1-stearoyl lysoPS=1-palmitoyl lysoPS >1-myristoyl lysoPS >1-lauroyl lysoPS. The relative hemolytic activity of these compounds was similar to that observed in the mast cell activation. Dilauroyl PS, which shows similar hemolytic activity to 1- myristoyl lysoPS, did not activate mast cells appreciably.
2. The relative activity of these phospholipids in the binding to mast cells was 1-stearoyl lysoPS >dilauroyl PS > 1-lauroyl lysoPS. Hemolytic activity, as well as activity on mast cells, of lysoPS analogs was well correlated to mast cell membrane incorporation, whereas such a correlation was not found with PS analogs. Dilauroyl PS could be accumulated in the mast cell membrane and showed hemolytic activity, but did not activate histamine secretion.

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Lysophosphatidylserine (lysoPS) strongly enhances degranulation of rat mast cells induced by concanavalin A (Con A). In the present paper, the metabolism of exogeneous lysoPS in intact mast cells was investigated. Incubation of mast cells with 1-stearoyl-sn-glycero-3-phospho-[3-³H]serine resulted in the rapid binding of lysoPS to mast cells and the time-dependent formation of a considerable amount of[³H]phosphatidylserine. No other radiolabeled lipid metabolites were detected. These results suggest that phosphatidylserine (PS) is synthesized through acylation of lysoPS incorporated into mast cells. Most of the lysoPS associated with mast cells was removed by washing with bovine serum albumin, whereas PS newly formed from lysoPS was not. The cells washed with albumin showed no appreciable histamine release upon subsequent addition of Con A. A different set of experi-ments was performed using lysoPS analogs which were modified at the hydroxyl group at position 2 of glycerol to avoid acylation. 1-Stearoy1-2-0-methyl-glycero-3-phosphoserine showed almost the same potentiating activity as 1-stearoyl-lysoPS, although the former does not have the free hydroxyl moiety at position 2 of the glycerol residue. The enhancing activity of another lysoPS analog, 1-stearyl-propanedio1-3-phosphoserine, which lacks the hydroxyl group altogether, was quite similar to that of 1-stearyl-lysoPS. From these results we conclude that the acyla-tion of lysoPS bears no relation to its potentiating activity and that lysoPS acts toward mast cells as lysoPS itself without any conversion to PS. The effect of replacement of an ester bond at position 1 of glycerol in lysoPS with an ether bond, and the phospholipid composition of rat mast cells are also discussed.

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Calcitonin (CT) stimulated phosphorylation of two liver cytosolic proteins whose molecular weights are 67, 000 and 93, 000. Stimulation of 67, 000-M_r protein phosphorylation began shortly after subcutaneous injection of CT, reaching a maximum at 5 min and decreasing to below the control level at 30 min. The reaction was independent of cyclic AMP or Ca²⁺, and was not influenced by a calmodulin antagonist, W7. Stimulation of 93, 000-M_r protein phosphorylation became evident by 30 min. This reaction was also stimulated by administration of vasopressin or epinephrine, which is known to cause increased phosphorylation of glycogen phosphorylase having the same molecular weight. The phosphorylation of 93, 000-M_r protein, stimulated by CT, was dependent on Ca²⁺ but not on cyclic AMP, and appeared to be inhibited by W7. In addition, CT did not influence the phosphorylation of 61, 000-M_r protein, a major protein phosphorylated in a cyclic AMP-dependent manner. These results suggest that CT may exert its effect on liver cells through protein phosphorylation, most probably in a cyclic AMP-independent manner.

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Monoamine oxidase was purified from pig liver mitochondria to homogeneity. The enzyme sample contained a large amount of phospholipids. Depletion of lipids from the enzyme sample resulted in a decrease in its activity, while activity was restored by the binding of the lipid-depleted enzyme to phosphatidylcholine, phos-phatidylethanolamine, or mitochondrial lipids. Upon binding the lipid-depleted enzyme to the mixture of phosphatidylcholine and phosphatidylethanolamine (molar ratio, 1 : 1), the enzymatic activity toward serotonin was elevated over that of the purified enzyme, but not toward benzylamine, suggesting a change in substrate specificity. Upon lipid depletion, inhibition by deprenyl became weaker, while that by clorgyline became stronger. This alteration was reversed by the binding to lipids. By the binding of the lipid-depleted enzyme to some lipids such as the mixture of phosphatidylcholine and phosphatidylethanolamine (molar ratio, 1 : 1), inhibition by clorgyline became even weaker than for the original enzyme sample.

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The effects of epidermal growth factor (EGF) on isoproterenol (IPR)-stimulated DNA synthesis and the activities of the rate limiting enzymes of polyamine syn-thesis (ornithine and S-adenosylmethionine decarboxylases) in parotid glands were investigated in vitro in cultured rat parotid explants and in vivo in submandibulec-tomized mice (mice after bilateral removal of the submandibular and sublingual glands).
When the explants were cultured on siliconized lens paper floating on chemically defined synthetic medium, IPR caused the increases of both tissue cAMP level and the two decarboxylase activities in the prereplicative period and the stimulation of DNA synthesis with similar time courses to those observed in vivo. Dibutyryl cyclic AMP (DBcAMP) also increased the enzyme activities, but not DNA synthesis. EGF (1-2 ng/ml) had little effect on the IPR- and DBcAMP-dependent increases of amylase secretion and the enzyme activities, but it markedly enhanced IPR-stimulated DNA synthesis. Moreover, increase in DNA synthesis by DBcAMP was clearly observed in the presence of EGF when the explants were treated with this nucleotide analogue only during the early prereplicative period.
In in vivo experiments, IPR-dependent increase in DNA synthesis was less in submandibulectomized mice than in intact animals. This decreased response to IPR of DNA synthesis was completely reversed by administration of EGF, though EGF alone did not induce either the enzymes or DNA synthesis. In submandibulectomized mice, although increases in the enzyme activities 8 h after injection of IPR were lower and they were significantly reversed by EGF, the activities at 12 h and the changes in polyamine levels at 8 and 12 h were almost the same as those in intact mice and were not affected by EGF treatment.
These results obtained in vitro and in vivo suggest that EGF participates in the maximal response of IPR-dependent DNA synthesis but is not involved in the change of polyamine synthesis induced by IPR in murine parotid glands.

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Multiple forms of the extracellular dextransucrase [EC 2.4.1.5] from Leuconostor mesenteroides NRRL B-512F strain were characterized by polyacrylamide gel electrophoresis. Based on the Rm (Relative mobility) values, a newly devised simple plot of log (Rm × 10/(1 - Rm)) vs. degree of association of the enzyme showed a good correlation with the results obtained by the Hedrick-Smith method. Both results indicated that the B-512F dextransucrase aggregates were a mixture of two types of forms, i.e., oligomers of a 65 kDa protomer and their charge isomers. Boiling and treatment of the enzyme at pH 10.5 suggested that enzyme aggregates contained dextran or its fragments bound to the enzyme and the enzyme-dextran complex showed the charge isomerism. Since the highly aggregated forms showed higher activity for dextran synthesis than the dissociated forms, the endogenous dextran may serve as a source of primer and may stabilize the enzyme molecule. Besides allosteric regulation of the activity, the occurrence of oligomeric forms of the enzyme may play an important role in the control of dextran synthesis in vivo.

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Increase in the density of liver lysosomes after leupeptin administration was marked in starved rats but only slight in starved-refed rats. The levels of several intracellular enzymes in the liver lysosome fraction purified from leupeptin-treated rats were about 10 to 30 times more in starved rats than in refed rats. However, there was no difference between the intralysosomal levels of endocytosed FITC-labeled asialofetuin in starved and refed rats, indicating that refeeding after starvation markedly suppressed autophagy but not heterophagy in vivo. Immunohistochemical studies with cathepsin B and asialofetuin Fab'-peroxidase conjugates showed that refeeding after starvation markedly altered the cellular distribution of cathepsin B in the liver, resulting in a linear arrangement of the enzyme only on the periphery of hepatocytes. In contrast, endocytosed asialofetuin was found only in the periphery of hepatocytes of both starved and starvedrefed rats. These results indicate that autophagy and heterophagy are regulated by different mechanisms in vivo.

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Calcium-activated neutral protease (rabbit mCANP), composed of large and small subunits, was converted to a lower-Ca²⁺-requiring form (derived μCANP) by limited autolysis in the presence of C²⁺. The NH₂-terminal regions of the two subunits of mCANP were cleaved by autolysis, but the COOH-termini remained intact after autolysis. When native mCANP or derived μCANP was dissociated into subunits, the proteolytic activity of the large subunit was reduced to 2-5 of that of the native dimeric enzyme. The Ca²⁺-sensitivity of one hybrid CANP reconstituted from the large subunit of derived μCANP and the small subunit of native mCANP was similar to that of derived μCANP. However, the other hybrid molecule composed of the large subunit of native mCANP and the small subunit of derived μCANP required a high concentration of Ca²⁺ for activity, like native mCANP. These results indicate that the Ca²⁺-sensitivity of derived μCANP is determined by the structural change of the large subunit resulting from loss of its NH₂-terminal region. The autolysis of the small subunit apparently has no effect on the reduction of the Ca²⁺-requirement.

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Human erythrocyte cytoskeletal ATPase was extracted with 0.2mM ATP (pH 8.0) from Triton X-100 treated ghosts. The ATPase fraction contained mainly spectrin, actin, and band 4.1. When the ATPase fraction was applied to a Sepharose 4B column, 90 % of the ATPase activity was recovered in a spectrin, actin, and band 4.1 complex fraction and none was detected in the spectrin fraction. A specific activity of the complex ATPase was 60-120 nmol/(mg protein•h). No ATPase activity was detected in the presence of EDTA. The presence of magnesium in the incubation medium was essential for the ATPase activity. The activity was activated by KC1 and was almost completely inhibited by 10^-5M free calcium in the presence of 0.2mM MgCl₂. The K₁ for Ca²⁺ was 7 × 10^-7 M. Phalloidin and DNase 1, which affect actin, inhibited this K, Mg-ATPase activity by 95%, but cytochalasin B did not inhibit it. N-Ethylmaleimide activated the ATPase 1.6-fold. The order of affinity for nucleotides was ATP > ITP>CTP, ADP, AMP-PNP, GTP. A specific ATPase activity of purified actin was 50 nmol/(mg•h). These results suggest that the cytoskeletal ATPase is actin ATPase and the actin ATPase is activated by spectrin and band 4.1.

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Delineation of the location(s) of antigenic activity in CNBr peptide 126-194 from porcine skeletal muscle adenylate kinase (AK) was attempted. Peptide 126-194 was digested with chymotrypsin, Staphylococcus aureus V8 protease and trypsin, and several short peptides were purified from the digests by reverse-phase high-performance liquid chromatography (HPLC). Inhibition of the binding of radioiodinated peptide 126-194 to goat antibody to porcine skeletal muscle AK (anti-AK antibody) by the peptides obtained by the enzymatic cleavages was examined by solid phase radioimmunoassay (RIA). At least two antigenic determinants have been identified from the results. One is in the amino (N)-terminal half region 126-154, especially in the vicinity of 131-144, and the other is in the carboxyl (C)-terminal half region 165-183, especially in the vicinity of 165-171. Both of them seem to correspond to exposed and accessible regions in the three-dimensional structure of AK. The correlation between antigenicity and high mobility of the loop in the estimated antigenic region 131-144 is also discussed.

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Adenine nucleotide binding sites on the coupling factor ATPase of thermophilic bacterium PS3 (TF₁) were investigated by UV spectroscopy and by equilibrium dialysis.
When ADP was mixed with TF₁ in the presence and in the absence of Mg²⁺, an UV absorbance change was induced (t_1/2-1 min) with a peak at about 278 nm and a trough at about 250 nm. Similar spectral changes were induced by ADP with the isolated β subunits in the presence and in the absence of Mg²⁺, and with the isolated α subunits in the presence of Mg²⁺ although the magnitudes of the changes were different.
From equilibrium dialysis measurement we identified two classes of nucleotide binding sites in TF₁ in the presence of Mg²⁺, three high-affinity sites (K_d = 61 nM) and three low-affinity sites (K_d = 87μM). In the absence of Mg²⁺, TF₁ has one high-affinity site (K_d <10 nM) and five low-affinity sites (K_d=100μM). Moreover, we found a single Mg²⁺-dependent ADP binding site on the isolated α subunit and a single Mg²⁺-independent ADP binding site on the isolated β subunit.
From the above observations, we concluded that the three Mg²⁺-dependent high-affinity sites for ADP are located on the α subunit in TF₁ and that the single high-affinity site is located on one of the β subunits in TF₁ in the absence of Mg²⁺.

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Phosphoenolffruvate carboxykinase of chicken liver cytosol was purified to homo-geneity by procedures including affinity chromatography with GTP as a ligand. The purified enzyme showed a molecular weight of 68, 000 on gel electrophoresis in the presence of dodecyl sulfate. Comparative studies on this enzyme and its isozyme purified from chicken liver mitochondria were performed. As regards amino acid composition, the cytosolic enzyme w as quite different from the mito-chondrial enzyme, but was rather similar to rat liver cytosolic phosphoenolpyruvate carboxykinase. Specific activities of the cytosolic enzyme were 30-100% higher than those of the mitochondrial enzyme for oxaloacetate-CO₂ exchange, oxaloacetate decarboxylation, and phosphoenolpyruvate carboxylation reactions, though the relative rates of the activities were similar, decreasing in the order given. Apparent Michaelis constants for oxaloacetate in the oxaloacetate decarboxylation reaction were 11.6 and 17.9μM for the cytosolic and the mitochondrial enzyme, respectively, but the values for GTP, GDP, phosphoenolpyruvate, and CO₂ in the oxaloacetate decarboxylation and phosphoenolpyruvate carboxylation reactions were 1.3-2.2 times higher for the cytosolic enzyme than for the mitochondrial enzyme. Thus, the fundamental catalytic properties of the chicken liver phosphoenolpyruvate carboxykinase isozymes were rather similar, despite the marked difference in amino acid compositions.

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Effects of temperature and monovalent cations on the activity and the quaternary structure of tryptophanase of Escherichia coli were studied. The conversion of the apoenzyme into the active holoenzyme was attained at 30°C in Tris-HC1 buffer (pH 8.0) containing pyridoxal-P and K⁺, while no conversion occurred at 5°C. The active holoenzyme thus formed was stable even at 5°C, as long as the cation was present. When K⁺ was absent, however, the active enzyme gradually lost the activ-ity upon chilling to 5°C. The HPLC gel filtration analysis of the active holoenzyme and the low temperature-inactivated enzyme species revealed that the tetrameric holoenzyme dissociated into the dimeric apoenzyme concomitant with the low temperature-induced inactivation at 5°C. The results of HPLC experiments together with other available evidence also suggest that the inactive tetrameric holoenzyme was first formed from the dimeric apoenzyme and pyridoxal-P prior to the formation of the active holoenzyme and that the cation promoted the conversion of the inactive holoenzyme into the active holoenzyme rather than being involved in the conversion of the apoenzyme and pyridoxal-P into the holoenzyme. Among various cations tested for the above effects, NH₄⁺ exhibited the largest effect and K+ the second.

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We previously reported that sera from various kinds of animals contain a protein(s) capable of inhibiting the growth of the non-malignant epithelial cell line derived from Buffalo rat liver (BRL). In the present study, a similar epithelial cell-specific growth inhibitor (EGI) was purified to homogeneity from an acid-ethanol extract of human platelets. During purification, EGI was separated from the major com-ponent of type &beta; transforming growth factor (TGF-&beta;), which can stimulate the colony formation of the non-malignant fibroblastic cell line derived from rat kidney (NRK) in soft agar in the presence of epidermal growth factor (EGF). The purified EGI had an Mr of 27, 000, and was composed of two subunits identical in M_r. It significantly inhibited the growth in monolayer cultures of three non-malignant epithelial cell lines, BRL, MDCK (from Madin-Darby canine kidney) and BSC-1 (from African green monkey kidney), at doses lower than 40 pg/ml in medium con-taining 10 % fetal calf serum. Its inhibitory activity was stable against heating at 90°C for 3 min, but not against treatment with 50mM dithiothreitol. In addition, TGF-&beta; was also partially purified from the same extract. The purified TGF-&beta; did not show any inhibitory activity toward the growth of BRL, MDCK, BSC-1, or NRK.

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The aspartase gene (aspA) of Pseudomonas fluorescens was cloned and the nucleotide sequence of the 2, 066-base-pair DNA fragment containing the aspA gene was determined. The amino acid sequence of the protein deduced from the nucleotide sequence was confirmed by N- and C-terminal sequence analysis of the purified enzyme protein. The deduced amino acid composition also fitted the previous amino acid analysis results well (Takagi et al. (1984) J. Biochem. 96, 545-552). These results indicate that aspartase of P. fluorescens consists of four identical subunits with a molecular weight of 50, 859, composed of 472 amino acid residues. The coding sequence of the gene was preceded by a potential Shine-Dalgarno sequence and by a few promoter-like structures. Following the stop codon there was a structure which is reminiscent of the Escherichia coli rho-independent terminator. The G+ C content of the coding sequence was found to be 62.3%. Inspection of the codon usage for the aspA gene revealed as high as 80.0% preference for G or C at the third codon position. The deduced amino acid sequence was 56.3 % homologous with that of the enzyme of E. coli W (Takagi et al. (1985) Nucl. Acids Res. 13, 2063-2074). Cys-140 and Cys-430 of the E. coli enzyme, which had been assigned as functionally essential (Ida & Tokushige (1985) J. Biochem. 98, 793-797), were substituted by Ala-140 and Ala-431, respectively, in the P. fluorescens enzyme.

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Two GNn-fl-galactosidases, f-galactosidases I, and II, have been highly purified from bovine brain by procedures including acetone and butanol treatments, and chromatographies on Con A-Sepharose, PATG-Sepharose, and Sephadex G-200. /3-Galactosidase I was purified 30, 000-fold and fl-galactosidase II 19, 000-fold. Both enzymes appeared to be homogeneous, as judged from the results of polyacrylamide disc gel electrophoresis. Enzyme I had a molecular weight of 600, 000-700, 000 and enzyme II one of 68, 000, as determined on gel filtration. On sodium dodecyl sulfate polyacrylamide slab gel electrophoresis under denaturing conditions, enzyme II gave a single band with a molecular weight of 62, 000, while enzyme I gave two minor bands with molecular weights of 32, 000 and 20, 000 in addition to the major band at 62, 000. Both enzymes liberated the terminal galactose from GM1 ganglioside and lactosylceramide but not from galactosylceramide. Enzyme I showed a pH optimum of 4.0 and was heat stable, while enzyme II showed a pH optimum of 5.0 and lost 50% of its activity in 15 min at 45'C. Enzyme I showed a pI of 4.2 and enzyme II one of 5.9.

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We isolated over 20 phage clones carrying the ornithine transcarbamylase (OTC) [EC 2.1.3.3] gene, from two independently constructed human genomic DNA libraries, using as probes either a rat OTC cDNA or several nuclear DNA fragments derived from some of these isolated clones. These clones, classified into 10 different groups, overlapped and spanned a region of more than 85 kilobase pairs of the human genomic DNA. Restriction mapping and Southern blot analyses demon-strated that one of the clones covers the 5'-end region of the OTC gene. We se-quenced the 5'-end region of the OTC gene and found that it covered 665 base pairs of the 5'-flanking region, the complete first exon and a part of the first intron (150 base pairs). In the 5'-flanking region, there were two pairs of putative CAAT and TATA boxes and one enhancer core-like sequence, GTGGAAAG. The first exon contained a coding region for most of the OTC presequence, i.e. 26 out of 32 amino acid residues of the presequence, including the initiation methionine.

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We cloned human lymphotoxin (LT) cDNA from a cDNA library prepared from phorbol myristate acetate (PMA) and Con A-stimulated human T cell hybridoma (AC5-8) cells. The nucleotide sequence of the cDNA insert in the plasmid, pLT13, was determined and compared with those of peripheral blood mononuclear cell derived LT cDNA and the LT gene. Four stretches, containing 14 nucleotides in total, were different among the three sequences. The differences included one base change from C to A in the coding region. Because this change was expected to result in a change in the amino acid at position 26 from Thr to Asn, we constructed an E. coli expression plasmid (pLTl3tac6-8.2) and a mammalian cell expression plasmid (pSV2-LT) in order to confirm that pLT13 contains the coding sequence of human LT. Both plasmids were found to synthesize active LT molecules after transfection into JM105 and COS-1 cells, respectively, and their lytic activities were found to be completely neutralized by anti human LT serum. Using an insertion mutant and a deletion mutant, we examined the role of the C terminal domain in the LT activity. The results obtained strongly suggested that the intactness of the C terminal less than 10 residues is required for the LT activity.

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Cytochrome oxidase vesicles catalyzed the peroxidatic oxidation of ferrocytochrome c. The maximal peroxidase activity in the absence of an uncoupling agent was 9.8 mol ferrocytochrome coxidized/(s•mol heme a), indicating a 5-fold activation compared with the soluble enzyme system. The peroxidase activity was further enhanced 1.2 to 2.1 times upon addition of an uncoupler, carbonyl cyanide p-trifluoromethoxyphenyl hydrazone. The stoichiometry of the reduction of hydrogen peroxide by ferrocytochrome c was established to be 1 : 2, indicating water formation. Potassium cyanide (0.14mM) completely inhibited the peroxidase activity. The inhibition by 1mM CO was 40-77% depending on the energized state of cytochrome oxidase vesicles, but in contrast, 85% inhibition was observed with the soluble enzyme. In the energized state the enzyme showed a slightly lower affinity for CO than in the deenergized state. Coupled with the peroxidase activity, a membrane potential of 72mV was registered transiently; this may be physiologically significant in relation to the energy transduction mechanism.

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A unique and simple set of proteins (S1 proteins) has been detected in the cell nuclei of various tissues of the rat (Inoue et al. (1983) Eur. J. Biochem. 135, 61-68). They were liberated from the nuclei by digestion with DNA-hydrolyzing enzymes under the conditions where transcriptionally active chromatin is preferentially digested and released into the reaction supernatant. S1 proteins account for 20% of the total supernatant proteins (Higashi et al. (1984) Biochem. Int. 9, 697-704). The present study demonstrates that S1 proteins occur at the sites sensitive to RNase A as well as DNase I, and are rapidly liberated from the nuclei by digestion with either enzyme.

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A high-molecular-mass protease, ingensin, was purified to homogeneity from rabbit reticulocytes by DEAE-cellulose, HPLC gel filtration, and hydroxyapatite chromatographies. By these procedures, ingensin activity was separated from the activities of two other unique aminopeptidases, one of which is activated by ATP. Ingensin had the following properties: the optimum activity was seen around pH 9.0 and at 50°C; addition of 0.04% SDS and 1mg/ml linoleic acid resulted in 8- and 4-fold increases in peptide-hydrolyzing activity, respectively. The molecular mass was found to be 700, 000±100, 000 daltons on gel filtration, but SDS electrophoresis revealed that the enzyme is composed of several subunits with molecular weights of less than 35, 000. The N-terminal-blocked tyrosine- and arginine-MCA derivatives, but not Arg-MCA, were hydrolyzed rapidly by ingensin. The approximate Km values for the reaction of ingensin with Suc-Leu-Leu-Val-Tyr-MCA and Z-Ala-Arg-Arg-MCA were 0.32 and 0.12mM, respectively. The degradation of several proteins in the reticulocyte extract was stimulated by the addition of SDS and linoleic acid. The activator concentrations necessary for stimulation of the protein hydrolysis are similar to those of the purified reticulocyte ingensin for synthetic substrates. Ingensin did not associate with either right-side-out or inside-out red cell membranes. These results suggest that ingensin is a cytosolic fatty acid-stimulated protease, which is involved in the protein turnover in reticulocyte extracts.

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The effects of various divalent cations on the Ca²⁺ uptake by microsomes from bovine aortic smooth muscle were studied. High concentrations (1mM) of Co²⁺, Zn²⁺, Mn²⁺, Fe²⁺, and Ni²⁺ inhibited neither the Ca²⁺ uptake by the microsomes nor the formation of the phosphorylated intermediate (E_??_P) of the Ca²⁺, Mg²⁺-ATPase of the microsomes. The cadmium ion, however, inhibited both the Ca²⁺ uptake and the E_??_P formation by the microsomes. Dixon plot analysis indicated that Cd²⁺ inhibited (K₁=135μM) the Ca²⁺ dependent E_??_P formation in a non-competitive manner. The inhibitory effect of Cd²⁺ was lessened by cysteine or dithiothreitol. The strontium ion inhibited the Ca²⁺ uptake competitively, while the E_??_P formation increased on the addition of Sr²⁺ at low Ca²⁺ concentrations. At a low Ca²⁺ concentration (1μM), Sr²⁺ was taken up by the aortic microsomes in the presence of 1mM ATP. It is thus suggested that Sr²⁺ replaces Ca²⁺ at the Ca²⁺ binding site on the ATPase.

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The substrate specificity of endo-β-galactosidase of Pseudomonas sp. was found to differ from that of Flavobaeterium keratolyticus or Escherichia freundii, based on the following experimental results. (a) The endo-β-galactosidases from these three bacteria released 6-O-sulfo-GlcNAcβ1-3Ga1 as one of the major products from keratan sulfates from different sources. In addition to the sulfated disaccharide, Flavobacterium and Escherichia enzymes produced GIcNAcβ1-3Gal, which is also an integral repeating unit of keratan sulfate, whereas the Pseudomonas enzyme did not release any non-sulfated disaccharide. (b) Tetrasaccharides were prepared from the teleost skin keratan sulfate by digestion with Pseudomonas enzyme followed by gel filtration on Sephadex G-50 chromatography. A part of the tetrasaccharide fraction was hydrolyzed by Flavobacterium enzyme to produce 6-O-sulfo-GlcNAcβ1-3Gal and GlcNAcβ1-3Gal, whereas the fraction was completely resistant to retreatment with the Pseudornonas enzyme. (c) Endo-β-galactosidases from F. keratolyticus and E. freundii hydrolyzed the internal β-1, 4-galactosyl linkage of various neolacto-type glycosphingolipids to produce glucosylceramides. However, these glycosphingolipids were completely resistant to the Pseudomonas enzyme. These findings clearly show that the sulfation on the N-acetylglucosamine adjacent to galactose in the lactosaminoglycans is essential for expression of the Pseudomonas enzyme, but not for that of the Flavobaeterium or Escherichia enzyme.

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The saccharide binding ability of the low affinity (LA-) binding site of ricin D was abrogated by N-bromosuccinimide (NBS)-oxidation, while in the presence of lactose the number of tryptophan residues eventually oxidized decreased by 1mol/mol and the saccharide binding ability was retained (Hatakeyama et al., (1986) J. Biochein. 99, 1049-1056). Based on these findings, the tryptophan residue located at the LA-binding site of ricin D was identified. Two derivatives of ricin D which were modified with NBS in the presence and absence of lactose were separated into their constituent polypeptide chains (A- and B-chains), respectively. The modified tryptophan residue or residues was/were found to be contained in the B-chain, but not in the A-chain. From lysylendopeptidase and chymotryptic digests, peptides containing oxidized tryptophan residues were isolated by gel filtration on Bio-Gel P-30 and HPLC. Analysis of the peptides containing oxidized tryptophan revealed that three tryptophan residues at positions 37, 93, and 160 on the B-chain were oxidized in the inactive derivative of ricin D, in which the saccharide binding ability of the LA-binding site was abrogated by NBS-oxidation. On the other hand, the modified residues were determined to be tryptophans at positions 93 and 160 in the active derivative of ricin D which was modified in the presence of lactose, indicating that upon binding with lactose, the tryptophan residue at position 37 of the B-chain was protected from NBS-oxidation. From these results, it is suggested that tryptophan at position 37 on the B-chain is the essential residue for saccharide binding at the LA-binding site of ricin D.

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Bilirubin and its photoisomers in the biological fluids of a hyperbilirubinaemic newborn infant before and during phototherapy were analyzed by a recently improved HPLC method. In the serum, the percentages of (EZ)- and (ZE)-bilirubin in the total bilirubin concentration before phototherapy were approximately 10% and on average increased over 1.5-fold at 2h after initiation of phototherapy. The percentage of the (EZ)-cyclobilirubin in the serum bilirubin was under 1%. In the bile, the mean concentration of (ZZ)-bilirubin, derived mainly from (ZE)-bilirubin, nearly tripled during phototherapy. The (EZ)-cyclobilirubin concentration in the bile was very low before phototherapy, increased nearly ten-fold at 3 h after initiation of phototherapy, and was 5- to 6-fold as high as that of (ZZ)-bilirubin. In the urine, upon exposure to light, the urinary concentration of (EZ)-cyclobilirubin is apparently equivalent to half of the biliary concentration of (ZZ)-bilirubin and one-fifth of that of (EZ)-cyclobilirubin. It was concluded that during phototherapy of neonatal hyperbilirubinaemia the structural photoisomer [(EZ)-cyclobilirubin] predominates considerably over the geometric photoisomer [(ZE)-bilirubin].

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A division arrest mutant, cdaA, of Tetrahymena thermophila is known to have a ts-defect in the formation of the fission zone which determines the position of the fission plane. A protein (M_r=85, 000; pI=4.7, designated as p85) has recently been identified in our laboratory as a possible gene product of the cdaA locus by two-dimensional gel electrophoresis and genetic experiments (Ohba et al., submitted). In the present research, we have isolated p85, prepared its antibody, and demonstrated that in wild-type cells or in cdaA cells at permissive temperature, immunofluorescence for p85 appears on the equatorial basal bodies at the predicted fission zone just before formation of the zone. In such a case, the fission zone appears to be formed just anterior to the fluorescence-associated basal bodies, and then constriction of the division furrow occurs at the zone. However, in cdaA cells at the restrictive temperature, the equatorial p85 deposit and subsequent fission zone formation and furrowing do not occur at all. Thus, we conclude that p85 plays a key role in the formation of the fission zone and in the positioning of the equatorial fission line.

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Histidinoalanine, a cross-linking component of connective tissue proteins, was detected in the acid hydrolysate of human urine. The concentrations in urines from newborn babies, children, and adults were 1.33±0.27, 0.77±0.23, and 0.89±0.33nmol/mg creatinine, respectively. Possible origins of urinary histidinoalanine are discussed.

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The effects of oncogene activation on glycosphingolipid (GSL) synthesis by a mouse fibroblast clonal cell line were studied. A transfectant that expressed the activated ras gene showed a definite change in the composition of acidic GSLs, probably an increase in polysialoganglioside, while one that expressed the myc gene showed only a slight change. Neither transfectant grew in soft agar. However, another trans-fectant, which expressed both the myc and ras genes, and grew in soft agar, showed a more dramatic increase in the acidic GSL component. Thus, activations of the myc and ras oncogenes have a synergistic effect on GSL synthesis during transformation.

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The 45K protein from sea urchin eggs forms a complex with actin in the presence of Ca ²⁺. The fraction was not readily dissociated on depletion of Ca²⁺ by gel filtration, but was dissociated on dialysis against an EGTA-containing solution. The free 45K protein and the 45K protein-actin complex affect F-actin in different manners in the presence of Ca²⁺. The former severs F-actin in the presence of Ca²⁺ but not in its absence, while the latter caps the barbed end of F-actin in a Ca²⁺-independent manner thereby lowering the viscosity of F-actin slowly.

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Troponins which confer Ca-sensitivity to skeletal actomyosin ATPase were successfully isolated from striated and smooth adductor muscles of “Akazara” scallop (Chlamvs nipponensis akazara). SDS-gel electrophoresis showed that striated and smooth adductor troponins were composed of three components having molecular weights of about 52K (52, 000), 40K, and 20K, and about 40K, 21K, and 20K, respectively. The Mg-ATPase activity of actomyosin reconstituted from rabbit actin and either Akazara striated adductor myosin or smooth adductor myosin, along with the respective tropomyosin and troponin, indicated that the Ca²⁺ concentration required for the activation of actomyosin ATPase appeared to be favorable to myosin-linked regulation.

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