When sea urchin spermatozoa were treated with a Triton X-100 solution, cAMP-dependent protein kinase (cA-kinase) activity was extracted. Further extraction with Triton X-100 of axonemes isolated from the Triton-extracted sperm again released a considerable amount of the cA-kinase activity. The activity which remained after extraction three times with Triton X-100 was released by treatment with a low salt solution. These activities found in the various extracts were likely to be due to the same cA-kinase, which was a mammalian type II-like enzyme. The cA-kinase activity that remained in the axonemes after the first Triton X-100 extraction may be involved in the regulation of flagellar movement in the Triton-extracted sperm.

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The phosphorylation of glyoxalase I was observed when the phosphatase-treated enzyme was incubated in the presence of [γ-³²P] ATP and a cell extract prepared from a-type yeast cells which had been treated with the culture supernatant of a-type yeast cells. The phosphorylated protein was identified as glyoxalase I by using anti-glyoxalase I rabbit immunoglobulin G.

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Although Cys-14 (human numbering) of cytochrome c was conserved during its molecular evolution and it is supposed to be essential for most cytochromes c to retain heme c via two thioether bonds, a site-directedly mutated human cytochrome c which has an alanine residue at this position and only one thioether bond through Cys-17 turns out to be functional. This shows that Cys-14 is not essential. The absorption spectrum of the atypical cytochrome c is red shifted, and similar to those of Euglena and Crithidia cytochromes c, which also have only one thioether bond [Pettigrew, G. W., Leaver, J. L., Meyer, T. E., & Ryle, A. P. (1975) Biochem. J. 147, 291- 302].

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Hydroperoxide decomposition by the NADP-glutathione system in rat liver mitochondria was analyzed. Mitochondria were found to contain high concentrations of the reduced form of glutathione (GSH) (4.32±0.50 nmol/mg) and NADPH (4.74±0.64 nmol/mg), and high activities of glutathione peroxidase and reductase. In the initial phase of the reaction, the rate of hydroperoxide decomposition was proportional to both the GSH level and the activity of GSH peroxidase. However, in the later steady state, the step of NADP reduction was rate-limiting, and the overall reaction rate was independent of the initial concentration of GSH, and activities of glutathione peroxidase and reductase. Some GSH was released from mitochondria during incubation, but the rate of the decomposition could be simply expressed as k [GSH] /2, where k is the first-order rate constant of the peroxidase and [GSH] is the intramitochondrial level of GSH in the steady state. The rate of the reaction in the steady state was also dependent on the NADPH level, its reciprocal being linearly correlat-ed with [NADPH]^-1 The rate of decomposition of hydroperoxide was influenced by the respiratory state. During state 3 respiration, the rate was greatly depressed, but was still considered to exceed by far the rate of physiological generation of hydroperoxide.

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Yeast mitochondrial ATP synthase has three regulatory proteins, ATPase inhibitor, 9K protein, and 15K protein. The 9K protein binds directly to purified F₁-ATPase, as does the ATPase inhibitor, but the 15K protein does not [Hashimoto, T. et al. (1987) J. Biochem. 102, 685-692]. In the present study, we found that 15K protein bound to purified F₁F₀-ATPase, forming an equimolar complex with the enzyme. The apparent dissociation constant was calculated to be 1.4×10^-5M. The ATPase inhibitor and 9K protein also bound to F₁F₀-ATPase in the presence of ATP and Mg²⁺, and the dissociation constants of their bindings were about 3×10_-6M. They bound to the enzyme competitively in the absence of 15K protein, but in its presence, they bound in equimolar amounts to the enzyme. The ATP-hydrolyzing activity of the enzyme-ligand complex was greatly influenced by the order of bindings of ATPase inhibitor and 9K protein: when the ATPase inhibitor was bound first, the activity of the enzyme was inhibited completely and was not restored by 9K protein, but when 9K protein was added first, the activity was inhibited only partially even after equimolar binding of the ATPase inhibitor to the enzyme. These observations strongly suggest that the 15K protein binds to the F₀ part and functions to hold the ATPase inhibitor nr giC orntein on the F1 subunit.

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The pH dependence of kinetic parameters for the hydrolysis of mixed micelles of 1, 2-dipalmitoyl-sn-glycero-3-phosphorylcholine (diC₁₆PC) with Triton X-100, catalyzed by the intact and the N-terminal α-NH₂-modified phospholipases A₂ (PLA2s) of Agkistrodon halys blomhoffli, was studied at 25°C and ionic strength 0.1 in the presence of saturating amounts of Ca²⁺. The pH dependence of the kinetic parameters for the hydrolysis of monodispersed diC₆PC, catalyzed by the modified enzyme, was also studied under the same conditions, and the data were compared with the previous results for the intact enzyme [Teshima, K. et al. (1986) J. Biochem. 100, 1655-1662]. The pK values of the catalytic group, His 48, and Tyr 52 were found to shift from 5.55 to 7.00 and from 10.50 to 11.50, respectively, on binding of the micellar substrates to the enzyme. On the other hand, no participation of these ionizable groups was observed for the binding of the monodispersed substrate. On the basis of the present finding and the X-ray crystallographic studies on bovine pancreatic PLA₂ [Dijkstra, B. W. et al. (1981) J. Mol. Biol. 147, 97-123] and on a PLA₂ of Crotalus atrox venom [Brunie, S. et al. (1985) J. Biol. Chem. 260, 9742-9749], the hydrogen-bonding of Tyr 73, which is involved in the lipid-water interface recognition site, to His 48 and Tyr 52 in the active center was strongly suggested to be important for the hydrolysis of micellar substrates.

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The synthesis of 34-kDa stress protein was enhanced, with a simultaneous increase in heme oxygenase activity, when mouse macrophages were exposed to diethylmaleate or sodium arsenite. After 7 h of exposure to the sulfhydryl agents, the 34-kDa protein was the most actively synthesized protein. Immunoblot analysis showed that the induced 34-kDa protein reacted with an antibody raised against bovine heme oxygenase. Cadmium ions or 1-chloro-2, 4-dinitrobenzene also induced the 34-kDa protein which reacted with the antibody. Treatments of the cells with buthionine sulfoximine or hydrogen peroxide weakly induced the protein, while diamide treatment or heat shock was without effect. These results are consistent with our previous findings that heavy metal ions including arsenite and cadmium ions induce heme oxygenase (32-kDa stress protein) in human cell lines [Taketani, S., Kohno, H., Yoshinaga, T., & Tokunaga, R. (1989) FEBS Lett. 245, 173-176], and also suggest that the formation of glutathione conjugate with sulfhydryl-reactive agents may mediate the induction of the stress protein in mouse peritoneal macrophages.

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The pulmonary cytochrome P-450, P450 L-2, was purified 460-fold from pulmonary microsomes of untreated male rats. Its specific content was 10.6 nmol/mg of protein. The monomeric molecular weight was 54, 000 on SDS-polyacrylamide gel electrophoresis. The CO-reduced absorption maximum of P450 L-2 was at 451 nm, and the oxidized heme iron appeared to be in the low-spin state, as deduced from the Soret maximum at 421 nm. P450 L-2 had high lauric acid ω- and (ω-1)-hydroxylation activities, but low prostaglandin A1 ω- and (ω-1)-hydroxylation activities. It catalyzed the O-dealkylation of 7-ethoxycou-marin, but was not efficient in the hydroxylation of testosterone or the N-demethylation of aminopyrine. The NH₂-terminal amino acid sequence of P450L-2 was V-L-N-F-L-X-P-X-L (X being an unidentified residue). The catalytic properties of P450 L-2 resembled those of P450 K-5, the major rat renal cytochrome P-450. However, anti-P450 K-5 antibody did not cross-react with P450 L-2, and these forms had different NH₂-terminal sequences. To judge from the results of NH₂-terminal sequence analysis, P450 L-2 seems to be placed in the IVB gene family. Also, P-450 IIB1 was detected by immunoblotting in one of the peaks on ion-exchange HPLC during the purification of P450 L-2, suggesting the presence of P-450 IIB1 in rat pulmonary microsomes.

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The activity of an ATP-dependent protease, Clp, was examined in Escherichia coli SG1110 (lon-) in various growth phases. The ATP-dependent proteolytic activity (Clp activity) in a crude extract of the cells changed with the growth phase. Cells in the early exponential growth phase showed the lowest activity, but then the activity increased dramatically with cell growth. The highest Clp activity was found in the cells in the late exponential and early stationary phases, however, the activity returned to the original level on prolonged culturing. These changes in Clp activity were closely correlated to the amount of one of the components of Clp, Clp A, which was quantitated immunochemically with antibodies against the Clp A protein. However, the amount of the other component of Clp, Clp P, did not change with the growth phase. These results suggest that the activity of Clp in the cells is regulated by the amount of Clp A in various growth phases. We next examined the effect of the cellular ATP level on Clp activity, because ATP is a cofactor for Clp protease in vitro. The addition of dinitrophenol (DNP) and sodium azide reduced the intracellular concentra-tion of ATP, but had no effect on the Clp activity or the level of the Clp A protein when these drugs were added to the culture at the stationary phase. On the other hand, these drugs elevated both the Clp activity and the Clp A amount in exponentially growing cells, whose cellular ATP level was also reduced. In this case, cell growth was inhibited by the drugs, suggesting that the reduction in the cellular ATP level is not directly correlated with elevation of the Clp A amount and the Clp activity, but indirectly through interruption of cell growth. These results suggest that Clp activity is regulated by the amount of the Clp A protein in vivo in E. coli cells, and that the Clp A amount varies with the cell growth phase.

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We examined the inhibitory effect of three pseudo-aminosugars (validamine, valienamine, and valiolamine), which were isolated from the broth of Streptomyces hygroscopicus, on the oligosaccharide-processing glucosidases I and II involved in glycoprotein biosynthesis in rat liver. Both glucosidases I and II were inhibited to the same extent by the pseudo-aminosugars, and valiolamine had a more potent inhibitory activity than validamine or valienamine. A 50% inhibition of valiolamine was observed at 12μM for glucosidase I and glucosidase II activities acting respectively on the substrates Glc3Man9GlcNAc2 and p-nitrophenyl α-n-glucopyranoside. Further, in order to investigate further the ability of valiolamine to inhibit glucosidase I, reaction products were analyzed by gel filtration on a Bio-Gel P-4 column. We also compared the inhibitory action of these pseudo-aminosugars on the acid α-glucosidase of rat liver lysosomes. They competitively inhibited the hydroly-sis of both substrates, maltose and glycogen. Valiolamine again had a more potent lysosomal α-glucosidase inhibitory activity than the other two. The Ki values of valiolamine for the hydrolysis of maltose and glycogen were 8.1 and 11μM, respectively. Valiolamine is a particularly effective inhibitor of oligosaccharide glucosidases I and II and of lysosomal α-glucosidase. Hence valiolamine might be useful as a research tool in investigations of carbohydrate metabolism.

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An allergenic pentasaccharitol, Gp-1β-b6, was isolated as a minimum structural unit responsible for the allergic reaction in skin of patients with sea squirt allergy from a saccharitol fraction, Gp-1β-b, that had been liberated by β-elimination from a glycopep-tide in a Pronase digest of a sea squirt antigen, Gi-rep. Methylation/GC-MS and FAB-MS analyses indicated the sugar sequence of Gp-1β-b6 to be GalNAc1→2Fucl→(GalNAc1→) 3, 4G1cNAc1→3GalNAc-ol. To analyze the structure in more detail, Gp-1β-b6 was labeled with p-aminobenzoic acid ethyl ester (ABEE), i.e., the reducing terminal 3-O-substituted Ga1NAc-ol of the saccharitol was oxidized to 2-O-substituted L-ThrNAc with equimolar periodate, and the resultant aldehyde was labeled with ABEE by reductive amination. The ABEE-labeled Gp-1β-b6 was subjected to sequential exoglycosidase digestion with α-N-acetylhexosaminidase, α-N-acetylgalactosaminidase, and α-fucosidase, and the digests were chromatographed on an HPLC column of TSK gel Amide 80. From the results of the HPLC, methylation/GC-MS, and FAB-MS analyses of the digests of the labeled substrate, the structure of Gp-1β-b6 was determined to be GalNAcα1→2Fucα1→3(GalNAc β1→4)GlcNAcβ1→3GalNAc-ol. Enzymatic elimination of either the non-reducing terminal α-Ga1NAc or the non-reducing terminal α-Ga1NAc led to inactivation of the allergenic pentasaccharitol. Accordingly, it is possible that the allergenic saccharitol contains two disaccharide units as the allergy-specific epitopes, one GalNAcα1→2Fucα1→ and the other GlicNAcβ1→4GlcNAcβ1→.

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Escherichia coli cells harboring an altered Qβ RNA replicase which has amino acid substitutions of the glycine residue at position 357 in the conserved sequence Tyr³⁵⁶-G1y³⁵⁷- Asp³⁵⁸-Asp³⁵⁹ of the β-subunit protein lost the replicase activity but interfered with proliferation of Qβphage [Inokuchi and Hirashima (1987) J. Virol. 61, 3946-3949]. To examine the mechanism of the interference, we further analyzed various mutants lacking the carboxy-terminal region of the β-subunit protein. The cells expressing the β-subunit gene with up to 17% deletion from the carboxy-terminus of the protein prevented the proliferation of Qβphage. However, in the case that the deletion extended beyond 25% from the carboxy-terminus, the cells showed no interference. In addition, when the interference took place, the phage coat protein synthesis was inhibited. These results indicate that the region between amino acids 440 and 487 of the β-subunit protein is involved in the interference and suggest that the defective replicase inhibits the phage coat protein synthesis by competing with the ribosomes at the initiation site of the coat gene.

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The molecular heterogeneity and tissue specificity of crustacean tropomyosin were inves-tigated, using muscle and nonmuscle tissues from the crayfish, Cambarus clarki. In muscle, three types of tropomyosin isoforms were found on two-dimensional gel electrophoresis. One of them was specific to cardiac muscle, and the other two were shared by skeletal and visceral muscles. In nonmuscle tissues, four types of isoforms were found on two-dimensional gel electrophoresis and in immunoreplica tests using an antiserum against crayfish skeletal muscle tropomyosin. Two of them were common to the muscle isoforms, but the other two were not detected in muscles. Furthermore, nonmuscle tissues contained several peculiar isoforms, the electrophoretic mobilities of which were consider-ably higher than those of the other isoforms mentioned above. When tropomyosin was purified from the mid-gut gland, these isoforms with high mobilities were found in the crude tropomyosin preparation. These results showed that the crayfish tropomyosin was heterogeneous and that the isoforms were distributed in a tissue-specific manner, like vertebrate tropomyosin. However, the results did not coincide with those of our previous study on horseshoe crab tropomyosin, which showed molecular heterogeneity but no tissue specificity. In view of the difference in the isoform distributions between the two major groups (Crustacea and Merostomata) of Arthropoda, the significance of the tissue specificity of tropomyosin isoforms was discussed.

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Guinea pig polymorphonuclear leukocytes (PMNs) possess two distinct types of Fcγ receptor (FcγR): Fc γ_1/γ₂R for both IgG1 and IgG2, and Fcγ₂R, for IgG2 alone. The Fc γ₂R was previously shown to differ antigenically from homologous macrophage (Mφ) Fcγ₂R by the use of a monoclonal antibody to Mφ Fcγ₂R (VIIAI IgG1), though the Fcγ_1/γ₂R cross-reacts with a monoclonal antibody to homologous Mφ Fcγ_1/γ₂R (VIA2 IgG1). Recent-ly, we obtained a monoclonal antibody (MP-2) secreted by a hybridoma prepared by fusion of the splenic cells of mice immunized with guinea pig PMNs with a myeloma cell line. This antibody completely inhibited both the Fcγ₂R-mediated rosette formation of PMNs with IgG2 antibody-sensitized sheep erythrocytes and the Fcγ₂R-mediated binding of oval-bumin (OA)-complexed IgG2 antibody to PMNs. When the antigen of MP-2 was isolated by affinity chromatography with the antibody-Sepharose, it gave a single band with a molecular weight of 120, 000 on SDS-PAGE. The number of antigen molecules per PMN was estimated to be 9×10⁴ by measuring the binding of ¹²⁵I-MP-2 Fab. This value was essentially the same as that obtained by measuring the binding of OA-complexed IgG2 antibody to the PMNs treated with the Fab' of VIA2 IgGl. These results strongly suggest that MP-2 is a monoclonal antibody to PMN Fcγ₂R.

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Recombinant human single-chain urokinase-type plasminogen activator (suc-PA) (SM0: wild type) and its variants resistant to plasmin and/or thrombin (SM1: Lys¹³⁵ to Gln; SM3: Phe¹⁵⁷ to Asp; and SM4: Lys¹³⁵ to Gln and Phe¹⁵⁷ to Asp) have been constructed by site-directed mutagenesis with the aim of producing more efficient thrombolytic agents [Miyake, T. et al. (1988) J. Biochem. 104, 643-647]. In the present study, we characterized the recombinant variant scu-PAs expressed in Escherichia coli. They appeared to have structural integrity because their heat-stabilities, immunological reactivities, and circular dichroism spectra were essentially identical to those of each other and of native scu-PA (nscu-PA). In the presence of thrombin, SM3 and SM4 showed efficient clot lysis by all of the assays used, compared with SM0, SM1, and nscu-PA. While in the absence of thrombin, when measured by a fibrin plate method in a purified system, SM3 and SM4 had lower specific activities than SM0, SM1, and nscu-PA, because of their catalytic constants for conversion to the two-chain form (tcu-PA) by plasmin are lower. However, SM4 lysed clots as efficiently as SM0 in plasma by retaining the single-chain form, whereas SM0 was partly converted to the two-chain form.

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Rabbit α-l-antiproteinases S and F were treated with trypsin, chymotrypsin, Staphylococcus aureus protease V8, and thermolysin, and the liberated peptides encompassing the reactive region of the respective inhibitors were separated and sequenced. The reactive center of the F form was methionine, and the residues from P₃ to P'₁ (11e-Pro-Met-Ser) were the same as those of human α-1-antiproteinase. The S form, on the other hand, was found to be a mixture of two distinct proteins (S-1 and S-2), and their reactive centers (P₁-P'₁) were Ser-Ser and Tyr-Ser, respectively. Seven out of 17 amino acids in the F form and 7 out of 16 in the S-1 form were the same as the corresponding residues of human α-1-antiproteinase, while 5 of 10 residues in the S-2 form were the same as those of the human inhibitor. Ten out of 16 residues were the same between the F and the S-1 forms, whereas the sequence P₁ to P'₃ of the S-2 form (Tyr-Ser-Met-Pro) was the same as the corresponding residues of mouse α-l-antiproteinase.

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Both Escherichia coli and Streptococcus faecalis grew in an alkaline medium containing 100 μM carbonyl cyanide m-chlorophenylhydrazone (CCCP). Our data suggested that CCCP functioned as a protonophore at a high pH and that the proton-motive force was dissipated almost completely under such conditions. The pH gradient measured with dimethyl-oxazolidine-2, 4-dione and acetylsalicylic acid was very small in both bacteria at a high pH above 8, and was not affected significantly by the addition of CCCP. Based on these findings, we propose that the maintenance of neutral cytoplasmic pH is not obligatory for the growth of E. coli and S. faecalis in an alkaline medium.

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We previously reported a method of high-performance liquid chromatography-fast atom bombardment mass spectrometry (HPLC/FAB/MS) for the structural characterization of molecular species of GlcCer and IV³ βGal-Gb₄Cer [M. Suzuki et al. (1989) J. Biochem. 105, 829-833]. In this paper, we report a modification of this HPLC/FAB/MS method, which was used for the separation and characterization of neutral glycosphingolipids (G1cCer, LacCer, Gb₃Cer, Gb₄Cer, and IV³ αGalNAc-Gb₄Cer) and monosialogangliosides [GM3(NeuAc or NeuGc), GM2 (NeuAc or NeuGc), and GM1 (NeuAc or NeuGc)]. Mixtures of the purified neutral glycolipids and monosialogangliosides were subjected to HPLC on a silica gel column, with programmed elution with isopropanol-n-hexane-water, with or without ammonium hydroxide. In order to obtain mass spectra and mass chromatograms of individual components, effluent from the HPLC column was mixed with a methanol solution of triethanolamine, which was used as the matrix for the FAB ionization, and one-thirtieth of the effluent mixture was introduced into a mass spectrometer through a frit interface. A mixture of the five neutral glycolipids, 5 μg of each, gave five peaks on a mass chromatogram obtained by monitoring of the corresponding major pseudo-molecular ions. A mixture of the six monosialogangliosides, 5 μg of each, gave six peaks on a mass chromatogram obtained by monitoring of the major pseudo-molecular ions, indicating that GM3, GM2, and GM1 were clearly separated, and that separation due to differences in sialic
acid species was also achieved. In the mass spectra of the neutral glycolipids and monosialogangliosides, pseudo-molecular ions and fragment ions due to the elimination of sugar moieties were clearly detected. We applied this method to the analysis of a mixture of neutral glycolipids isolated from human erythrocytes and that of a mixture of monosialogangliosides isolated from the liver of a mouse, GlcCer, LacCer, Gb₃Cer, Lc₃Cer, Gb₄Cer, and nLc₄ Cer and GM3(NeuAc), GM3(NeuGc), GM2(NeuGc), and GM(NeuGc) being detected, respectively. This method is of great advantage for the structural characterization of neutral glycolipids and monosialogangliosides, especially when they are in mixtures and/or in small amounts. The method will also be quite promising for other neutral glycolipids and gangliosides with the establishment of suitable separation conditions on HPLC.

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To determine the role of cAMP-dependent protein kinase in the exocytosis of amylase from rat parotid acini, I studied the effects of various cAMP analogues on amylase release and protein phosphorylation in saponin-permeabilized parotid cells. The dose-dependent responses of amylase release and protein phosphorylation evoked by cAMP, dibutyryl-cAMP, and 8-chlorophenylthio-cAMP were closely correlated. Furthermore, when the permeabilized cells were incubated with a combination of site-selective cAMP analogues, such as N⁶-benzoyl-cAMP plus 8-thiomethyl-cAMP (RII-directed) or N⁶-benzoyl-cAMP plus 8-aminohexylamino-cAMP (RI-directed), synergistic stimulation of amylase release was clearly observed. As reported previously [T. Takuma (1988) Biochem. J. 256, 867-871], however, the protein phosphorylation evoked by 8-chlorophenylthio-cAMP was markedly inhibited by H-8 without a significant decrease in amylase release. These results suggest that cAMP-dependent protein kinase is involved in the regulation of amylase exocytosis, although the role of its catalytic subunit is still uncertain.

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We reported the polymorphic expression of GL-Y (IV⁶β[Ga1β1-4(Fucα1-3)G1cNAc]-Gb₅Cer in kidneys of inbred strains of mice in previous papers [J. Biochem. 101, 553-562 and 563-568 (1987)]. DBA/2 mice express a large amount of GL-X (Gb₅Cer), but not GL-Y, in their kidneys, because of a defect on a single autosomal gene (Gsl-5). This suggested that DBA/2 mice lack the ability to transfer GIcNAc onto the C-6 position of GaINAc of Gb₅ Cer or GL-X. In this study, we characterized UDP-G1cNAc:GL-X β1-6N-acetylglucosaminyl-transferase (β1-6GleNAc transferase) in the microsomal fraction of mouse kidney. Maximum activity was detected with an incubation mixture containing sodium cacodylate buffer (pH 6.4), 0.1% Zwittergent 3-16 and 1mM EDTA. Divalent cations were not required. The apparent K_m values for UDP-GlcNAc and GL-X were 0.42 and 0.12mM, respectively. The product of the enzymatic reaction was identified as IV⁶βGlcNAc-Gb₅ Cer by means of ¹H-NMR spectroscopy and permethylation analyses. Then, we measured the β1-6GicNAc transferase activity in the microsomal fractions of kidneys of inbred strains of mice and progeny obtained on mating. WHT/Ht, C57BL/10, BALB/c, and C3H/He mice, which express GL-Y in their kidneys, exhibited detectable amounts of activity, whereas CBA and DBA/2 mice, which do not express GL-Y, did not exhibit detectable activity. Analyses of the glycolipid expression and β1-6GleNAc transferase activity in the kidneys of backcross mice obtained on mating between (WHT/Ht×DBA/2)F₁ and DBA/2 mice indicated that the expression of GL-Y essentially requires the expression of the transferase activity. Thus, we conclude that Gsl-5 controls the expression of GL-Y through regulation of the β1-6G1cNAc transferase activity, and consequently DBA/2 mice cannot express IV⁶βGlcNAc-Gb₅ Cer and IV⁶β(Galβ1-4GlcNAc)-Gb₅ Cer either.

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We previously reported the binding specificities of two anti-ganglioside GD2 murine monoclonal antibodies (MAbs), A1-425 and A1-267, both of which are of IgG3 isotype. A1-425 reacts specifically with ganglioside GD2, whereas A1-267 binds preferentially to GD2 but also reacts with GD3 [Tai, T., Kawashima, I., Tada, N., & Dairiki, K. (1988) J. Biochem. 103, 682-687]. In this paper, they were used for comparative analyses of antibody-mediated cytotoxicity, i.e., antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) against human melanoma and neuroblastoma cell lines. Melanoma cells were found to contain GD2 and/or GD3, whereas neuroblastoma cells expressed only GD2. Both antibodies induced high levels of ADCC and CDC to GD2/ GD3-positive cells with human peripheral large granular lymphocytes (LGL) as effector cells and in the presence of human serum, respectively. A good correlation was obtained between the contents of disialogangliosides and the binding level of the antibodies; both melanoma and neuroblastoma cells with larger amounts of GD2/GD3 showed a higher level of antibody binding than did the cells with a smaller amount of GD2/GD3. Surprisingly, ADCC did not correlate well with the binding level of the antibodies. Thus, A1-425 showed stronger lytic activity than A1-267 in spite of the binding level of A1-425 being similar to or lower than that of A1-267 on the cell surfaces. Antigen-antibody complexes composed of GD2 and A1-425 showed higher binding levels to LGL than complexes of GD2 and A1-267. In contrast, free MAb molecules gave minimum binding to LGL. An anti-human Fc-receptors (III) MAb specifically inhibited both the binding of the antigen-antibody complex to LGL and the ADCC by the MAbs with LGL. These findings demonstrate that MAbs having high binding levels to Fcreceptors (III), as well as having specificities towards multiple ganglioside antigens, possess the strongest cytotoxicity against human tumor cells in ADCC.

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Random mutations were introduced into the B. subtilis glutamine synthetase gene by using nitrous acid, and a high temperature-sensitive mutant was selected. DNA sequencing of the restriction fragment containing the mutation revealed a single base-pair change resulting in the substitution of Leu 318 with Phe. The mutant enzyme was purified, and its kinetic and physical properties were characterized. The Mg²⁺-dependent activity and Mg²⁺ plus Mn²⁺-dependent activity of the mutant were less than 5% of those of the wild-type at 37°C, and these activities decreased above 15°C, whereas the Mn²⁺ dependent activity was nearly normal. Affinity of the mutant enzyme for glutamate was extremely decreased although the K_m values for NH₃ or ATP were almost the same as those of the wild-type. The mutant enzyme was more susceptible than the wild-type enzyme to digestion with chymotrypsin in the presence of glutamate, ATP, and Mg²⁺, although addition of glutamate, ATP, and Mn²⁺ completely protected both enzymes. These results and circular dichroism analyses suggested that Leu 318 is at the glutamate-binding site and that the substitution of Leu 318 for Phe reduces the ability of the enzyme to form the enzyme-substrate complex, probably supported by Mg²⁺.

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3-Ketosteroid-Δ¹-dehydrogenase from Nocardia corallina catalyzes transhydrogenation of 3-keto-4-ene-steroid to 3-keto-1, 4-diene-steroid e.g., progesterone to 1, 4-androstadiene-3, 17-dione. The reaction proceeded linearly at first and then soon slowed down owing to equilibration. The turnover number of this reaction was of the same magnitude as that of the dehydrogenation of 3-keto-4-ene-steroid. The pH optimum was 8.4, which is lower than that of the dehydrogenase reaction. The enzyme has a wide specificity for hydrogen acceptor steroids. The K_m' and K_max' values for these steroids and the values of the corresponding 3-keto-4-ene-steroids were compared. Kinetic studies of the steroid transhydrogenase reaction demonstrated a typical ping-pong mechanism. The enzyme oxidized 1, 2-tritiated progesterone and transferred the tritium atoms to the reaction product, 4-androstene-3, 17-dione, and water. Transhydrogenation in D₂O resulted in the incorporation of a deuterium atom into the C2-position of 4-androstene-3, 17-dione. The results indicate that the enzyme catalyzes C1, C2-trans axial abstraction of hydrogen atoms from progesterone, transfer of the 1α-hydrogen to the C1-position of 1, 4-androstadiene-3, 17-dione and release of the 2β-hydrogen to water. Reaction schemes based on the experimental results are proposed. The enzyme also catalyzes the reduction of 3-keto-1, 4-diene-steroids with reduced benzyl viologen.

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Sapecin is an antibacterial protein purified from the culture medium of NIH-Sape-4, an embryonic cell line of Sarcophaga peregrina [Matsuyama, K. & Natori, S. (1988) J. Biol. Chem. 236, 17112-17116]. As this protein inhibited the growth of Gram-positive bacteria better than that of Gram-negative bacteria, we studied its mode of action with special reference to its effects on S. aureus and Escherichia coli. Results showed that sapecin had high affinity for cardiolipin, which is a major phospholipid of S. aureus. Moreover, a mutant of E. coli with a defect in cardiolipin synthesis was more resistant to sapecin than wild type E. coli, suggesting that cardiolipin is a target for sapecin. Lipopolysaccharide of E. coli was also found to be a barrier for the antibacterial activity of sapecin.

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The complete amino acid sequence of rat kidney ornithine aminotransferase [EC 2.6.1.13] is presented. The 404-residue sequence was determined by analysis of peptides generated by digestion of the S-carboxyamidomethylated protein with CNBr, Achromobacter protease I, arginylendopeptidase, or Staphylococcus aureus V8 protease. Mueckler and Pitot have reported the amino acid sequence of the rat liver enzyme (440 residues) as predicted from the nucleotide sequence of the cDNA [Mueckler, M. M. & Pitot, H. C. (1985) J. Biol. Chem. 260, 12993-12997]. The amino acid sequence of the rat kidney enzyme presented herein coincides with residue 36 (Gly) through 440 (Phe) of the predicted precursor protein, indicating that the liver and kidney enzymes are identical, and that the enzyme is processed at the amino-terminal region after translation.

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The complete amino acid sequence and the location of disulfide bonds of a lectin from Japanese frog (Rana japonica) eggs, which specifically agglutinates transformed cells, are presented. The sequence was determined by analysis of peptides generated by digestion of the S-carboxyamidomethylated protein with Achromobacter protease I, or chymotrypsin, and by chemical cleavage with BNPS-skatole or cyanogen bromide. The lectin is a single-chain protein consisting of 111 residues, with a pyroglutamyl residue at the amino terminus. Four disulfide bonds link half-cystinyl residue 19 to 72, 34 to 82, 52 to 97, and 94 to 111. The sequence and the location of the disulfide bonds are highly homologous to those of bull frog (Rana catesbeiana) egg S-lectin. They are also homologous to human an-giogenin, a tumor angiogenesis factor, and a family of pancreatic ribonucleases.

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