Rat erythrocytes contained ganglio-series gangliosides, GM1, fucosyl GM1, and GDla, in a high concentration. The concentrations of GM1, fucosyl GM1, and GDIa in rat erythrocyte ghosts were 889.0 nmol, 470.6 nmol, and 462.0 nmol per g dry weight, respectively, and the molar ratio of lipid-bound sialic acid, cholesterol and lipid-bound phosphorus was 3.1:73.9:100.0. The reactions of fucosyl GM1 and GM1 on rat erythrocytes with rabbit anti-fucosyl GM1 and anti-GM1 antisera were measured by means of haemolysis in the presence of complement and a binding assay of antibodies with a FACS after staining erythrocytes by the indirect membrane immunofluorescence technique. When measured by ELISA, antifucosyl GM1 antiserum was found to react almost exclusively with fucosyl GM1 with a slight cross-reaction with GM1, but anti-GM1 antiserum cross-reacted to a significant extent with asialo GM1. Rat erythrocytes were haemolyzed specifically with anti-fucosyl GM1 antiserum, but not with antisera to GM1, asialo GM1, asialo GM2, Forssman and globoside, and the haemolysis was proved to be definitely caused by the specific recognition of fucosyl GM1 on rat erythrocytes by anti-fucosyl GM1 antibody according to the haemolysis inhibition reaction using various glycosphingolipid-containing liposomes as inhibitors. In addition, although the binding of anti-fucosyl GM1 antibody on rat erythrocytes was clearly demonstrated with a FACS, anti-GM1 antibody did not bind. The observations that the haemolysis of rat erythrocytes and the binding of antibody to rat erythrocytes were found only with anti-fucosyl GM1 antiserum, and not with anti-GM1 antiserum, but that nevertheless the titer of anti-GM1 antiserum was higher than that of anti-fucosyl GM1 antiserum and GM1 on rat erythrocytes was more abundant in concentration than fucosyl GM1, seem to be a matter of great importance in assessing the specificity of anti-ganglioside antibody and the surface distribution of gangliosides on the cell.

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A simple and reproducible method for the determination of serum pseudo-cholines-terase activity was developed by making use of a stable substrate, p-hydroxyben-zoylcholine, with p-hydroxybenzoate hydroxylase as a linked enzyme. The method is based on spectrophotometric measurement of the decrease of NADPH. p-Hydroxybenzoate released from p-hydroxybenzoylcholine is hydroxylated by the action of p-hydroxybenzoate hydroxylase in the presence of NADPH and O₂ to produce 3, 4-dihydroxybenzoate and NADP⁺. This method is superior to the conventional methods in that this substrate is extremely stable up to pH 9.0, which is close to the optimum pH for the assay (pH 8.0). Serum interference was resolved by the use of p-hydroxybenzoate hydroxylase as a linked enzyme. The K_m value of pseudo-cholinesterase for p-hydroxybenzoylcholine is 1×10^-5 M. The results of our method and Garry's method (Clin. Chem. 11, 91-96, 1965) correlated well (r=0.962). The within-run and between-run C. V. values were 2.1 and 2.7, respectively.

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The presence of an endopeptidase hydrolyzing succinyl trialanine-p-nitroanilide [Suc(Ala)₃-pNA] to Suc(Ala)₂ and Ala-pNA in human kidney and its partial characterization have been reported (Ishida et al. (1981) Biochem. Int. 3, 239-246).
This neutral metallo-endopeptidase was separated into two fractions (A and B) on Sephacryl S-300 and fraction B was further purified to an electrophoretically pure state.
The fraction B enzyme had a molecular weight of 100, 000 and was inhibited by metal chelators such as EDTA, o-phenanthroline and phosphoramidon, but not by serine protease inhibitors. The enzyme was found to hydrolyze peptide bonds preferentially at the amino sides of hydrophobic amino acids such as Leu and Phe, when its specificity was studied using insulin B chain and angiotensin I. Fraction A seems to be a tetramer of fraction B, judging from its molecular weight, pI, substrate specificity and immunological properties.

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The 20, 000 dalton light chain (L2) was isolated from rabbit and chicken striated muscle myosins, and the Ca²⁺-induced conformational changes of these proteins were investigated. 1) The reaction of thiol groups of L2 with dithiobisnitrobenzoic acid (DTNB), 2) measurements of the UV difference absorption spectrum, 3) measurements of Stokes radius (R^s) by gel filtration and 4) measurements of the ESR spectrum of L2 whose cysteine or tyrosine residues were spin-labeled were used for the structural studies. The effect of Ca²⁺ on phosphorylated L2 was also investigated.
The long axis of chicken L2 was calculated as 136A from the Stokes radius, suggesting that the L2 is an asymmetrical molecule. After the addition of Ca²⁺ the long axis was reduced to 104 A. The same effect of Ca²⁺ has been reported with rabbit L2 (Alexis, N. M. & Glatzer, W. B. (1978) Biochemistry 17, 2319-2325). Besides this large shape change, the addition of Ca²⁺ to L2 induced environmental changes around tyrosine residues and also changes in the reactivity of cysteine residues with DTNB. Ca²⁺ is supposed to be bound to the N-terminal region of the molecule, while the tyrosine and cysteine residues are located at the C-terminal region, which is probably sterically remote from the N-terminal region. The reason for the remote effect of Ca²⁺ may be related to the structural rigidity of the L2 molecule. The functions of two properties of L2, Ca²⁺-binding and phosphorylation, are discussed in relation to muscle contraction.

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The physicochemical and immunochemical properties of a thereto-labile antigen (TLA a) which is located on the cell surface of Saccharomyces cerevisiae were studied. The sedimentation constant (S20, w) and molecular weight (sedimentation equilibrium method) were 6.26S and 68, 800, respectively. The circular dichroic (CD) spectrum of TLA a had negative maxima at 210 and 221 nm, indicating the presence of α-structure of a polypeptide chain. The molar ratio of antibody to antigen which gave maximum precipitation was 2.7. Approximately 50% of the antigenic activity of heat-denatured TLA a was recovered when denatured molecules were dissolved in 6 M guanidine hydrochloride followed by 25-fold dilution with H₂O. The amount of TLA a existing on the yeast cell surface was estimated to be 37.5 μg per 10.5 mg of fresh cells, corresponding to 0.36% by weight of the fresh yeast.

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Ovomucoid-trypsin association complex was prepared by incubating chicken egg white ovomucoid with bovine trypsin. The reactivity of ovomucoid-trypsin complex was investigated by immunodiffusion, quantitative precipitation and enzymelinked immunosorbent assay (ELISA). It was demonstrated that the association of trypsin with ovomucoid hindered the binding of the specific antibody at some antigenic sites of ovomucoid by lowering the antibody-binding affinity of these sites. The anti-ovomucoid antiserum was absorbed with ovomucoid-trypsin complex, and non-absorbed antibody was collected by immunoaffinity chromatography of ovomu-coid-coupled Sepharose 4B. The antibody blocked the trypsin-inhibitory activity of ovomucoid in a molar ratio (antibody/ovomucoid) of about 1.2:1. The findings suggested that at least one antigenic site is located near the reactive site of trypsin inhibition (Arg⁸⁹ Ala⁹⁰) of ovomucoid.

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The aromatic region of the NMR spectrum of bovine pancreatic ribonuclease A was analyzed in order to clarify the nature of the microenvironments surrounding the individual histidine, tyrosine, and phenylalanine residues and the interactions with inhibitors. The NMR titration curves of ring protons of six tyrosine and three phenylalanine residues as well as four histidine residues were determined at 37°C between pH 1.5 and pH 11.5 under various conditions. The titration curves were analyzed on the basis of a scheme of a simple proton dissociation sequence and the most probable values were obtained for the macroscopic pK values and intrinsic chemical shifts. The microenvironments surrounding the residues and the effects of inhibitors are discussed on the basis of these results.
Based on the titration curves of ring protons, the six tyrosine residues were classified into the following four groups: (1) titratable and different chemical shifts for C(δ) and C(ε) protons (two tyrosine residues), (2) titratable but similar chemical hifts for C(δ) and C(ε) protons (two tyrosine residues), (3) not titratable and different chemical shifts for C(δ) and C(ε) protons (one tyrosine residues), and (4) not titratable and similar chemical shifts for C(δ) and C(ε) protons (one tyrosine residue). The resonance signals of ring protons were tentatively assigned to tyrosine and phenylalanine residues.
The NMR titration curves of His-48 ring protons were continuous in solution containing 0.2 M sodium acetate but were discontinuous in solution containing 0.3 M NaCl because the NMR signals disappeared at pH values between 5 and 6.5. The effects of addition of formate, acetate, propionate, and ethanol were investigated in order to elucidate the mechanism of the continuity of the titration curves of His-48 in the presence of acetate ion. The NMR signal of His-48 C(2) protons was observed at pH 6 in the presence of acetate and propionate ions but was not observed in the presence of formate ion or ethanol. This indicated that both the alkyl chain and the anionic carboxylate group are necessary for the continuity of the titration curves of His-48 ring protons. Based on the results, the mechanism of the effects of acetate ion is discussed.

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In order to investigate the role of carboxyl groups of a base non-specific ribonuclease from Aspergillus saitoi [EC 3. 1. 27. 1] (RNase M, molecular weight 36, 000), the modification of RNase M with a water-soluble carbodiimide, 1-cyclohexyl-3- (2-morpholinyl- (4) -ethyl)carbodiimide (CMC), was studied. The inactivation of RNase M proceeded almost linearly with the incorporation of about 9.5 CMC moieties. The peptide backbone structure of the modified RNase M was practically the same as that of the native RNase M, as assessed from the CD spectra in the region of 200-250 nm. In the presence of competitive inhibitors, adenosine, and cytidine, inactivation of RNase M by CMC was partially inhibited. In the presence of cytidine (1M), the modification of about 4 carboxyl groups of RNase M proceeded with a slight loss of enzymatic activity (ca. 20%). Further modification inactivated RNase M with the incorporation of ca. 4-5 CMC without any detectable intramolecular peptide bond formation. Therefore, it was concluded that carboxyl groups responsible for enzymatic activity were included among these carboxyl groups protected by cytidine.
The logarithm of the half-live of the inactivation of RNase M by CMC was a linear function of log[CMC] with a slope of minus one, indicating that at least one carboxyl group among the modified ones may be essential for catalysis. The digestion of CMC-modified RNase M with carboxypeptidase A eliminated the carboxyl terminal group from the site of CMC modification.

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An endoribonuclease existing as a complex with inhibitor in the cytosol of rat liver has been purified about 128, 000-fold after inactivation of the inhibitor with CdCl₂. The enzyme had a molecular weight of 16, 000 and produced 3'-CMP via 2', 3'-cyclic phosphate of cytidine from poly (C). The breakdown of poly (U) by the enzyme was less than 5% of poly (C) breakdown. Poly (A) was not hydrolyzed by the enzyme. The enzyme had a pH optimum of 7.5-8, was heat-stable and had a K_m of 952 μg yeast RNA and a K_m of 198 μg poly (C) per ml. The maximal velocities for yeast RNA and poly (C) degradation were 3, 970 A₂₆₀/min/mg protein and 1, 890 A₂₆₀/min/mg protein, respectively. The enzyme was slightly stimulated by polyamines or monovalent and divalent cations except Mn²⁺, but was inhibited by nucleoside triphosphate, poly (G) and rat liver RNase inhibitor. Inhibition of the enzyme by rat liver RNase inhibitor was not prevented by monovalent and divalent cations or polyamines, although inhibition by poly (G) was prevented by these ions.

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Dissociation of highly purified EF-lαβγ (a high molecular weight form of polypeptide chain elongation factor-1) from pig liver into EF-1α and EF-1βγ at various temperatures was examined and the following results were obtained. (i) When dissociation of EF-1αβγ was analyzed by gel filtration with Sephacryl S-200, it was found that in the absence of GTP, it did not dissociate at any temperature between 4 and 37°C, whereas in the presence of GTP, it tended to dissociate with elevation of the temperature, and almost complete dissociation was observed at 32°C. This indicated that the dissociation constant of EF-1αβγ into EF-1α and EF-1βγ in the presence of GTP increased with increase in the temperature. (ii) When gel filtration was performed in the presence of both GTP and [¹⁴C] Phe-tRNA, the formation of a ternary complex of EF-1α•GTP•[¹⁴C] Phe-tRNA from EF-1αβγ was noted, and its amount was found to increase with elevation of the temperature. (iii) The amount of [¹⁴C] Phe-tRNA bound to ribosomes dependent on added EF-1αβγ similarly increased with increase in the temperature, as in the case of ternary complex formation, whereas the binding of [¹⁴C] Phe-tRNA to ribosomes dependent on free EF-1α proceeded fairly well even at 0°C. From these results we concluded that among the reaction steps in the binding of [¹⁴C] Phe-tRNA to ribosomes dependent on EF-1αβγ, dissociation of EF-1αβγ to form EF-1α•GTPandEF-1βγ in the presence of GTP is the step which is strongly influenced by temperature.

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1. Methyl hydroperoxy-epoxy-octadecenoate (ML-X) inhibited state 3 respiration of rat heart and liver mitochondria with glutamate and malate as substrates. It also inhibited the NADH oxidase and NADH-ubiquinone-1 reductase activities of rat heart and liver submitochondrial particles (SMP).
2. In liver mitochondria and SMP, these inhibitory effects of ML-X were transient, whereas in heart mitochondria and SMP, recovery of the respiratory activities did not occur.
3. Results from high pressure liquid chromatography and ¹³C-NMR studies indicated that ML-X was converted to hydroperoxy-epoxy-octadecenoic acid (L-X) by incubation with liver mitochondria or SMP but not with heart mitochondria.
4. Purified L-X apparently inhibited state 3 respiration of heart and liver mitochondria with glutamate and malate as substrates, but the amount required for 50 inhibition was 2-3 times larger than that of ML-X.
5. Heart mitochondria adsorbed 36% of the added ML-X, while only 10% of the added L-X was adsorbed.
6. These findings suggest that the recovery of the ML-X-inhibited respiratory activities of liver mitochondria and SMP occurs by conversion of ML-X to L-X, which is only weakly adsorbable on the mitochondrial membrane.

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The role of polyamine in the proliferation of cultured mouse L cells was investigated using DL-α-hydrazino-δ-aminovaleric acid (DL-HAVA), a potent and competitive inhibitor of ornithine decarboxylase [EC 4.1.1.17].
When confluent mouse L cells were reseeded, the intracellular concentration of polyamines increased sharply, and the maximal levels of putrescine, spermidine, and spermine were 3.3, 2.2, and 1.8 times their initial values, respectively, one or two days after inoculation. DL-HAVA produced prompt depletion of the intracellular putrescine and spermidine contents and a further increase of the spermine level to 30-90% more than that of the control throughout the experiment. The total level of the three polyamines was reduced to a great extent in DL-HAVA-treated cells. Concomitant with the disappearance of the two polyamines, cell proliferation, measured as the total cell number and DNA accumulation, was greatly suppressed by the inhibitor. Addition of exogenous putrescine or spermidine with or after DL-HAVA restored the inhibited cell growth in a dose-dependent manner. Putrescine administered to inhibitor-treated cultures was rapidly incorporated into the cells and effectively converted to spermidine. Addition of spermidine to the culture medium also normalized the intracellular spermidine content, but the putrescine level remained unchanged. Neither cadaverine nor 1, 3-diaminopropane, structural analogs of putrescine, overcame the inhibition under the same conditions.
Thymidine kinase [EC 2. 7. 1. 21] activity and the pools of triphosphates of thymidine and deoxyadenosine were appreciably reduced in DL-HAVA-treated cells, whereas DNA polymerase [EC 2. 7. 7. 7] activity was not changed significantly.
These findings suggest that spermidine might play essential roles in the metabolism of deoxyribonucleoside triphosphates and growth of mouse L cells in culture. Recently, striking increases in the activities of the enzymes of polyamine biosynthesis, ornithine decarboxylase [EC 4. 1. 1. 17] and S-adenosylmethionine decarboxylase [EC 4.1.1.50], and enhanced cellular levels of polyamines have been observed in a variety of mammalian cells when they are induced to proliferate (reviewed in Refs. 1 and 2). With use of inhibitors of the polyamine biosynthetic pathway, considerable evidence has been accumulated that elevated levels of polyamines are necessary for growth and DNA synthesis of various tissues in vivo and of cultured cells (reviewed in Ref. 2, 3-10).
Previously, we showed that DL-α-hydrazino-δ-aminovaleric acid (DL-HAVA), a competitive inhibitor of ornithine decarboxylase, depressed growth and DNA synthesis by reducing the cellular putrescine level in isoproterenol-stimulated mouse parotid glands (11), mouse sarcoma-180 (12), ethylphenylpropiolate-treated mouse skin (13), regenerating rat liver (3), and cultured mouse mammary explants (6). Administration of putrescine with DL-HAVA greatly reduced the inhibitory effect of DL-HAVA in all these cases. However, the effectiveness of this inhibitor on cultured mammalian cells has not yet been described. Recently, Hölttä et al. (9) reported the antiproliferative effect of DL-HAVA on phytohemagglu-tinin-activated human lymphocytes and found that this inhibition was not prevented by adding low concentrations of polyamines to the cultures.
In the present work, we tested the effect of DL-HAVA on mouse L cells in culture and found that DL-HAVA greatly reduced the growth of mouse L cells and that this inhibition was reversed by exogenous putrescine and spermidine.

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Cyclohexadextrin and maltose bound to soybean β-amylase and affected the environments of tryptophan and tyrosine residues, producing characteristic difference spectra in the ultraviolet region. The difference spectrum produced by cyclohexadextrin, a competitive inhibitor, had peaks at 285, 292, and 299 nm, while that by maltose, a reaction product, had peaks at 285 and 292 nm and a small trough at around 300 nm. By using the peaks at 292 and 299 nm, the dissociation constants of enzyme-cyclohexadextrin and enzyme-maltose complexes were calculated to be 0.35mM and 8.1mM, respectively. The effects of modification of SH groups of β-amylase on the interaction of the enzyme with these sugars were examined by using β-amylase carboxymethylated at the SH1 site and the enzyme modified at SH1 and SH2 sites with iodoacetamide or with 5, 5'-dithiobis- (2-nitrobenzoic acid) (DTNB). The dissociation constants of the enzyme-cyclohexadextrin and enzymemaltose complexes were not changed by the modification of these SH groups, but the modification of SH2, the so-called essential SH group of soybean β-amylase, strongly affected the difference spectra produced by maltose. The spectropho-tometric titration of β-amylase by cyclohexadextrin in the presence of maltose showed that cyclohexadextrin and maltose bind to the enzyme competitively, regardless of the modification of SH2. These results indicated that SH2 is located near the binding site of cyclohexadextrin and maltose, but is not involved in the binding of these sugars.

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The experimental time-courses of eight avian lysozymes, seven hen-type lysozymes and one goose-type lysozyme, were measured with a substrate of chitopentaose (GlcNAc)₅ at pH 5.0 and 50°C. Chitooligosaccharides in the reaction mixture were analyzed by high-performance gel-filtration. From the experimental timecourses, the overall reaction rates represented by the disappearance of the initial substrate and the values of reaction parameters were estimated by computer analysis. With taking hen lysozyme as the reference, the values of reaction parameters estimated were correlated to the replaced amino acid residue in the binding site of the lysozyme, and the roles of some amino acid residues in the binding site were discussed.

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A new chromogenic substrate, N^α-benzoyl p-guanidino-L-phenylalanine p-nitroanilide (Bz-GPA-pNA), was synthesized. This compound is a good substrate for bovine trypsin (K_m =1.56×10^-5M, k_cat=0.081s^-1, at pH 8.2) and was hydrolyzed as fast as N^α-benzoyl-L-arginine p-nitroanilide (Bz-Arg-pNA) with much the same k_cat/K_m values. But the values are two orders of magnitude smaller than those for ester substrates, N^α-benzoyl p-guanidine-L-phenylalanine ethyl ester (Bz-GPA-OEt) and N'-benzoyl-L-arginine ethyl ester (Bz-Arg-OEt). Substrate activation behavior was observed on tryptic hydrolysis of this new substrate in a substrate concentration range higher than about 5.0×10^-4M in analogy with the trypsin-Bz-Arg-pNA system.

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Tryptophan residues in thermolysin (3 Trpjmolecule) and in its specific inhibitor, talopeptin (1 Trp/molecule), were modified with N-bromosuccinimide (NBS). The decrease in the absorption at 280 nm and the fluorescence intensity above 310 nm (excited at 280 nm) accompanying the modification were followed by the stoppedflow method as a function of time. When the sole tryptophan residue of talopeptin was modified with NBS, its inhibitory activity against thermolysin was almost completely destroyed. For thermolysin, the decrease in molar absorptivity corresponds to the modification of one of its three tryptophan residues, and the enzyme activity does not decrease significantly with the modification (remaining activity was 96% at [NBS]/[E]=6). The results obtained for the modification of EI complex suggested that the formation of El complex remarkably reduces the rate constant for the modification of the tryptophan residue in talopeptin, but does not affect that of the tryptophan residue (s) in thermolysin.

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Human pancreatic phospholipase A₂ was purified to homogeneity from pancreatic juice and a reliable radioimmunoassay for the enzyme was developed. The molecular weight of the enzyme as estimated by sodium dodecyl sulfate polyacrylamide gel electrophoresis was 14, 000. Phosphatidylcholine was hydrolyzed well in an alkaline pH range, and the optimum activity was obtained at pH 9. Calcium ion was indispensable for activity. The enzyme was stable to heat treatment at 60°C for 5min.
The radioimmunoassay system was highly sensitive, reproducible and specific. The dilution curves for the sera of patients with acute pancreatitis were parallel to the standard curve. In healthy individuals, serum phospholipase A₂ concentrations ranged from 2.0 to 7.9 ng/ml, the average being 5.1 ng/ml (S. D.:1.7). In patients with acute pancreatitis, significant elevations of serum phospholipase A₂ contents were observed, and the highest value found was 4, 000 ng/ml.

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Warburganal, a unique dialdehyde sesquiterpene isolated from East African Warburgia plants, showed a strong antifungal activity. However, this growth inhibition in Saccharomyces cerevisiae was reversed with L-cysteine. In addition, warburganal inhibited the alcoholic fermentation of S. cerevisiae while L-cysteine reversed this inhibiton. When alcohol dehydrogenase, a sulfhydryl enzyme, was incubated with warburganal, the enzyme activity decreased with time. The decrease was more rapid at alkaline pH. L-Cysteine prevented this enzyme inhibition by warburganal but could not restore the enzyme activity lost already due to warburganal. Warburganal lost its characteristic ultraviolet absorption spectrum in the presence of L-cysteine. The change in absorbance was favored at alkaline pH, indicating Michael reaction type addition of L-cysteine to warburganal. Based on these observations, a variety of physiological activities due to warburganal appear to result from its irreversible reactivity with sulfhydryl groups.

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The orientation of thyroid peroxidase in hog thyroid microsomes was studied by trypsin treatment, gel filtration, binding to Concanavalin A Sepharose and iodination of thyroglobulin. Trypsin treatment of microsomes did not solubilize the thyroid peroxidase activity completely but solubilized the NADPH-cytochrome c reductase activity almost completely. The apparent molecular size of thyroid peroxidase was not altered by trypsin treatment of microsomes. It was, however, decreased by the same treatment of deoxycholate-treated microsomes. On the other hand, the apparent molecular size of NADPH-cytochrome c reductase was reduced by trypsin without prior deoxycholate treatment. Thyroid peroxidase of microsomes did not bind to Concanavalin A Sepharose. Thyroglobulin added exogenously was not iodinated by microsomes, but endogenous thyroglobulin, which had been associated with microsomes, was iodinated. Similar results were obtained with rough microsomal membranes prepared from crude microsomes by sucrose density gradient centrifugation. These results suggest that thyroid peroxidase is oriented toward the luminal side of the microsomal vesicles.

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Bovine liver tRNA was adsorbed on silicone-coated porous glass in 5M NaCl, 10mM Tris-HCI (pH 7.6) and fractionated by elution with decreasing NaCI concentrations. tRNA^Pro, tRNA^Val, tRNA^Ile, tRNA^Thr, ttRNA^Ser, and tRNA^Phe were eluted in this order. tRNA which had been digested with ribonuclease A was not adsorbed. Qβ RNA (adsorbed onto the glass in 5M NaCl) was eluted with 1.5M NaCl. RNA species in a crude rRNA fraction from Escherichia coli were separated into tRNA, 5S rRNA, and high molecular weight rRNA on siliconized porous glass. A half of calf thymus DNA was adsorbed on the glass in 5M NaCl and the residual part passed through the column. The CD spectra showed that DNA and tRNA took the C-form and the A-form in 5M NaCl, respectively. Therefore, the discrepancies of behavior of the DNA and RNA on siliconized porous glass may be related to the occurrence of these forms. The recovery of these nucleic acids from the column was 83-100%. Adsorption chromatography on siliconized porous glass may be a useful method for the separation of tRNA, rRNA, and mRNA.

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A tropomyosin has been purified from porcine kidney and its properties compared with those of rabbit skeletal muscle tropomyosin. Kidney tropomyosin was separated from contaminating vascular smooth muscle tropomyosin by hydroxylapatite chromatography. Kidney tropomyosin resembles tropomyosin from other nonmuscle cells in regard to subunit size, mobility on SDS-polyacrylamide gels in the presence and absence of 6M urea, amino acid composition and morphology. The binding of tropomyosin to F-actin is strongly dependent on the Mg²⁺ concentration. With kidney tropomyosin, binding begins at 1mM Mg²⁺ and is complete at about 4-5mM, while with muscle tropomyosin, binding is initiated at 1mM Mg²⁺ and reaches saturation at 2-3mM Mg²⁺. Both kidney and muscle tropomyosins bind to actin in a similar ratio of 1 tropomyosin/6-7 actin monomers at saturation. Both kidney and skeletal muscle tropomyosins prevent “severing” of F-actin filaments induced by gelsolin/villin-like protein purified from kidney. These results suggest that, in non-muscle cells, tropomyosin may protect microfilaments from Ca²⁺-dependent solation at the site where they may interact with myosin.

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When rat liver mitochondria were incubated with L-[³⁵S]cystine or L-[¹⁴C]cystine in the presence of γ-cystathionase [EC 4.4.1.1], the mitochondrial proteins incorporated ³⁵S, but not ¹⁴C, showing that only the S-atom was incorporated from L-cystine into the mitochondrial proteins by the action of γ-cystathionase.
The incorporation of ³⁵S into the mitochondrial proteins was not effectively inhibited by dilution with the cold sulfide ion which was added to the reaction mixture at a concentration 5 times higher than that of L-[³⁵S] cystine used as a substrate. Therefore, the S-atom of L-cystine may not be incorporated into mitochondrial proteins through the sulfide ion produced by γ-cystathionase.
The S-atom incorporated into mitochondrial proteins was fairly stable under acidic conditions, but was released as sulfide by incubating the proteins with thiols such as dithiothreitol. The ³⁵S-labeled mitochondrial proteins were digested by pronase (nonspecific proteinase), and a ³⁵S-labeled compound obtained in the hydrolysate was identified as cystine trisulfide by Dowex 50 column chromatography and high-voltage paper electrophoresis. It was assumed, therefore, that the ³⁵S-atom of [³⁵S] cystine is incorporated to form a cystine trisulfide structure with two cysteine residues in the mitochondrial protein.
The subfractionation of the ³⁵S-labeled mitochondria was performed by using digitonin and lubrol, revealing that the outer membranous, intermembranous space and inner membranous proteins were highly labeled.
The mitochondrial protein incorporating the S-atom from L-cystine was capable of converting the inactive form of δ-aminolevulinate synthetase of Rhodopseudomonas spheroides to the active form. Therefore, the mechanism of this activation reaction seems to be the same as that of the “regulator” protein found in R. spheroides cells.

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Further structural features of the sulfated polysaccharide-peptidoglycan complex, which is produced by an Arthrobacter sp. and contains phosphorus as its minor component, were investigated. Phosphoric acid esters such as D-glucose 6-phos-phate, glycerol 1-phosphate and muramic acid phosphate were isolated from the acid hydrolysate of the complex. On mild acid treatment, the complex became positive for both the Morgan-Elson reaction and acid phosphatase digestion. The mild acid hydrolysate readily formed the Morgan-Elson chromogen on heating at pH 7, indicating release of terminal reducing N-acetylglucosamine substituted on C-3 by adjacent sugars, and its release was accompanied by that of phosphomonoester. The complex released peptidoglycan fragments on the mild acid treatment, together with acid-degraded, sulfated polysaccharide chains with terminal reducing N-acetylglucosamine. A large proportion of phosphorus in the complex was shown to occur in the sulfated polysaccharide chains, and the rest as muramic acid phosphate in the peptidoglycan fragments. After mild acid treatment of the complex, 50% of total phosphorus was released as inorganic phosphate on phosphatase digestion of the hydrolysate.
These results suggest that the sulfated polysaccharide chains, which are additionally phosphorylated to a low degree, are linked to the peptidoglycan fragments through acid-labile phosphodiester linkages, probably between (1-3)-linked N-acetylglucosamine 1-phosphate and muramic acid.

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The effects of sugars and polyols on the thermal denaturation temperature, T_m, of acid-soluble collagen from calf skin were studied at pH 4.0 under atmospheric pressure as well as high pressures up to 4, 000 atm. Addition of these compounds invariably raised T_m with increases in their concentration over the whole range of pressure. The extent of stabilization by different sugars and polyols is discussed in terms of their different influences on the structure of water. The hydroxymethyl chain length of polyols and equatorial OH groups of the sugars were found to be decisive factors for their stabilizing effect on collagen structure. The similarity in their stabilizing effects on collagen and globular proteins suggests that our stabilization mechanism proposed for globular proteins can be essentially extended to fibrous proteins: such protein stabilization would be dominantly mediated through a preferential hydration of protein, originating in the water-structure-making character of sugars and polyols.

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Isolation of linkage-region glycopeptides from corneal peptidokeratan sulfate was attempted under mild conditions. Peptidokeratan sulfate, which had been found in advance of the present study to contain three mannose residues per chain as a major component of the carbohydrate-protein linkage region, was digested with Pseudomonas endo-β-galactosidase. The disaccharide-repeating chain was partially hydrolyzed, and almost all the galactose and N-acetylglucosamine residues were found in oligosaccharides of various sizes. The resulting linkage region-enriched glycopeptides were separated by gel filtration from these oligosaccharides and then fractionated by DEAE-cellulose and Dowex 50 chromatography with the guidance of the mannose content. The glycopeptides obtained were highly enriched in the linkage region and a large portion of them was free from sulfate groups, suggesting that they could be used to elucidate the structure of the linkage region.

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In order to elucidate the chemical structure of the carbohydrate-protein linkage region of bovine corneal keratan sulfate, glycopeptides which had been highly enriched in the linkage region under mild conditions were subjected to glycosidase digestion in combination with methylation analysis. After removal of the peripheral N-acetylglucosamine and galactose residues by exhaustive glycosidase digestion, the residual glycopeptide (GP-A) was found to contain 0.6 mol of fucose, 3 mol of mannose, and 2 mol of N-acetylglucosamine per mol. GP-A was then serially digested with exoglycosidases, and the released sugars and the composition of the residual glycopeptides isolated by gel filtration were analyzed by gas-liquid chromatography. Further, the dansyl derivative of GP-A was digested with endo-β-N-acetylglucosaminidase D and the products were analyzed to explore the location of the fucose residue. The results obtained, combined with those from methylation analysis of all the glycopeptides, revealed that the carbohydrate structure of the linkage region and its environs of corneal keratan sulfate is as follows.
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Bindings of cobra venom phospholipases A₂ to micelles of n-hexadecylphosphoryl-choline were studied by the tryptophyl fluorescence method at 25°C and ionic strength 0.1. The data were analyzed by assuming that the micellar surface has multiple binding sites for the enzyme and these sites are identical and mutually independent. The enzyme binding site was found to accommodate a constant number of substrate (monomer) molecules, N=10, 5 or 13 for N. naja atra apoenzyme and its Ca²⁺ complex, and N. naja kaouthia apoenzyme, respectively. The binding constant of the enzymes to the micelle, K_mic=0.18-3.1×10⁶ M^-1, was 9-160 times greater than that to the monomeric substrate, K_mon=2×10⁴ M^-1 (Teshima et al. (1981) J. Biochem. 89, 1163-1174). This was interpreted in terms of the presence of an additional substrate-binding site in the enzyme molecule. The binding constant of the enzyme-Ca²⁺ complex to the micelle was smaller than that for the apoenzyme over a wide range of pH.
The pH dependence of the binding constant of the apoenzyme to the micelle was well interpreted in terms of pK shifts of two ionizable groups from 5.4 to 5.53 and 7.55 to 7.95. The pH dependence curve for the Ca²⁺ complex, which lacked the former transition, was interpreted in terms of the pK shift of only a single ionizable group from 7.25 to 7.55. The former ionizable group was assigned as Asp 49, to which Ca²⁺ can coordinate, and the latter as His 48 in the active site on the basis of the reported pK values of these ionizable groups in the apoenzyme and Ca²⁺ complex (Teshima et al. (1981) J. Biochem. 89, 13-20 and Teshima et al. (1982) J. Biochem. 91, 1777-1788). No participation of the a-amino group with a pK value of 8.55 was observed.

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High performance liquid chromatography was performed by an ion-pair reversedphase method of six standard unsaturated disaccharides derived from heparan sulfate and heparin. Separation of ΔDi-GlcNAc, ΔDi-GlcN (2S), ΔDi-GlcNAc (6S), ΔDi-GlcN (2, 6- or 2, 2'-diS) and ΔDi-GlcN(2, 6, 2'-triS) was achieved on a column of Jasco SC-02 with 10mM tetrabutylammonium phosphate (pH 7.0) containing 30 or 47% methanol as a mobile phase. ΔDi-GlcN (2, 6-diS) and ΔDi-GlcN (2, 2'-diS) were separated on the same column with 35mM triethylamine phosphate (pH 5.3). Four preparations (BL-1.0-1, BL-1.0-2, BL-1.0-3, and BL-1.25-1) separated from crude bovine lung heparan sulfate, a standard bovine lung heparan sulfate (BL-ST), bovine kidney heparan sulfate 1.0M Fr and 1.25M Fr (BK-1.0 and BK-1.25), and porcine kidney heparan sulfate 1.0M Fr (PK-1.0) were digested with a mixture of heparinase, and heparitinases 1 and 2. The resulting foregoing unsaturated disaccharides in the digests were analyzed by the above HPLC procedures. The proportions of the unsaturated disaccharides in the digests of BL-1.25-1 and BL-ST were similar, but those of the others differed from each other. It is noteworthy that ΔDi-GlcNAc plus ΔDi-GlcNAc (6S) in the digest of BL-1.0-1 was approxi-mately 95% of the total unsaturated disaccharides. Small amounts of ΔDi-GlcN (2, 6, 2'-triS) were found in all the samples. It was found that ΔDi-GlcN (2, 2'-diS) was a prominent component in the disulfated unsaturated disaccharides from BL-1.25-1 and BK-1.25.

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The nucleotide sequence of a cloned section of the Escherichia coli chromosome containing the promoter regions of the malB divergent operons was determined. The region of the proximal gene, malE of the malEFG operon, was identified on the basis of the known amino acid sequence of the precursor molecule of maltosebinding protein. The region of malK, the proximal gene of the malKlamB operon, was deduced from the observation that a cloned segment contains an aminoterminal portion of the malK gene. The non-coding region between malE and malK is 299 base pairs long and contains two long GC clusters. Another feature of this region that may be related to the regulation of gene expression is the presence of two palindromic structures between the GC clusters.
The DNA regions binding to cyclic AMP binding protein were determined by a method using polyacrylamide gel electrophoresis. The sites are thought to be located close to GC clusters.

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Structural elucidation, including molecular weight, carbohydrate sequence and molecular species of the ceramide portion, of gangliosides and asialo gangliosides from bovine brain, was successfully performed by negative ion fast atom bombardment mass spectrometry (NEG-FAB-MS). Ceramide monohexoside, ceramide dihexoside, asialo GM2 (GA2) and asialo GM1 (GAl), all of which were prepared from bovine brain gangliosides by treatment with 1 M formic acid and monosialo-gangliosides, GM3, GM2, and GM1, were analyzed without any derivatization by NEG-FAB-MS. They clearly gave the intensive molecular ion species, (M-H)-, and the fragment ions cleaved at glycosidic linkage sequentially from the nonreducing end with or without the ceramide portion. The spectra were quite simple, easily obtained without expansion of ion intensity, and extremely useful for the structural elucidation of underivatized glycosphingolipids.

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The stopped flow and flash photolysis methods were applied for the kinetic study of the reaction of carbon monoxide with myoglobin in solution, in an amorphous state, and in crystals. From the flash photolysis data, the reactivity of myoglobin was concluded to be essentially the same in all three states. When the reaction was started with a stopped flow apparatus, the rate became slower as the state of myoglobin was changed from solution to amorphous precipitate, and to crystals. The bigger the crystals, the slower the reaction became. Therefore, the change of the rate could be explained in terms of a diffusion layer formed on the crystals.
In the reaction of crystalline myoglobin, the effective concentration of CO was increased locally by about 20 μM after flash photolysis and approached the bulk concentration during the reaction. In contrast, in the reaction of amorphous precipitate of myoglobin, such an increase in the CO concentration was apparently dispersed homogeneously just after flash photolysis.

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Porcine liver aminopeptidase was inactivated by various sulfhydryl-reactive reagents, whose inactivation rates were in the order: p-chloromercuribenzoate (PCMB)>HgCl²>2, 2'-dithiodipyridine>5, 5'-dithiobis (2-nitrobenzoic acid) (DTNB). The processes of inactivation by these reagents did not follow pseudo-first-order kinetics, and prolonged incubation did not alter the level of maximum inactivation. The substrates provided no protection against the inactivation by DTNB, and the numbers of sulfhydryl groups titrated with the reagent were not influenced by the presence or absence of puromycin (a competitive inhibitor). The modification of sulfhydryl groups caused a slight increase in the K_m value for the enzyme and a significant decrease of the V_max value. There are two ionizable groups (pK_e 6.2; 7.8 and pK_es, 6.0; 7.8) in the catalytic action of the enzyme. From the pK₁ vs. pH profile of inhibition with PCMB, the pK value of 7.8 does not correspond to the ionization of a sulfhydryl group. The thiol-modified enzyme was activated by cobalt ion, as was the native enzyme (Kawata, S., et al. (1982) J. Biochem. 92, 1093-1101). But in contrast with the native enzyme, the thiol-modified enzyme was activated about 2.5-fold and the maximum activation remained almost constant during prolonged incubation with cobalt ion. These results suggest that the sulfhydryl groups of the enzyme are located apart from the binding site of cobalt ion and do not participate directly in the catalytic process.

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The interactions of putrescine, the major diamine in E. coli, with E. coli DNA as a model of phage DNA were studied by melting temperature analysis, equilibrium dialysis and X-ray diffraction with the aid of molecular model building.
1. The chemical analysis of the DNA-putrescine complex shows that the molar binding ratio of putrescine to DNA (phosphate) is nearly 1 to 2.
2. The equilibrium (or reversible) binding of putrescine to DNA was suggested by the fact that the melting temperature increased according to the concentration of added putrescine, and its elevation was not saturated even at the molar ratio of 6 to 1.
3. The equilibrium dialysis experiments indicate that the association constant for the complex is a little smaller than, but in the same order (10³ liter/mol) as, that of DNA-spermine complex.
4. The binding of putrescine stabilizes the B-form of DNA fiber, which is well preserved even at 66% relative humidity.
5. The distance between the neighboring DNA helices in the wet fiber increased with the increasing degree of hydration, as in the case of native DNA.
6. Unlike spermine, putrescine does not form precipitate upon mixing with DNA in the concentration range for UV measurement, suggesting that the cross-bridge formed by putrescine is intra-double helical.
The equilibrium binding of putrescine to DNA, seems to be important for the life cycle of λ-phage.

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ATPase inhibitor protein, which blocks mitochondrial ATPase activity by forming an enzyme-inhibitor complex, was found to be synthesized as a larger precursor in a cell-free translation system directed by yeast mRNA. Other protein factors, which stabilize latent ATPase by binding to the enzyme-inhibitor complex, were also found to be formed as larger precursors. The precursor of ATPase inhibitor protein was transported into isolated yeast mitochondria and was cleaved to the mature peptide in the mitochondria. Impaired mitochondria lacking phospho-rylation activity could not convert the precursor to the mature form. Neither antimycin A nor oligomycin alone exhibited a marked effect on the transport-processing of the precursor by intact mitochondria. However, when antimycin A was added with oligomycin, the transport-processing was markedly inhibited. The processing was also strongly inhibited by an uncoupler, carbonylcyanide p-trifluoro-methoxyphenyl hydrazone. The inhibition by the uncoupler was not relieved by ATP added externally. It is concluded that the transport-processing of precursor proteins requires intact mitochondria with a potential difference across the inner membrane.

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Messenger RNA coding mitochondrial ATPase inhibitor protein, a small peptide comprised of 63 amino acid residues, was separated from a large quantity of mRNAs of larger molecules by high speed gel permeation chromatography. Messenger RNA coding a small stabilizing factor of inactivated F₁F₀-ATPase complex, which is also comprised of 63 amino acids, was recovered in the same fraction as the ATPase inhibitor, whereas mRNA for a large stabilizing factor with an apparent molecular weight of 15, 000 was recovered in a fraction of slightly larger molecules. ATPase inhibitor precursor labeled with various kinds of radioactive amino acids was prepared separately by cell-free translation with the purified mRNA, and the amino terminal sequence of the precursor was examined. It was demonstrated that an extra peptide of 21 amino acid residues, including 5 leucine, 4 serine, 1 glycine, and 1 methionine residues, is located at the amino terminus of the ATPase inhibitor precursor.

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Crystal structure analysis of a [2Fe-2S] ferredoxin I from Aphanothece sacrum, a blue green alga, was carried out at 2.5 Å resolution. The phase angle of each reflection was determined by the single isomorphous replacement method coupled with the anomalous dispersion effect for the uranium derivative. The four molecules in an asymmetric unit were clearly seen in a 3.9 Å electron density map. The main chains of three molecules were traced at 2.5 Å resolution. The structure of the remaining one molecule, however, remains unknown because of the poor electron density of the corresponding region. The three main chain folds exhibit the same topology as that in Spirulina platensis. The structural similarity between A. sacrum and S. platensis ferredoxins, whose amino acid sequences are different from each other by about 30%, strongly suggests that all plant-type ferredoxins have the same main chain fold.

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To clarify the role of gangliosides in the morphological and biochemical differentiation of neuronal cell cultures, the model cell culture system represented by two neuroblastoma cell lines, GOTO and NB-1, which were established from adrenal gland and metastatic neck lymph node, respectively, was examined. We found that the total ganglioside fraction from human brain had two remarkable effects on these cell lines, which are similar to those of nerve growth factor (NGF): (a) an increase in the cell number, and (b) an increase in the neurite number and the total length of neurites. In these cases, the genuine effector in total gangliosides could not be ascribed to a possibly contaminating NGF-like protein, but rather to a particular molecular species of the gangliosides, GQ1b, which could completely replace the effector function not only qualitatively but also quantitatively. Our results provide direct evidence for the participation of gangliosides in such functions.

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Two different hybrid myosins were obtained by combining “desensitized” myosin (DM) of Akazara scallop striated adductor with rabbit skeletal DTNB-light chains (DTNB-LC) and with chicken gizzard regulatory light chains (GR-LC). Using the two hybrid myosins, the following were found: (a) DTNB-LC has an inhibitory effect on the Mg-ATPase activities of Akazara DM and acto-DM both in the absence of calcium and in its presence. (b) DTNB-LC also has an enhancing effect on the superprecipitation activity of acto-DM. (c) The Mg-ATPase activities of DM and acto-DM are made sensitive to calcium by GR-LC, regardless of whether GR-LC is phosphorylated or unphosphorylated. (d) However, the Mg-ATPase activity of acto-myosin hybridized with phosphorylated GR-LC is definitely higher than that of acto-myosin hybridized with unphosphorylated GR-LC.

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Experimental acute degenerative changes in skeletal muscle accompanied by a drastic increase in cathepsins B&L were induced in rats by intramuscular injection of a local anesthetic, bupivacaine. Cathepsins B&L have been implicated in the rapid disappearance of muscle fibers. Degenerating muscle showed a spotty fluorescence when stained with antibodies against cathepsin B, indicating that the increased cathepsin B did not originate from the muscle itself, but from invading phagocytes. We report here results showing that cathepsin B of nonmuscle cell origin is involved in the breakdown of myofibrillar proteins in acute bupivacaineinduced muscle degeneration.

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The amino acid sequence of a stabilizing factor, 9, 000 dalton protein, of protontranslocating ATPase (F₁F₀-ATPase) -inhibitor complex isolated from yeast mitochondria was established. It was highly homologous with that of yeast intrinsic ATPase inhibitor and several structural characteristics were noted. They were compared with those of E. coli ε and bovine δ subunits to point out the similarity among them.

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In contrast to high molecular weight forms of elongation factor 1 (EF-1_H) from animal sources which contain three subunits, EF-la, EF-lb, and EF-1c, EF-1_H from wheat embryo consisted of four subunits, EF-la, EF-lb, EF-lb', and EF-1c, in an equimolar ratio. The molecular weights of EF-1a, EF-1b, EF-1b', and EF-1c from wheat embryo were 52, 000, 29, 000, 28, 000, and 48, 000, respectively. In the animal system, EF-1a and EF-1b correspond functionally to EF-Tu and EF-Ts, respectively. In the wheat system, however, both EF-1b and EF-1b' had the EF-Ts-like activity to stimulate EF-1a-dependent binding of aminoacyl-tRNA to ribosomes. EF-1b and EF-1b' from wheat embryo gave 21 and 20 tryptic peptides, respectively. Twenty peptides were common.

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Calcium ions inhibited perfringolysin O-induced hemolysis at a concentration lower than 1mM, but not the hemolysis by digitonin at 10mM. The introduction of calcium ions into ghosts inhibited the lysis more strongly than the addition of calcium ions outside ghosts. When erythrocytes were treated with perfringolysin O in the presence of 1mM CaCl containing ⁴⁵CaCl₂, the radioactivities inside cells rapidly increased during incubation. On the other hand, when perfringolysin O-treated erythrocytes were incubated in a calcium-free medium, the erythrocytes released calcium ions at a 3.3-fold higher rate than untreated cells. These results suggested that perfringolysin O accelerated both the calcium influx into and efflux from erythrocytes.

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A new method for purification of anti-glycosphingolipid antibodies has been devel-oped. N-Glycolylneuraminyl (α2-3) lactosylceramide [hematoside (NeuGc)] could be hydrophobically bound on octyl-Sepharose 4B in the presence of 0.1M KCl. The Sepharose gel coated with hematoside (NeuGc) was used as immunoadsorbent for affinity column chromatography to purify avian anti-hematoside (NeuGc) antibody. The procedure is very simple, reproducible and applicable to purification of almost all anti-glycosphingolipid antibodies. The glycosphingolipid used for the affinity chromatography could be recovered without any destruction by successive extraction of the gel with methanol and methanol/chloroform (1:2, v/v).

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