The self-association of subunits of D-amino acid oxidase holoenzyme was studied by high-speed gel filtration with a short column of TSK-GEL G3000 SW in 0.1M sodium pyrophosphate (pH 8.3) at 25°C. Over the range of the peak concentrations of 0.009-4.45mg/ml in the presence of FAD the apparent Stokes radii increased with an increase of the concentrations and did not level off. The largest value obtained in this study was 61.5 Å. This would correspond to that calculated for the hexamer with linear subunit arrangement which has the largest Stokes radius among the various arrangements. These results provide the first gel chromatographic evidence that the higher polymers greater than the dimer participate in the self-associating system of the enzyme.

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Wild mice are divided into two groups with different ganglioside compositions in the liver. Most Japanese and a few Chinese wild mice have GM2 (NeuGc) as a major ganglioside, whereas all wild mice caught at other places distributed all over the world other than Japan and China express GM1 (NeuGc) and GD1a (NeuGc) in addition to GM2 (NeuGC). We recently reported that inbred strains of laboratory mice were also grouped into the same two types based on the ganglioside composition in the liver, and that the expression of GM1 (NeuGc) and GD1a (NeuGc) was regulated by a gene located at the left outside the H-2 complex on chromosome 17 (Hashimoto, Y., Suzuki, A., Yamakawa, T., Miyashita, N., & Moriwaki, K. (1983) J. Biochem. 94, 2049-2054). The present study suggests that oriental wild mice would be a donor of a defective gene for expression of GM1 (NeuGc) and GD1a (NeuGc) in mice of laboratory stocks which are commonly used for biochemical and immunological studies, such as C57BL/6, C57BL/10, BALB/c, DBA/2, C3H/He, and CBA mice.

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The holoenzyme of D-glucosaminate dehydratase [EC 4. 2. 1. 26] from Agrobacteriwn radiobacter showed absorption peaks at 280 and 415 nm with a shoulder in the region of 320 to 330 nm. The treatment of the enzyme with hydroxylamine followed by dialysis led to disappearance of both the absorption peak at 415 nm and the shoulder, giving the apoenzyme.
The fluorescence excitation maximum of the holoenzyme was at 320 nm with a shoulder at 420 nm (emission at 510 nm), and the emission maxima were at 420 nm with a shoulder at 370 nm (excitation at 320 nm) and at 510 nm (excitation at 420 nm). The holoenzyme showed a negative circular dichroic band at 418 nm and a positive shoulder at around 320 nm.
Reduction of the holoenzyme with sodium borohydride caused a loss of the absorption peak at 415 nm with a concomitant increase of 325 nm absorbance and an irreversible loss of the activity. The occurrence of ε-N-pyridoxyllysine in the acid hydrolysate of the reduced enzyme showed that D-glucosaminate dehydratase contains a catalytically essential lysine residue whose ε-amino group binds the 4-formyl group of pyridoxal 5'-phosphate to form a Schiff base.
The plots of absorption of the apoenzyme against the amount of pyridoxal 5'-phosphate added showed that four and two molar equivalents of the cofactor bind to the apoenzyme and subunit, respectively. The biphasic nature of the spectrometric titration curve of the apoenzyme with pyridoxal 5'-phosphate and the two K_m values obtained for the cofactor suggest the occurrence of two distinct types of binding sites for pyridoxal 5'-phosphate in the enzyme.

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To deepen our understanding of Ca²⁺-activation in calmodulin-dependent reactions, the properties of Ca binding to calmodulin were examined by dual-wavelength spectrophotometry using a Ca indicator, tetramethylmurexide.
1. The Scatchard plot for Ca binding to calmodulin was linear, indicating the occurrence of homogeneous independent binding sites.
2. The apparent binding constant of calmodulin for Ca²⁺ (K) was (1.96±0.05)×10⁵M^-1 (20), and the number of binding sites (n) was 3.36±0.04 (20) mol/mol in reaction medium containing 100mM KCl, 20mM MOPS-KOH (pH 6.80), and 0.12mM tetramethylmurexide at 20°C.
3. K was strongly dependent on the concentration of KCI, while n was independent of it. On the assumption that this is due to the effect of ionic strength (I), K may be described as follows: log K=6.73 -3.2 [2√I/(1+√I)-0.4×I] (0.006≤I≤0.256, pH 6.80, at 20°C).
4. Mg²⁺ decreased K, which could be expressed as K/(1+130 [Mg]) ([Mg] in mol (M)) at the ionic strength of 0.106, pH 6.80 at 20°C. n was also decreased in the presence of Mg²⁺. This suggests that Mg²⁺ may have effects other than simple competition.
5. The pH of the medium affected K or n very little at around neutral pH. This may correspond to the fact that calmodulin is an acidic protein with pI_??_4.3.
6. K or n was only slightly dependent on the temperature. This corresponds to the calorimetric result that the enthalpy change on Ca binding was very slight and endothermic.
Based on these findings, much of the discrepancy among the results already reported on Ca-binding can be explained. Activation of a calmodulin-dependent reaction is discussed on the basis of Ca binding to calmodulin under physiological conditions.

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It has previously been reported that abnormally enlarged high density lipoproteins (HDL) appear in rats with extrahepatic cholestasis induced by ligation of the common bile duct. To see whether similar changes in HDL occur in intrahepatic cholestasis in rats, we studied HDL alterations in rats treated with α-naphthyliso-thiocyanate (ANIT), which is known to produce a cholestatic response in rats similar to intrahepatic cholestasis in man. Findings were obtained which indicated changes in HDL similar to those in bile duct-ligated rat serum: HDL from ANIT-treated rats were separated into two subfractions, enlarged particles and smaller ones, on Bio-Gel A5m column chromatography. In electron micrographs, the two subfractions appeared spherical and the diameters of the enlarged particles and the other ones were 15.0±2.6 nm and 11.5±2.2 nm, respectively. Both subfractions showed slow α-mobility in agarose gel electrophoresis. The enlarged HDL had apoE as their major apoprotein, while apoA-I was the major apoprotein in the other HDL subfraction. The enlarged HDL contained less protein and more cholesterol than the other HDL subfraction. The two HDL subfractions were also separated b) heparin-Sepharose affinity chromatography.

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The mechanism of hormonal induction of serine dehydratase [EC 4. 2. 1. 13, SDH] was studied in primary cultures of adult rat hepatocytes by measuring the rates of syntheses of the enzyme protein and its translatable mRNA. The rate of synthesis of enzyme protein, measured as incorporation of [³H] leueine into the enzyme protein in hepatocytes, was increased 4-5 times by dexamethasone (Dex) plus glucagon. Neither hormone alone increased the rate. The increased rate induced by the two hormones was suppressed by insulin and epinephrine. The decay curves of [³H] -leucine-labeled SDH showed that these hormones did not affect the rate of enzyme degradation. The level of translatable mRNA was determined by measuring cell-free synthesis of SDH in a reticulocyte lysate system. Dex plus glucagon increased the level of mRNA of SDH in hepatocytes. Insulin and epinephrine suppressed this increase without changing the rate of mRNA degradation. The level of mRNA changed in parallel with that of the rate of synthesis of the enzyme protein. These results suggest that these hormones regulate transcription of SDH, rather than its translation.
After pretreatment of hepatocytes with Dex, further addition of glucagon caused more rapid induction of mRNA of SDH than addition of both hormones together. The effect of glucagon after pretreatment with Dex was inhibited by actinomycin D and α-amanitin, suggesting that glucagon does not affect post-tran-scription, but transcription per se. The requirement for both Dex and glucagon for induction of this enzyme is discussed in comparison with the requirements for either hormone alone for inductions of other gluconeogenic enzymes.

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The neutral glycosphingolipids of ova of the fresh-water bivalve, Hyriopsis schlegelii were characterized. The most abundant glycolipid was ceramide monosaccharide, followed by ceramide trisaccharide, ceramide tetrasaccharide, and ceramide disaccharide. More complex neutral glycolipids accounted for almost one-third of the total. The total amount of these glycolipids was 0.59mg/g of dry weight of the ova preparation, a yield which was one-seventh of that of spermatozoa neutral glycolipids. Structural analyses were performed by enzymatic hydrolysis of the glycolipids with exoglycosidases, permethylation experiments, and also immuno-chemical assays. The proposed structures are as follows: ceramide monosac-charides, Gal-Cer and Glc-Cer; ceramide disacharides, Gal (β1-4) Gal-Cer, Gal (β1-4) Glc-Cer, and Man (βl-4) Glc-Cer; ceramide trisaccharide, Man (α1-3) Man (β1-4) Glc-Cer; ceramide tetrasaccharides, Man (αl-3) [Xyl (βl-2)] Man (β1-4) Glc-Cer, GlcNAc (β1-2) Man (αl-3) Man (β1-4) Glc-Cer, Man (α1-3) [Gal (β1-2)] Man (β1-4) Glc-Cer, and Man (αl-2?) Man (α1-3) Man (β1-4) Glc-Cer. The latter two ceramide tetrasaccharides were new types of glycosphingolipids. The spectrum of ova glycolipids appeared to be more complicated than that of the spermatozoa glycolipids. The ova glycolipids characterized here, with the exception of ceramide tetrasaccharides, contained considerable amounts of 2-hydroxy fatty acids, which were not observed in the spermatozoa glycolipids. The major sphingosine base was C₁₈-sphingenine in all the ova glycolipids as well as in the spermatozoa glycolipids. However, the content of anteiso type of sphingosine base was 2- to 3-fold higher in the ova than in the spermatozoa.

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On treatment with 10mM EDTA at 30°C, protein of 18, 000 daltons was released from myofibrils, thin filaments and myosin B prepared from the smooth muscle of an ascidian, Halocynthia roretzi. This protein was purified from the EDTA extract of myofibrils by differential centrifugation, freeze-drying and gel-filtration. Based on its molecular weight, electrophoretic mobilities in the presence and absence of Ca²⁺ and other properties, it was identified as troponin C.
By EDTA treatment, ascidian myosin B lost the Ca²⁺-sensitivity of Mg²⁺-ATPase, and EDTA-treated myosin B recovered the sensitivity by mixing with the EDTA extract of myosin B in the presence of Mg²⁺. Gel-electrophoretic patterns indicated that desensitization and resensitization of ascidian myosin B were accom-panied by the removal and binding of troponin C. These results indicate that ascidian smooth muscle is regulated by a troponin-tropomyosin system, and desensitization induced by EDTA treatment is due to the removal of troponin C but not the release of the light chains of the myosin molecule.
Based on these findings, we have established a simple method for the purification of troponin C from ascidian smooth muscle.

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An acid phosphatase species which is activated by Fe²⁺ was purified 3, 700-fold from rat spleen by chromatography on columns containing Blue-Sepharose, concanavalin A-Sepharose, Sephadex G-100, and CM-Sephadex. The enzyme hydrolyzed aryl phosphates, nucleoside di- and triphosphates, phosphoproteins, and thiamine pyrophosphate with K_m values of 10^-4 to 10^-3 M at an optimal pH of 5.0-5.8. Copurification of the acid phosphatase and acid phosphoprotein phosphatase indicated that they were identical. The purified enzyme was glycoprotein in nature, showing four heterogeneous forms on acid polyacrylamide gel electrophoresis (pI values, 7.8, 8.0, 8.3, and 8.5), but it gave a molecular weight of 33, 000 on sodium dodecyl sulfate-gel electrophoresis and gel permeation chromatography. The enzyme had a purple color (λmax 545 nm) and contained 2 iron atoms per enzyme molecule.
Among reductants, ascorbic acid and Fe²⁺ were the best activators, although their combined effect was not additive. Fe²⁺ and ascorbic acid both changed the purple enzyme into the same active form (λmax 515 nm), giving almost the same kinetic constants for substrates and for inhibitors such as molybdate, phosphate and fluoride. However, low concentrations of Fe²⁺, from 0.01mM to 1.0mM, immediately and reversibly activated the enzyme, whereas high concentrations of ascorbic acid over 1mM were required for maximal activation, which was slow and irreversible.

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A crystallin was isolated from the homogenate of the Squid (Loligo pealii) lens by gel filtration on a Sepharose CL-6B (2.5×170cm) column. Biochemical characterization showed it is a dimeric protein with a molecular weight of (5.1±0.4)×10⁴ and a Stokes' radius of 26Å. Electrophoresis on a cellulose acetate membrane indicated it is a basic protein with an isoelectric point higher than 8.6. High resolution two-dimensional gel in 8M urea/2% NP-40 resolved this crystallin into 6 charge isomers, each with a major subunit of molecular weight 29, 000 daltons and a minor subunit of 27, 000 daltons in a molar ratio of 3:1. The extreme susceptibility of the protein to denaturation and precipitation even at low temperature hampered further characterization of this crystallin under nondenaturing conditions. Amino acid analysis indicated it contains an unusually high content of methionine (12.8 mol%) which may have some bearing on the instability of this crystallin in vitro. Biochemical comparison of the squid crystallin with mammalian lens crystallins shows that it is a crystallin distinguishable from all reported vertebrate lens crystallins. A detailed study of this protein may shed light on the evolution of lens crystallins in general.

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TSK-GEL SW was found to be useful as a packing in high performance liquid chromatography for the separation of double-stranded DNA restriction fragments. DNA fragments smaller than 300 base pairs were separated as discrete peaks depending solely upon difference in chain length. The recovery of DNA fragments was higher than 90%.

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A secretion vector system in Bacillus subtilis was constructed from the α-amylase promoter and signal sequence coding region of an α-amylase hyperproducing strain, B. subtilis NA64, and the major part of the plasmid pTUB4 which was derived from pUB110. When an Escherichia coli β-lactamase gene, lacking its own promoter and signal sequence coding region, was introduced into the secretion vector system, β-lactamase was expressed in B. subtilis. In addition, more than 95% of the enzyme synthesized was secreted into the culture medium via the secretion vector system. Secreted β-lactamase crossreacted with rabbit antiserum raised against the E. coli enzyme.

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Calcium-activated neutral proteases (CANPs) were purified from rabbit skeletal muscle and chicken skeletal muscle, and compared as to their electrophoretic properties, metal requirements, subunit amino acid compositions and immunological cross-reactivities. Two kinds of CANPs (μCANP and mCANP) were isolated from rabbit but the chicken tissue lacked one corresponding to μCANP. They were acidic in the order of chicken mCANP, rabbit mCANP, and rabbit μCANP but the difference between the former two was very small. All of them were composed of two subunits, so-called 80K and 30K subunits. The molecular weight of the 30K subunit was the same for these CANPs (28K) but those of the 80K subunit were different (79K for rabbit μCANP, 75K for rabbit mCANP and 81K for chicken mCANP). The calcium-sensitivity of chicken mCANP was very high when compared with that of rabbit mCANP and close to that of rabbit μCANP. Antisera against chicken CANP and those against rabbit CANP cross-reacted with rabbit CANP and chicken CANP, respectively, when examined by immunoelectrotransfer blot techniques.

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Normal human sera contain heterophile hemagglutinins to rabbit erythrocytes which are different from anti-B isoantibody and other heterophile antibodies such as Hanganutziu-Deicher antibody or Paul-Bunnell antibody. The antigen to this antibody was purified from rabbit erythrocyte stroma, and identified as pentaglycosyl ceramide, Gal (α1-3) Gal(β1-4) GlcNAc (β1-3) Gal (β1-4) Glc-Cer.

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The binding to yeast alcohol dehydrogenase of NAD⁺ and its five derivatives (N⁶-[2-[N-[2-[N-(2-methacrylamidoethyl)carbamoyl]ethyl]carbamoyl]ethyl]-NAD (I), N⁶-[N-[2-[N-(2-methacrylamidoethyl)carbamoyl]ethyl]carbamoylmethyl]-NAD (II), copolymer of I with acrylamide (PA-I), copolymer of II with acrylamide (PA-II), and copolymer of I with N, N-dimethylacrylamide (PDMA-I)) were studied statically and kinetically by the stopped-flow method by using the quenching of the enzyme fluorescence in the presence of pyrazole. Apparent dissociation constants and apparent rate constants were determined therefrom. It was concluded that (1) the N⁶-CH₂CH₂CO group (of I) is effective in making the derivative bind more strongly as well as faster than NAD⁺, while the N⁶-CH₂CO group (of II) is not; and (2) the binding of the polymer derivatives of NAD⁺ to the enzyme is not essentially weaker and slower than that of native NAD⁺, but is even faster in some cases. The coenzymic activities of the above compounds were also determined with yeast alcohol dehydrogenase, pig heart malate dehydrogenase, and rabbit muscle lactate dehydrogenase.

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A high mobility group (HMG) nonhistone protein fraction HMG (1+2), composed of HMG1 and HMG2, was prepared from pig thymus chromatin. In order to examine a possibility that the HMG (1+2) participates in the unwinding of the DNA double-helix, DNA hydrolysis assay systems with the endonucleases specific for single-stranded DNA were employed. In the presence of HMG (1+2), the hydrolysis of double-stranded DNA by N. crassa endonuclease was markedly promoted, while the hydrolysis of single-stranded DNA was hardly enhanced. The reaction kinetic data showed that the stimulation of the hydrolysis of double-stranded DNA in the presence of HMG (1+2) was due to the unwinding of the DNA doublehelix by the HMG(1+2), and not due to stimulation of enzyme activity of the endonuclease by the protein. The unwinding reactions were dependent on the HMG protein concentration at low weight protein to DNA ratios and reached a maximum at the ratio of 0.025. The region unwound in the whole DNA was partial. Similar results were obtained for experiments with nuclease S1. Isolated HMGI and HMG2 fractions showed DNA unwinding activity of similar extents. The association constant obtained by fluorescence quenching analysis showed that the HMG (1+2) has higher affinity to single-stranded DNA than to double-stranded DNA. The susceptibility to the unwinding differed with the DNA source. These results suggest that HMG (1+2) at a low weight protein to DNA ratio binds to some limited doublestranded region in DNA and unwinds the DNA partially.

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RNA-RNA interactions between 18S ribosomal RNA and noncapped influenza cRNA were detected by psoralen photochemical cross-linking in reticulocyte 80S ribosome-cRNA complexes. In vitro transcripts of type A influenza virus synthesized by endogenous RNA polymerase with adenylyl-(3'→5')-guanosine primer formed 80S complexes with rabbit reticulocyte ribosomes. The extent of the complex formation by these noncapped cRNAs was less than that by the m⁷G-capped reovirus in vitro transcripts, but the former RNAs in the 80S complexes were cross-linked to 18S rRNA as efficiently as the latter RNAs by photoreaction with an RNA cross-linking agent, 4'-aminomethyl-4, 5', 8-trimethylpsoralen. These results suggested that mRNA with or without the cap structure on the 5'-terminal can form complexes with the ribosomes in a eukaryotic cell-free translation system by basepair formation with 18S rRNA. Correspondingly, sequences capable of forming extensive base-pairs including four- to five-base complementarities were found between the 3'-terminal of rabbit reticulocyte 18S ribosomal RNA and the 5'-non-coding regions of either influenza virus transcripts or reovirus mRNA.

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A previously found yeast-mitochondrial protein fraction stabilizing the inactivated complex between mitochondria) ATPase and intrinsic ATPase inhibitor (Hashimoto, T., et al. (1983) J. Biochem. 94, 715-720) was separated into two proteins by high performance liquid chromatography on a cation exchanger. The molecular weights of the factors were estimated to be 9, 000 and 15, 000 daltons by sodium dodecyl sulfate (SDS)-gel electrophoresis. Both factors were required to stabilize a complex of inhibitor and proton-translocating ATPase (F₁F₀-ATPase) either in its purified form or in mitochondrial membranes. On the other hand both factors together could not stabilize a complex of the inhibitor and F₁-ATPase, suggesting that both factors act together with the F₀-portion. The factors also facilitated very efficiently the binding of ATPase inhibitor to F₁F₀-ATPase in the presence of ATP and Mg²⁺. Both the 15, 000 and 9, 000 dalton stabilizing factors were hardly distinguishable from δ- and ε-subunit, respectively, on an SDS-gel electrophoregram, but immuno-diffusion assay showed that neither factor was present in the purified F₁-ATPase containing the δ- and ε-subunit.

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Phospholipase B bound tightly to the membrane fraction of baker's yeast (Saccharomyces cerevisiae) was purified approximately 226-fold by extraction with sodium deoxycholate (DOC), acetone precipitation, ammonium sulfate fractionation, and column chromatographies on Phenyl-Sepharose CL-4B, DEAE-Sepharose CL-6B, and Sepharose 4B. Only one major activity peak with an apparent molecular weight of 330, 000 was detected by gel filtration on Sepharose 4B. However, two glycoprotein bands, one major and one minor, were evident on SDS-polyacrylamide gel electrophoresis in the absence of 2-mercaptoethanol. Isoelectric focusing also revealed two activities, pI 3.4 (major) and pt 3.0 (minor). The purified enzyme had phospholipase B activity (in the presence of DOC) and lysophospholipase activity in a ratio of 1:4.8. Both activities had an optimum pH of 3.5-4.0. Phospholipase B activity was appreciably stimulated only by DOC among bile acids, but lysophospholipase activity was markedly inhibited by them. Both activities were not stimulated by Ca²⁺ and inhibited by SDS, Triton X-100, Fe³⁺ and Al³⁺. The purified enzyme hydrolyzed the acyl ester bonds of phosphatidylcholine sequentially, first the 2-acyl and then the 1-acyl groups. It preferentially hydrolyzed 1-acyl-sn-glycero-3-phosphocholine and, to a lesser degree, 2-acyl-sn-glycero-3-phosphocholine.

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The previous study showed that the H2A histone of the protozoan Tetrahymena pyriformis comprises equimolar amounts of two variants, H2A(1) and H2A (2), and their sequences of 137 and 132 residues, respectively, are blocked at the N-terminal and possibly partially modified in the homogeneous N-terminal and heterogeneous C-terminal regions [Fusauchi, Y. & Iwai, K. (1983) J. Biochem. 93, 1487-1497]. Now, the N-terminal blocking group was identified as a-N-acetyl by 1H NMR spectroscopy of the N-terminal blocked serine residue isolated by carboxypeptidase digestion of the N-terminal tryptic peptide (residues 1-5). Two ε-N-acetylated (16 and 14%) lysine residues positioned at 5 and 12, respectively, were found on carboxypeptidase digestion and subsequent amino acid analysis of a blocked N-terminal tryptic peptide (res. 1-8) and on aminopeptidase digestion analysis of another tryptic peptide (res. 11-17), both containing two lysine residues. The C-terminal BrCN fragment (res. 119-137) of H2A (1) containing three, two, one, and no phosphoserine, and that (res. 119-132) of H2A (2) containing two, one, and no phosphoserine were isolated by ion-exchange chromatography and determined by carboxypeptidase digestion analyses. Similar analyses of the tryptic peptides derived from these BrCN fragments showed that H2A (1) was phosphorylated at serine residues 122 (76%), 124 (15%), and 129 (25%); and H2A (2) at serine residues 122 (81%) and 128 (44%). This is the first time that C-terminal phosphorylation has been found in nucleosome-core histones.

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The ganglioside composition of chromaffin granule membranes of bovine adrenal medulla was investigated. Two major gangliosides, which accounted for over 95% of total gangliosides, were characterized as G_M3 (NeuAC) and G_M3 (NeuGc) by thin layer chromatography (TLC) and gas-liquid chromatography (GLC).
Chromaffin granule membranes contained a relatively higher concentration of gangliosides than the membranes of other subcellular organelles; three times higher than microsomal membranes and seven times higher than mitochondrial membranes. This high concentration of ganglioside in chromaffin granule membranes could contribute to the high concentration to the high concentration of gangliosides in the adrenal medulla as compared to other extraneural tissues.

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The effects of nucleotides on the Ca²⁺-gated cation channel in sarcoplasmic reticulum (SR) vesicles were studied by measuring choline influx. The choline influx was measured by following the change in scattered light intensity using the stopped flow technique. ATP enhanced the Ca²⁺-induced choline influx. The activation followed a single-site titration curve with a dissociation constant of 1.0±0.5mM, independent of the Ca²⁺ concentration. ATP seems to increase the pore radius or number of channels without affecting the gating mechanism of the Ca²⁺-gated cation channel. ADP, AMP, and adenine enhanced the choline transport in a manner similar to ATP, but cAMP, ITP, UTP, CTP, and GTP did not. The apparent dissociation constants and the maximal activations were as follows: ATP 1.0mM, 28-fold; ADP 0.9mM, 18-fold; AMP 0.6mM, 7-fold, and adenine 0.4mM, 4-fold. Adenine and AMP behaved as a competitive inhibitor for the activation by ATP. These results are consistent with the Ca²⁺-induced Ca²⁺ release observed in skinned muscle fiber and isolated SR.

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Amino acid sequences of the regulatory light chains of striated adductor muscle myosin from Ezo giant scallop (Patinopecten yessoensis) and akazara scallop (Chlamys nipponensis akazara) were determined. Tryptic peptides of each light chain were isolated and sequenced. The alignment of the tryptic peptides in each chain was deduced from the amino acid compositions and the partial sequences of peptic peptides of the Ezo giant scallop light chain. The light chains both consist of 156 residues. Heterogeneous residues, glutamic acid and aspartic acid were observed at the 155 th position in the sequence of the akazara scallop light chain. A comparison of the Ezo giant scallop light chain with the glutamic acid-containing and aspartic acid-containing akazara light chains revealed 6 and 7 amino acid substitutions, respectively. When the presented sequences were compared with those of the regulatory light chains of gizzard, cardiac and skeletal muscle myosins, a strong homology was observed in the calcium binding region, but there were considerable heterogeneities in the N- and C-terminal regions.

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The effect of vitamin A deficiency on rat testis was studied biochemically and histologically. The earliest change in nuclear basic proteins was a decrease and disappearance of the testis-specific protein (TP); the disappearance approximately coincided with cessation of growth. At later stages of deficiency, the testis-specific histone TH2B was markedly reduced and in some cases disappeared, as did the TH1 histone band. Histological studies indicated that the loss of TH2B and TH1 requires a substantial degree of testicular degeneration, because they could still be detected to some extent in testes containing mainly Sertoli cells, reduced numbers of spermatogonia and very few spermatocytes. The effect of retinol in maintaining the germ cells and the testis-specific basic proteins TP and TH2B was very specific, in that it could not be replaced by retinoic acid, which can maintain normal body growth. Prolonged supplementation of vitamin A-deficient animals with retinyl palmitate partially restored the levels of TP, TH2B, and TH1.

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Protein C inhibitor isolated from human plasma inhibited thrombin, factor Xa, trypsin and chymotrypsin as well as activated protein C, but had very little effect on urokinase and plasmin. The inhibition constants (K₁) of protein C inhibitor for activated protein C, thrombin and factor Xa were 5.6×10^-8M, 6.7×10^-8M and 3.1×10^-7M, respectively. The second-order rate constant for inhibition of activated protein C by the inhibitor increased about 30-fold in the presence of an optimal heparin concentration (5-10 units/ml). The inhibition of activated protein C by plasma protein C inhibitor was also accelerated by heparin. When activated protein C (M_r=62.000) was incubated with protein C inhibitor (M_r=57, 000), enzyme-inhibitor complexes with apparent M_r=102, 000 and 88, 000 were observed in the nonreduced and the reduced samples, respectively, on SDS-polyacrylamide gel electrophoresis. In addition to these complexes, a band of unbound enzyme and a band with M_r=54, 000 were detected. When ¹²⁵I-labeled protein C inhibitor was exposed to activated protein C, the inhibitor band was converted to bands with apparent M_r=102, 000 and 54, 000 in the nonreduced samples, as determined by autoradiography after gel electrophoresis in SDS. The band with M_r=54, 000 also appeared when the inhibitor reacted with other serine proteases. The activated protein C was released from the inactive complex by treatment with 1M ammonia or hydroxylamine. This phenomenon was found by SDS-polyacrylamide gel electrophoresis to represent the dissociation of the enzyme-inhibitor complex by ammonia or hydroxylamine into the free enzyme and the proteolytically modified inhibitor. Thus, the results indicate that the protein C inhibitor inhibits activated protein C by forming an enzyme-inhibitor complex accompanied with the proteolytic modification of the inhibitor by the enzyme.

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The pyridylamination reaction of sugar chains from glycoproteins was re-investigated to raise the total yield of pyridylamino sugars. Sugar chains of glycoproteins were released by hydrazinolysis, and free amino groups were N-acetylated. The sugar chains were coupled with 2-aminopyridine, and the pyridylamino derivatives thus obtained were purified by Sephadex G-15 column chromatography and analyzed by high performance liquid chromatography. In this study, conditions for the coupling reaction (temperature, time, pH, concentration of 2-aminopyridine, and amount of the reducing reagent) were re-investigated. Under the conditions established in the present study, the total recovery of pyridylamino derivatives of sugar chains of glycoproteins was about 70%, and 0.15 nmol of a glycoprotein was enough to detect the pyridylamino derivatives of sugar chains. The stability of sialyl and fucosyl linkages under the present conditions was also studied.

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Previously, we isolated t-cinnamoyl-D-glucose as a possible intermediate in chlorogenic acid biosynthesis from sweet potato root. The enzyme which catalyzes the formation of t-cinnamoyl-D-glucose has been purified 539-fold from sweet potato root (Ipomoea batatas Lam.) and characterized. It required UDP-glucose as a glucosyl donor. Its molecular weight was estimated to be 45, 000 by gel filtration chromatography through Sephadex G-100. Its K_m values were 0.2mM for t-cinnamic acid and 0.1mM for UDP-glucose. It also showed activity toward various aromatic carboxylic acids other than t-cinnamic acid with the following relative activities at the concentration of 1.8mM: t-cinnamic acid, 100; p-coumaric acid, 57; o-coumaric acid, 52; caffeic acid, 15; benzoic acid, 71; ferulic acid, 27; 4-hydroxyl-3-methoxy-benzoic acid, 35. When p-coumaric acid was used as a substrate, the enzyme introduced the glucosyl group exclusively to a carboxyl group, not to a hydroxyl group on a benzene ring. It was inhibited by p-chloromercuribenzoate and HgCl₂. Its activity in the extract from sliced root decreased during the first 28 h after slicing, then increased to the original level by 75 h. The apparent decrease seemed to be caused by the appearance of an inhibitory factor of high molecular weight in the tissue extract.

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Two-dimensional changes of the redox level of NADH were measured by fluorescence photography with the aid of computer-processing in cross sections of rat brain that had been frozen in situ in liquid nitrogen. Structure-related heterogeneity was noted in the distribution of the redox level in normal brain of conscious rats. Using 2-deoxy-[¹⁴C] glucose autoradiography, the area showing relatively high NADH fluorescence was found to be the area of high glucose utilization. Quantitatively, however, the fluorescence intensity did not parallel the rate of glucose uptake.
Increase of NADH fluorescence was observed specifically in the nucleus caudatus putamen and the thalamus when the rats were anesthetized with pentobarbital. Chemical determination of the NADH content was also performed simultaneously, and the results were consistent with those obtained by fluorescence photography.

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Glucuronolactone reductase [EC 1.1.1.20] from rat kidney was purified over 300-fold by ammonium sulfate fractionation, chromatography on DEAE-cellulose and hydroxylapatite columns, and preparative isoelectric focusing. The substrate specificity of the enzyme in the reduction reaction was broad, and hexuronic acid was one of the best substrates among monosaccharides. K_m values for D-glucuronic acid, D-glucuronolactone, D-galacturonic acid, and L-iduronic acid were 6, 9, 4, and 6mM, respectively.
An investigation of the activity for aldose led to the finding that triose and tetrose served as good substrates for this enzyme. However, the activity for aldopentose or aldohexose was less than 1% of that for D-glucuronic acid at the same concentration. The enzyme was inactive towards most hexosamines (galactosamine, mannosamine, N-acetylglucosamine, N-acetylgalactosamine, and N-acetylmannosamine, but not glucosamine), meso-inositol, D-fructose, and tetrasaccharides from hyaluronic acid and chondroitin 4-sulfate.
Trisaccharides from hyaluronic acid and chondroitin 6-sulfate which possess glucuronic acid at the reducing end were poor substrates for the enzyme and the activity towards these 4-substituted glucuronic acids was less than 3% of that towards non-substituted glucuronic acid.

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An enzymatic method was devised for determination of free glucuronic acid by the use of glucuronolactone reductase from rat kidney. Free glucuronic acid in the range of 4 to 200 μg was determined quantitatively by this method even in the presence of neutral sugars and oligosaccharides of glycosaminoglycans. The interference due to 20 to 100 μg of aldohexose or aldopentose was less than 0.5% at the same concentration of free glucuronic acid. The activity of the enzyme towards substituted glucuronic acid was only 1% of that towards free glucuronic acid. The enzyme did not act on N-acetylhexosamines.
The method was applied to a study of glycosaminoglycan degradation by lysosomal enzyme. When hyaluronic acid and chondroitin 4-sulfate were incubated for 24 h with a glycoprotein fraction obtained from rat liver lysosomes by concanavalin A agarose chromatography, free glucuronic acid was liberated (showing a sigmoid curve as a function of incubation time) and reached 21 and 10% of total glucuronic acid, respectively. Tetrasaccharide from chondroitin 4-sulfate was degraded to equimolar amounts of free glucuronic acid and sulfated trisaccharide.

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A lectin was purified from the hemolymph of Allomyrina dichotoma larvae by affinity chromatography on acid-treated Sepharose 4 B. The purified lectin showed two protein bands on polyacrylamide gel electrophoresis. These two lectin bands (allo A-I and -II) were separated by DEAE-Cellulofine column chromatography. By gel filtration on Sephadex G-100, the molecular weights of alto A-I and -II were estimated to be 65, 000 and 66, 500, respectively. On the other hand, by SDS-polyacrylamide gel electrophoresis after cross-linking of subunits with glutaraldehyde, they are estimated to be 38, 000 and 39, 000, respectively. On SDS-polyacrylamide gel electrophoresis, it was proved that both allo A-I and -II lectin consisted of two subunits, respectively. The molecular weights were 17, 500 and 20, 000 for allo A-I, and 19, 000 and 20, 000 for allo A-II. The isoelectric points of alto A-I and -II were estimated to be 6.4 and 5.9, respectively.
On double immunodiffusion, allo A-I and -II gave single precipitin lines, which fused completely with each other, against the antibody to crude allo A. The hemag-glutinating activity of allo A-I and -II was inhibited only by β-linked D-galactose such as lactose and lactulose.

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Biosynthesis of N-acetylcysteine S-conjugates of xenobiotics, mercapturic acids, occurs via inter-organ metabolism of the corresponding glutathione derivatives (Inoue, M., Okajima, K., & Morino, Y. (1982) Hepatology 2, 311-316). To elucidate the mechanism of mercapturate biosynthesis and its urinary elimination, hepatorenal cooperation in the enzymic processing and membrane transport of cysteine derivatives was studied in isolated hepatocytes, perfused liver and renal cortical tubules. Isolated hepatocytes rapidly accumulated S-benzylcysteine by a carrier-mediated mechanism and metabolized it to the N-acetylcysteine conjugate. Experiments in perfused rat liver revealed that, upon infusion of S-benzylcysteine, N-acetyl-S-benzylcysteine appeared in the effluent perfusate and hepatic excretion of this mercapturate was inhibited by simultaneous infusion of probenecid, an inhibitor of the organic anion transport system. Isolated renal cortical tubules actively accumulated N-acetyl-S-benzylcysteine by a dinitrophenol-sensitive, carrier-mediated mechanism which was competitively inhibited by probenecid and hippuric acid.
These and other results indicate that a cysteine S-conjugate in plasma is rapidly taken up by the liver and converted to the N-acetyl derivative which is translocated into plasma via a probenecid-sensitive transport system in hepatic sinusoidal membranes. The mercapturic acid excreted in plasma is transferred to the kidney and finally excreted into urine by a probenecid sensitive transtubular transport system for organic anions in renal cortical tubules. Hepato-renal cooperation in metabolic conjugation and membrane transport of these intermediates appears to constitute an important process in mercapturate biosynthesis and urinary excretion of the final metabolites.

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The temperature-dependent precipitability of a monoclonal IgG3x cryoimmuno-globulin (Jir) without known antibody activity is shown to be affected by various physico-chemical factors, such as protein concentration, pH value and NaCl concentration. The molecular properties characterizing this protein (carbohydrate and amino acid compositions, peptide constitutions and susceptibility to enzymatic proteolysis) are described. The cryoprecipitability of the protein was completely lost upon papain hydrolysis, and none of the isolated fragments, Fab-Fc, Fc, and Fab, showed any precipitating activity. In the cryo-coprecipitation assay using the ¹²⁵I-labeled fragments, it was demonstrated that the association activity with intact Jir protein was still retained on the Fab-Fc and Fc fragments, but not on the Fab fragment. The evidence suggests that a specific interaction may be involved in the primary intermolecular association required to form the cryoprecipitate at temperatures below the critical point, and that one of the pairing sites resides on the Fc portion of the protein molecule.

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Potato lectin (Solanum tuberosum agglutinin, STA) was found to contain fluorescent tryptophan residues highly exposed to solvent. The binding of chitin oligosac-charides to STA induced fluorescence quenching, a shift of the fluorescence maximum to shorter wavelength, a decrease in the quenching constant of iodide ion and a decrease of the number of tryptophan residues modifiable by N-bromosuccinimide. The results suggested that one tryptophan residues is located at or near a sugar binding site of STA, and that its environment is altered from hydrophilic to relatively more hydrophobic upon interaction with specific sugars.
The binding constants of STA with chitin oligosaccharides were determined by measuring the peak-trough heights in the fluorescence difference spectra induced by various concentrations of sugars. The inhibition constants of chitin oligosac-charides for the hemagglutinating activity of STA were obtained by the method of Pitts and Yang [(1981) Biochem. J. 195, 435-439] and the results were in good agreement with those obtained by the fluorescence spectral method.
Standard and unitary free energy changes (ΔG° and ΔG_u) and standard enthalpy changes (ΔH_??_) were also obtained. These values decreased with sugar chain length up to at least the tetramer. Thus, it was assumed that there are at least 4 subsites, A, B, C, and D, in the sugar binding site of STA. The contributions to the binding energy (ΔG_uG) were -17.0, -12.6, -7.3, and -4.4 kJ/mol at subsites A, B, C, and D, respectively, and the bindings of chitin monomer (GlcNAc), dimer, trimer, and tetramer were assumed to occur at subsite A, AB, ABC, and ABCD, respectively. Methyl glycosides of N-acetyl-D-glucosamine, and oligomers larger than the dimer (but not the monomer) induced fluorescence quenching, suggesting that the tryptophan residue responsible for the quenching is located at subsite B or between subsites A and B. The negative values of ΔH° indicate that the binding is an exothermic reaction.

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To determine the steric course of the reaction of bacterial ornithine decarboxylase [EC 4. 1. 1. 17], we have carried out the decarboxylation of L-ornithine in ²H₂O and that of DL-[2-²H] ornithine in H₂O, and obtained putrescine bearing a single deuterium atom in the C-1 position. The stereochemistry of [1-²H] putrescine was established by conversion to 1-(2-pyrrolidinyl)-2-propanone with acetoacetate and the pro-S hydrogen-specific diamine oxidase from pea seedlings. Analysis of deuterium content by gas chromatography-mass spectrometry showed that the deuterium label was fully retained during the conversion of [1-²H] putrescine produced by the decarboxylation of L-ornithine in ²H₂O to 1-(2-pyrrolidinyl)-2-propanone, in contrast with the considerable loss of label from [1-²H]putrescine which was produced by the decarboxylation of DL-[2-²H] ornithine in H₂O. The extent of loss of the deuterium label was in good agreement with the estimated value based on the isotope effect in the diamine oxidase reaction. These results indicate that the introduced deuterium (or hydrogen) is in the pro-R position at C-1 of putrescine, and consequently the ornithine decarboxylase reaction proceeds with retention of configuration.

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The effect of epidermal growth factor (EGF) on collagen degradation in clonal osteoblastic MC3T3-E1 cells was investigated by measuring the activities of dipeptidyl-aminopeptidase (DAP) and collagenase-like peptidase (CL-peptidase). EGF at concentrations of 2 to 50 ng/ml markedly increased DAP and CL-peptidase activities in the cells. The same concentrations of this factor significantly decreased the cellular hydroxyproline content. Since DAP and CL-peptidase are thought to be enzymes involved in collagen degradation, these results suggest that a physiological concentration of EGF stimulates collagen catabolism in osteoblasts.

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The activities of S-adenosylmethionine synthetase isozymes in liver were measured after rats received a diet containing excess methionine. The activity of the α-form increased with increasing methionine content in the diet, and reached 4-5 fold after 6 days on a 3 methionine diet. However, the activity of the β-form showed only a 1.5 fold increase. The activity of the γ-form in kidney showed no significance change.

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Sensitive determination of anti-glycolipid antibody titer and glycolipid content by an enzyme-linked immunosorbent assay (ELISA) using polystyrene beads was achieved. Glycolipid-coated polystyrene beads were used as the immobilized antigen. As antigen glycolipids, gangliotetraosylceramide (GA₁), gangliotriosylceramide (GA₂) and neolactotetraosylceramide (paragloboside) were used. Concentrations of 1-500 ng glycolipid in liposomes/ml or 0.1-100 μg glycolipid/ml could be used for the glycolipid determination. Glycolipid determination by the competitive inhibition method was not influenced by the presence of other glycolipids. A great advantage of this method is that the glycolipid-coated beads can be used repeatedly by washing the used beads with 3M NaSCN solution. The method was applied to the detection of auto-antibody against GA₁ in ascitic fluid from cancer patients.

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In oxidative and photosynthetic phosphorylation, the release of ATP from the catalytic site of F₁-ATPase by proton flux was explained by the conformational change induced in a proton reacting acid base cluster in the ATPase.

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