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Toshio Hakoshima, Ken-ichi Oka, Shyoji Toda, Masahiro Tanaka, Keigo Go ...
1990 Volume 108 Issue 5 Pages
695-698
Published: 1990
Released on J-STAGE: November 18, 2008
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Ribonuclease T
1 and the mutant enzymes were cocrystallized with several ribonucleotides, including non-hydrolyzable substrate analogs of di- and triribonucleotides, which have a novel guanylate in which the 2'-hydroxyl group of the ribose is replaced by a fluorine atom. One of the mutant enzymes has a tryptophan residue, instead of Tyr45 of the wild-type enzyme, to enhance the binding of ribonucleotides to the enzyme and the other mutant enzyme has histidine and aspartate residues, instead of Asn43 and Asn44, respectively, to reproduce the natural substitutions found in ribonuclease M
s. Polymorphism of the crystals was observed for wild-type and mutant enzymes. However, orthorhombic crystals, which are virtually all isomorphous to each other, were successfully obtained from wild-type and mutant (Y45W) enzymes by the macroscopic seeding technique using mother crystals of the wild-type ribonuclease T
1 complexed with 2'GMP or 3'GMP. The diffraction patterns of these crystals extend beyond 2.5 Å resolution and the diffraction data were collected from some of the crystals on a diffractometer up to a range of 2.5 to 1.8 Å resolution.
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Tohru Yoshimura, Yutaka Matsushima, Katsuyuki Tanizawa, Moon-Hee Sung, ...
1990 Volume 108 Issue 5 Pages
699-700
Published: 1990
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The active site lysyl residue (K239) of the thermostable aspartate aminotransferase [EC 2.6.1.1] was replaced by cysteinyl residue by means of site-directed mutagenesis. The K239C mutant enzyme obtained was catalytically inactive. The reaction of the cysteinyl residue of the K239C mutant enzyme with ethylenimine led to the formation of
S-(β-aminoethylcysteinyl (SAEC) residue. The K239SAEC mutant enzyme obtained showed about 25% of the activity of wild-type enzyme, and absorbed at 375nm, which suggested the internal Schiff base formation.
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Atsushi Nakagawa, Yoshiki Higuchi, Noritake Yasuoka, Yukiteru Katsube, ...
1990 Volume 108 Issue 5 Pages
701-703
Published: 1990
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The three-dimensional structure of cytochrome
c-553 isolated from sulfate-reducing bacterium,
Desulfovibrio vulgaris Miyazaki F strain, has been determined by the multi-wavelength anomalous dispersion technique with use of synchrotron radiation. The result shows that bacterial S-class cytochromes
c have a variety of folding patterns. The relative location of two α-helices at amino- and carboxyl-terminals and the style of bonding to the heme group show “cytochrome
c folding, ” but other regions of the structure are different from those of other cytochromes
c previously reported. The results also give useful information about the location of sulfate-reducing bacterium on the phylogenetic tree of the bacterial cytochromes
c superfamily.
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Yasunori Kozutsumi, Takehiro Kawano, Tamio Yamakawa, Akemi Suzuki
1990 Volume 108 Issue 5 Pages
704-706
Published: 1990
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The activity of CMP-
N-acetylneuraminic acid hydroxylase, that converts CMP-
N-acetylneuraminic acid (CMP-NeuAc) to CPM-
N-glycolylneuraminic acid (CMP-NeuGc), in mouse liver was determined by a newly developed HPLC method using non-radioactive CMP-NeuAc as a substrate. The activity was detected in the cytosol fraction but not in the microsomal fraction. Either NADH or NADPH was used as an electron donor by the cytosol enzyme, but NADH was much more efficiently used than NADPH. An antibody against cytochrome
b5 markedly reduced the CMP-NeuAc hydroxylase activity when added to incubation mixture containing either NADH or NADPH as an electron donor. These data led us to postulate the following electron transport system, which is involved in the CMP-NeuAc hydroxylation in mouse liver cytosol:
NADH→X→cytochrome
b5→Z→_??_CMP-NeuAc
NADPH→Y→(soluble)→Z→CMP-NeuGc
where X, Y, and Z are components supposedly involved.
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Hiroshi Furuichi, Yukihiro Ozaki, Katsumi Niki, Hideo Akutsu
1990 Volume 108 Issue 5 Pages
707-710
Published: 1990
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Resonance Raman spectra of the soluble-domain of a membrane-bound hydrogenase from
Desulfovibrio vulgaris Miyazaki F were recorded in different oxidation states. In the oxidized state, the Raman band due to the totally symmetric stretching mode of the iron-sulfur cluster was observed at 341cm
-1, which was attributed to the 3Fe-4S cluster. In the hydrogen-reduced state, only a weak and broad band was observed in its vicinity. During the process of reoxidation, a Raman band assignable to the 4Fe-4S cluster was observed at 333cm
-1 in the first step. Then, the band at 341cm
-1 became stronger and eventually dominated the spectrum. Corresponding changes were observed in the visible absorption spectra of the same sample. It was concluded from these observations that this hydrogenase has both 3Fe-4S and 4Fe-4S clusters and takes on at least three oxidation states, namely, oxidized, intermediate, and hydrogen-reduced ones.
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Hiroyuki Takeya, Aya Onikura, Toshiaki Nikai, Hisayoshi Sugihara, Sada ...
1990 Volume 108 Issue 5 Pages
711-719
Published: 1990
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Crotalidae and Viperidae snake venoms contain several kinds of metalloproteinases which cause localized hemorrhage by direct action on blood vessel walls. We report here the entire amino acid sequence and the disulfide bridge locations of HT-2, one of the hemorrhagic toxins isolated from the venom of
Crotalus ruber ruber (red rattlesnake). The non-reduced protein was first cleaved at methionine residues to provide a set of 8 fragments, which covered the entire sequence of HT-2. The disulfide bridge locations of HT-2 were also determined by using these primary fragments. The unambiguous sequence for the whole protein was then established by conventional methods using lysyl endopeptidase and thermolysin digests. HT-2 consisted of 202 amino acid residues with two disulfide bridges, which were assigned to Cys-117-Cys-197 and Cys-157-Cys-164. HT-2 had a typical zinc-chelating sequence His-Glu- X- X-His (residues 142-146) found in thermolysin, and its overall sequence showed, respectively, 50, 52, and 53% identities to those of HR2a, H
2-proteinase, and the metalloproteinase domain of HR1B. However, the disulfide bridge locations of HT-2 were different from those in the other metalloproteinases. The primary structure of HT-2 was more closely related to that of Ht-d from
Crotalus atrox recently determined (81% identity). From the structural comparison with five metalloproteinases so far elucidated, six conservative amino acid residues, which may possibly be related to the induction of the hemorrhagic activity, were suggested to be present in these toxins.
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Atsushi Sato, Toshiyuki Suzuki, Hideo Kochi
1990 Volume 108 Issue 5 Pages
720-725
Published: 1990
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Administration of glucagon, epinephrine, or dibutyryl cAMP to chicks induced cytosol-specific phosphoenolpyruvate carboxykinase in liver.
In vitro translation assay with poly(A)
+RNA indicated that this induction was due to the increase in phosphoenolpyruvate carboxykinase-coding mRNA synthesis which resulted from an increased level of hepatic cAMP. Either hydrocortisone or α-adrenergic agonist was ineffective for the induction by itself, but showed a significant effect when administered together with one of the inducing agents given above. In particular, hydrocortisone enhanced the synthesis of phosphoenol-pyruvate carboxykinase-specific mRNA without changing the profile of the time courses of the induction and of hepatic cAMP level. Those observations suggest that the fundamental machinery required for induction of cytosol-specific phosphoenolpyruvate carboxykinase in liver is shared in common between rat and chick, and that the absence of appreciable induction of cytosol-specific hepatic phosphoenolpyruvate carboxykinase in starved chicks is due to neither lack nor impairment of the hormone-mediated induction mecha-nism, but is due to the difference in usage of the genetic information between the two animal species.
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Kosuke Abe, Kazuhiko Yamamoto, Hyogo Sinohara
1990 Volume 108 Issue 5 Pages
726-729
Published: 1990
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Mouse α-macroglobulin and murinoglobulin were labeled with
125I and utilized for plasma clearance studies performed with mice. Desialylated murinoglobulin was rapidly cleared from the circulation with a half-life of about 5min. On the other hand, desialylated α-macroglobulin showed a biphasic curve: about half was cleared at a rate similar to that of the intact molecule while the remaining half had a shorter half-life of about 20min which was prolonged by a simultaneous injection of a 200-fold excess of unlabeled asialoorosomucoid. Virtually no cross competition was observed between these asialoglobulins and formaldehyde-treated bovine serum albumin or trypsin-bound α-macroglobulin. These results suggest that the intravascular elimination of desialylated α-macroglobulin and murinoglobulin is independent of the clearance systems responsible for formaldehyde-modified proteins or proteinase-bound α-macroglobulins, and that the structure or spatial arrangement, or both, of oligosaccharide units of α-macroglobulin is somewhat different from that of murinoglobulin, resulting in a difference of avidity of interaction with the asialoglycoprotein receptor. The desialylated α-macroglobulin and murinoglobulin accumulated principally in the liver.
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Hsiao-Tung Sun, Yutaka Yoshida, Shoichi Imai
1990 Volume 108 Issue 5 Pages
730-736
Published: 1990
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A Ca
2+-ATPase with a high affinity for free Ca
2+ (apparent
Km of 0.13μM) was found and characterized in membrane fractions from porcine aortic and coronary artery smooth muscles in comparison with the plasma membrane Ca2
+-pump ATPase purified from porcine aorta by calmodulin affinity chromatography. The activity of the high-affinity Ca
2+-ATPase became enriched in a plasma membrane-enriched fraction, suggesting its localization in the plasma membrane. The enzyme was fully active in the absence of exogenously added Mg
2+, but required a minute amount of Mg
2+ for its activity as evidenced by the findings that it was fully active in the presence of 0.1μM free Mg
2+ but lost the activity in a reaction mixture containing trans-cyclohexane-1, 2-diamine-N, N, N', N'-tetraacetic acid as a divalent cation chelator which has, unlike EGTA, high affinities for both Ca
2+ and Mg
2+. It was able to utilize a variety of nucleoside di- and triphosphates as substrates, such as ADP, GDP, ATP, GTP, CTP, and UTP, showing a broad substrate specificity. The activity of the enzyme was not modified by calmodulin (5, 10μg/ml). Trifluoperazine, a calmodulin antagonist, had a partial inhibitory effect on the activity at 30 to 240μM, but this inhibition could not be reproduced by a more specific calmodulin antagonist, W-7, indicating that this inhibition by trifluoperazine was not specific. Furthermore, the high-affinity Ca
2+-ATPase activity was not modified either by low concentrations (0.5-9μM) of vanadate or by 1-100μM p-chloromercuribenzoic acid. Cyclic GMP, nitroglycerin, and nicorandil did not have any effect on the enzyme activity. In contrast, the plasma membrane Ca
2+-pump ATPase purified from porcine aorta required millimolar Mg
2+ for activity and could utilize only ATP as a substrate. Furthermore, the Ca
2+-pump ATPase activity was sensitive to stimulation by calmodulin and inhibition by low concentrations of vanadate. In the presence of 1-100μM p-chloromercuribenzoic acid, the purified Ca
2+-pump ATPase lost most of its activity. Therefore, it is concluded that the high-affinity Ca
2+-ATPase is a distinct entity from the Ca
2+-pump ATPase and coexists with the Ca
2+-pump ATPase in the plasma membrane of porcine vascular smooth muscles.
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Tsunenori Nozawa, Takumi Noguchi, Mitsuo Tasumi
1990 Volume 108 Issue 5 Pages
737-740
Published: 1990
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Resonance Raman spectra of chlorosomes isolated from the thermophilic green photosynthetic bacterium
Chloroflexus aurantiacus have been obtained with several excitation wavelengths from 441.6 to 514.5nm. Resonance Raman spectra of bacteriochlorophyll (BChl)
c isolated from
C. aurantiacus cells have also been observed. The C=C stretching frequencies of BChl
c in the chlorosomes were found to be at 1, 556 (strong) and 1, 544 (shoulder) cm
-1, which correspond to those expected for the 5-coordinated BChl
c. The C-9 carbonyl resonance Raman frequency was found at 1, 642cm
-1, indicating that this group is either hydrogen-bonded to an Mg-coordinated hydroxyl group or coordinated to the Mgion.
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Takeyuki Kohno, Ken'ichi Yamaguchi, Eiji Ishikawa
1990 Volume 108 Issue 5 Pages
741-747
Published: 1990
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Anti-angiotensin I IgG in serum was measured by immune complex transfer enzyme immunoassays using angiotensin I conjugates prepared by two different methods. In the first method, angiotensin I was conjugated to dinitrophenyl bovine serum albumin and β-D-galactosidase through covalent links. Anti-angiotensin I IgG in rabbit serum was reacted simultaneously with dinitrophenyl bovine serum albumin-angiotensin I conjugate and β-D-galactosidase-angiotensin I conjugate, and the complex formed of the three components was trapped onto (anti-dinitrophenyl group) IgG-coated polystyrene balls. After washing, the complex was eluted from the polystyrene balls with dinitrophenyl-L-lysine and transferred to goat (anti-rabbit IgG) IgG-coated polystyrene balls. β-D-Galactosidase activity bound to (anti-rabbit IgG) IgG-coated polystyrene balls was assayed by fluorometry. In the second method, biotinylated angiotensin I was coupled with dinitrophenyl bovine serum albumin-avidin conjugate and β-D-galactosidase-avidin conjugate and substituted for the two conjugates in the first method. The detection limits of anti-angioten-sin I IgG in serum were 10-30ng/liter (0.2-0.6pg/assay). These methods were 330 to 1, 000-fold more sensitive and much less affected by serum effect than the conventional enzyme immunoassay, in which an angiotensin I-bovine serum albumin-coated polystyrene ball was incubated with anti-angiotensin I IgG in serum and, after washing, with (anti-rabbit IgG) Fab'-peroxidase conjugate. The first method was more sensitive than the second method, but the second method may be superior in applicability to the first method.
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Mikiharu Yoshida, Eijiro Ozawa
1990 Volume 108 Issue 5 Pages
748-752
Published: 1990
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We found six groups of proteins, A0-A5, besides dystrophin itself in a dystrophin preparation obtained by the reported method [Campbell, K. P. & Kahl, S. D. (1989)
Nature 338, 259-262] with some modifications. Their molecular weights were 94, 62, 52, 43, 36, and 24 kDa, respectively. Their molar ratios to dystrophin were 0.14, 2.2, 0.88, 0.90, 1.7, and 0.34, respectively. Each of Al, A3, and A4 was split into several bands. But each group of bands except A3 seemed to behave like the same kind of protein. The doublet of A3 was subdivided into A3a and A3b in the decreasing order of molecular weight. All the A-proteins except A2 were cross-linked with dystrophin molecule by a cross-linker, bis(sulfosuccinimidyl)suberate, suggesting them to be dystrophin-associated proteins. When dystrophin preparation was treated with KI, which is known to break membrane cytoskeletal interactions, as described by Campbell and Kahl, A2, A3, and A4 were absorbed by wheat germ lectin (WGL) Sepharose, but the dystrophin molecule and Al were not absorbed. On the other hand, A2 and A3b reacted with biotinyl WGL but A3a and A4 did not in blotting analysis. This apparent discrepancy can be explained if we postulate that A3a and/or A4 would associate with A2 and/or A3b. On the basis of these results including stoichiometric considerations, we are of the opinion that the complex of A2. A4 among various possible ones is the most important to anchor dystrophin to sarcolemma. In this A2-A4 complex, A4 but not A2 is directly associated with dystrophin.
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Hideto Watanabe, Shunji Hattori, Shogo Katsuda, Isao Nakanishi, Yutaka ...
1990 Volume 108 Issue 5 Pages
753-759
Published: 1990
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Human neutrophil elastase was purified to homogeneity as two isozymes named E1 and E2. The isozymes degraded Type IV collagen, laminin, fibronectin, and heparan sulfate proteoglycan similarly to each other. The degradation of such basement membrane components by elastase may assist the extravasation of neutrophils in the process of inflammation. Among the substrates tested, only type V collagen, which is susceptible to neutrophil gelatinase, was resistant to elastase. This broad substrate specificity of the enzyme may also contribute to tissue destruction at the sites of inflammation. We produced a monoclonal antibody against the purified enzyme and applied it to immunohistochemical studies. In bronchopneumonia and polyarteritis nodosa, elastase was associated with the cleaved elastic fibers, indicating that the enzyme really destroys tissue
in vivo. In the exudates of rheumatoid joint, elastase was stained as diffuse fine granules. Immunohistochemical studies with the monoclonal antibody will provide a complementary way to disclose the mechanism of diseases related to neutrophil infiltration.
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Tetsuya Asakawa, Naomi Azuma
1990 Volume 108 Issue 5 Pages
760-765
Published: 1990
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Abalone myosin contains two kinds of light chain, regulatory light chain (LC2) and essential light chain (LC1) according to SDS-PAGE. Three distinct light chain bands were observed on polyacrylamide gel electrophoresis of purified abalone myosin in the presence of urea (urea-PAGE). The slower two components showed had mobility on SDS-PAGE and they also showed regulatory activity as the regulatory light chain. They were termed LC2-a and LC2-b in order of increasing mobility on urea-PAGE and isolated by DE-32 ion exchange column chromatography in the presence 8 M urea. The ratio of LC2-a and LC2-b in the central portion of adductor muscle of abalone (LC2-a:LC2-b=7:3) was different from that (1:1) in the peripheral portion. These results suggest that the two light chains are isoforms of the regulatory light chain. The amino acid compositions of LC2-a and LC2-b were very similar to each other except for the Cys content. The UV absorption spectra were also quite similar, as were the UV difference absorption spectra induced by Ca
2+. Phosphorylation was not detectable with the myosin light chain kinase of chicken gizzard. The Ca
2+ concentration dependencies of Mg-ATPase activity of LC2-a or LC2-b hybridized abalone myosin (a-myosin, b-myosin) were similar to each other in the absence of rabbit F-actin, but differed in the presence of actin. The b-myosin had a higher maximum value of actomyosin ATPase activity and a lower apparent binding constant of actin and myosin than a-myosin. The extent of superprecipitation of acto-b-myosin was higher than that of acto-a-myosin. These results indicate that the difference in the properties of the two RLC isozymes was not clear at the myosin level, but differences appeared at the actomyosin level.
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Jan Holgersson, Per-Âke Jovall, Bo E. Samuelsson, Michael E. Bre ...
1990 Volume 108 Issue 5 Pages
766-777
Published: 1990
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Total non-acid glycosphingolipids were isolated from the kidneys of single pigs serologically typed on their red blood cells as blood groups O and A. Glycolipid species were purified by HPLC and structurally characterized by thin-layer chromatography, mass spectrometry, proton NMIR, spectroscopy, degradation analysis, and reactivity with various monoclonal antibodies, Galαl-4Gal-specific
E. coli bacteria, and lectins. Glucosyl-, globotriaosyl-, and globotetraosylceramides were the predominant molecular species with lactosyl- and globopentaosylceramides (IV
3GralGb
4, Cer) as abundant constituents too. Small amounts of galactosyl- and digalactosylceramides were also present. In the blood group O pig kidneys, blood group H antigens based on four different core saccharides (types 1, 2, 4, and lactosyl core) were identified and the major blood group structure was V
2FucIV
3Gal-Gb4Cer. In the kidneys from the blood group A pig the corresponding blood group A antigens were found and in addition, a type 3 chain blood group A antigen was indicated by mass spectrometry and by its reactivity with a monoclonal antibody. Trace amounts of the type 2 chain-based X and Y antigens were found while blood group B antigens and the type 1 chain based Lewis antigens could not be detected. The ceramide part of the glycolipids was mainly composed of dihydroxy 18:0 long chain bases and non-hydroxy 16:0-24:0 fatty acids.
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Tamiko Matsuda, Tamio Noguchi, Kazuya Yamada, Masaru Takenaka, Takehik ...
1990 Volume 108 Issue 5 Pages
778-784
Published: 1990
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The regulation of the gene expression of two important glycolytic enzymes, glucokinase and L-type pyruvate kinase, by hormones and carbohydrates was studied, in primary cultures of adult rat hepatocytes. Insulin caused time- and dose-dependent increases in the amounts of the mRNAs of the two enzymes in hepatocytes, although glucokinase responded to this hormone faster than L-type pyruvate kinase. The induction of glucokinase mRNA by insulin did not require the presence of glucose itself, but that of the L-type isozyme was dependent on the glucose concentration. For this effect, fructose and glycerol could partially substitute for glucose, but pyruvate and 2-deoxyglucose, a nonmetabolizable glucose analog, could not. The time course of insulin induction in the presence of fructose, but not of glycerol, was similar to that in the presence of glucose. In the presence of glycerol, the mRNA increased in a diphasic manner: the first increase, which probably reflected the effects of fructose and glycerol in normal liver, reached a maximum after 3h, whereas the second increase corresponded to the increase in the presence of glucose. These results suggested that some metabolite of glucose was required for the insulin-induced increase in L-type pyruvate kinase mRNA. Cycloheximide inhibited the effects of insulin on the two mRNAs, suggesting that ongoing protein synthesis is required in both cases. The addition of 1-(5-isoquinolinesulfonyl)-2-methylpiperazine, an inhibitor of protein kinase C, also inhibited the effects of insulin. However, phorbol 12-myristate 13-acetate alone did not induce the two mRNAs. These results suggest that the mechanisms of the effects of insulin on the glucokinase and L-type pyruvate kinase mRNAs are partially the same, although they are largely different. Dexamethasone alone tended to decrease the levels of the two mRNAs, but enhanced their induction by insulin. Thyroid hormone had a permissive effect on the induction of glucokinase mRNA, but not on that of L-type pyruvate kinase mRNA, by insulin. 8-(4-Chlorophenylthio)-cAMP inhibited the induction of the two mRNAs by insulin.
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Tadashi Baba, Masayasu Takada, Ken Watanabe, Shin-ichi Kashiwabara, Yu ...
1990 Volume 108 Issue 5 Pages
785-791
Published: 1990
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In the present study, we identified cDNA clones of mouse acrosin from a testis λgt11 library. The deduced amino acid sequence indicates that mouse acrosin is initially synthesized as a single-chain polypeptide with a 16-residue signal peptide followed by a 23-residue light chain and then a 394-residue heavy chain; mouse acrosin zymogen contains 417 amino acid residues with a calculated molecular mass of 46, 993 Da. The cDNA-derived sequence of mouse proacrosin shows a high degree of similarity with human and porcine proacrosins and major portions of bovine trypsin, including the active site residues, the recognition site for substrate, the location of 12 cysteine residues, and two potential
N-glycosylation sites. The sequence homology suggests that mouse proacrosin is converted to a mature acrosin, which consists of the light and heavy chains with a combined molecular mass of 35, 587 Da, by cleavage of the peptide bond between Arg
23 and IIe
24, and sequential removal of 23-, 26-, and 50-residue COOH-terminal segments. Using Northern blot analysis of RNAs from various mouse tissues, the acrosin gene transcript was present only in testis. The 1, 800-base acrosin message was first detectable in 18-day-old testis. At the same time of testicular development, some of the acrosin mRNA was actually associated with polysomes. Also,
in situ hybridization analysis suggests that the acrosin gene is expressed only in the round spermatid. Therefore, it is most likely that transcription of the mouse acrosin gene and subsequent translation of its Mrna first occur in the early stages of the round spermatid, and that the acrosin message is not under translational control.
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Yukio Nisimoto, Hidetsugu Otsuka-Murakami
1990 Volume 108 Issue 5 Pages
792-797
Published: 1990
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A soluble protein containing very weak NADPH-dependent nitroblue tetrazolium reductase activity was partially purified from the cytosol of dormant human neutrophils by DEAE-5PW ion exchange chromatography. This preparation of cytosolic reductase exhibited three nitroblue tetrazolium-reducing bands with approximate molecular masses of 95, 45, and 40 kDa on non-denaturing gel electrophoresis in the presence of 35mM
n-octyl-glucoside, and two major bands with apparent masses of 45 and 40kDa along with a few variable minor bands on SDS-polyacrylamide gel electrophoresis. The 45 kDa protein is susceptible to endogenous proteases and is rapidly converted to proteolysis products at 36°C. The partially purified cytosolic protein(s) provided a concentration-dependent activation of NADPH oxidase in the cell-free system composed of the membrane, arachidonate and magnesium ion. In addition, polyclonal antibodies raised against rabbit hepatic NADPH:cytochrome P-450 reductase [EC 1.6.99.1] showed positive immunological reactivity toward cytosolic 45 kDa protein and also caused 30 to 40% inhibition of superoxide anion production in the cell-free system.
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Masato Nakai, Haruko Ishiwatari, Akiko Asada, Mayumi Bogaki, Kiyozo Ka ...
1990 Volume 108 Issue 5 Pages
798-803
Published: 1990
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The His-44 and Met-164 residues of yeast cytochrome
c1 are evolutionally conserved and regarded as heme axial ligands bonding to the fifth and sixth coordination sites of the heme iron, which is directly involved in the electron transfer mechanism. Oligonucleotide-directed mutagenesis was used to generate mutant forms of cytochrome
c1 of yeast having amino acid replacements of the putative axial ligands of the heme iron. When a cytochrome
c1-deficiency yeast strain was transformed with a gene encoding the Phe-44, Tyr-44, Leu-164, or Lys-164 protein, none of these transformants could grow on the non-fermentable carbon source. These results suggest that the His-44 and Met-164 residues have a critical role in the function of cytochrome
c1 in vivo, most probably as axial ligands of the heme iron. Further analysis revealed that the mutant yeast cells with the Phe-44, Tyr-44, or Leu-164 protein lacked the characteristic difference spectroscopic signal of cytochrome
c1. However, in the Lys-164 mutant cells, partial recovery of the cytochrome
c1 signal was observed. Moreover, the Lys-164 protein retained a low but significant level of succinate-cytochrome
c reductase activity
in vitro. The possibility that the nitrogen of Lys-164 served as the sixth heme ligand is discussed in comparison with cytochrome
f of a photosynthetic electron-transfer complex, in which lysine has been proposed to be the sixth ligand.
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Masao Nakamura, Isao Yamazaki, Sachiya Ohtaki
1990 Volume 108 Issue 5 Pages
804-810
Published: 1990
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Iodothyronines induced catalatic (H
20
2-decomposing) activity of thyroid peroxidase and lactoperoxidase, the effect increasing in the order of thyroxine (T
4) > triiodothyronine (T
3) > diiodothyronine (T
2). The iodothyronines served as electron donors in the peroxidase reactions, and during the reactions the catalytic intermediate of thyroid peroxidase was confirmed to be Compound II for T
4 and Compound I for T
3 and T
2 and from the Soret absorption spectra obtained by stopped-flow measurements. Rate constants for the reac-tions between T
4 and Compound II, T
3 and Compound I, and T
2 and Compound I were estimated at 1.9×10
5, 1.3×10
6, and 7.1×10
5 M
-1•s
-1, respectively. Unlike the case of thyroid peroxidase, the catalytic intermediate of lactoperoxidase observed during the oxidation of iodothyronines was invariably Compound II. From these and other data it was concluded that thyroid peroxidase catalyzed one-electron oxidation of T
4 and two-electron oxidations of T
2 and T
3 while lactoperoxidase catalyzed exclusively one-electron oxidation of the iodothyronines. Iodide was released during the enzymatic oxidation of iodothyronines, irrespective of the mechanism of one-electron and two-electron oxidations. The amount of released iodide increased in the order of T
4 > T
3 >T
2. The iodothyronines-induced catalatic activity of these peroxidases was ascribable to the release of iodide, but it was also found that the iodide-enhanced catalatic activity was stimulated by iodothyronines. In this case the effect of iodothyronines was greater in the order of T
2 > T
3> T
4 which was consistent with the order of iodothyronine activation for the iodinium cation transfer from enzyme to acceptor.
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Masahiro Tomita, Kenta Nakai, Akihiro Yamada, Ryo Taguchi, Hiroh Ikeza ...
1990 Volume 108 Issue 5 Pages
811-815
Published: 1990
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Of the total of 306 amino acids in the sequence of sphingomyelinase (SMPLC) from
Bacillus cereus, almost half (150) are expected to be involved in the formation of loop or turn structure, while 65 and 73 residues may participate in the formation of α-helix and β-structure, respectively. The helix content of SMPLC was calculated to be 0-5%, based on the CD spectra. The addition of divalent metal ions such as Mg
2+ or both Ca
2+ and Mg
2+ had no effect on the CD spectra of SMPLC, although the addition of these metal ions caused the breakdown of membranous SM and specific adsorption of SMPLC onto erythrocyte membranes. A hydropathy study showed that SMPLC has hydrophobic regions at the N-terminal domain which must be responsible for the binding of the enzyme to the membranes. The partial homologies between the amino acid sequences of SMPLC and
Clostridium perfringens α-toxin (phospholipase C) are discussed.
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Naoko Oda, Tomohisa Ogawa, Motonori Ohno, Hiroyuki Sasaki, Yoshiyuki S ...
1990 Volume 108 Issue 5 Pages
816-821
Published: 1990
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A cDNA clone of
Trimeresurus flavoviridis phospholipase A
2 was isolated from the venom gland cDNA library using DNA amplified by polymerase chain reaction with oligonucleotide primers corresponding to the N- and C-terminal sequences of this enzyme. The amino acid sequence deduced from the cDNA nucleotide sequence determined by the dideoxy termination method was found to be inconsistent in the segment comprising the 69th to 81st positions from that reported previously [Tanaka, S.
et al. (1986) J. Biochem. 99, 281-289]. Reinvestigation of the amino acid sequence of the peptide covering the sequence in question showed that the amino acid sequence predicted from the nucleotide sequence is correct. The sequence contains Asn-Asn-Gly (positions 69-71) and it was found that the Asn-Gly bond easily undergoes α-β transpeptidation when digested with
Achromobacter protease I at pH 9.0 but not seriously at pH 6.8. It is likely that the transpeptidation reaction caused a failure in the previous sequence determination. The cDNA clone obtained was 597 base pairs long and contained an open reading frame of 414 base pairs coding for 138 amino acid residues. A typical signal peptide sequence (16 amino acid residues long), located at the N-terminal moiety of the deduced sequence, was immediately followed by a polypeptide which corresponds to the mature enzyme. Northern blot analysis showed a single transcript only in the poly(A)
+ RNA fraction of the venom gland but not in those of many other organs tested.
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Akira Iwasaki, Tiee-Cherng Shieh, Yasuyuki Shimohigashi, Michinori Wak ...
1990 Volume 108 Issue 5 Pages
822-828
Published: 1990
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A coagulant enzyme, named okinaxobin I, has been purified to homogeneity from the venom of
Trimeresurus okinavensis (Himehabu) by chromatographies on Sephadex G-100 and CM-Toyopearl 650M columns. The enzyme was a monomer with a molecular weight of 37, 000 and its isoelectric point was 5.4. The enzyme acted on fibrinogen to form fibrin clots with a specific activity of 77 NIH units/mg. Fibrinopeptide B was released at a rate much faster than fibrinopeptide A. The enzyme exhibited 2 to 3 times higher activity toward tosyl-L-arginine methyl ester and benzoyl-L-arginine
p-nitroanilide than bovine throm-bin. The esterase activity was strongly inhibited by di isopropylfluorophosphate and phenylmethanesulfonyl fluoride, and to a lesser extent by tosyl-L-lysine chloromethyl ketone, indicating that the enzyme is a serine protease like thrombin. The N-terminal sequence was highly homologous to those of coagulant enzymes from
T. flavoviridis and
Bothrops atrox, moojeni venoms which preferentially release fibrinopeptide A. In order to remove most, if not all, of the bonded carbohydrates, the enzyme was treated with anhydrous hydrogen fluoride (HF), thereby reducing the molecular weight to 30, 000. The protein contained approximately 260 amino acid residues when computation was based on this value. The HF-treated enzyme retained about 50% of the clotting and esterolytic (TAME) activities and preferentially released fibrinopeptide B from fibrinogen. The carbohydrate moiety is not crucial for enzyme activity but might be necessary for eliciting full activity.
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Tomoko Kuroiwa, Masao Sakaguchi, Katsuyoshi Mihara, Tsuneo Omura
1990 Volume 108 Issue 5 Pages
829-834
Published: 1990
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Co-translational translocation of proteins across the membrane of rough endoplasmic reticulum (ER) is interrupted by particular amino acid sequences, which are functionally termed “stop-transfer sequence.” We analyzed the structural requirements for the inter-ruption of the peptide translocation. By the manipulation of the cDNA of interleukin 2 (IL2), which passes through ER membrane co-translationally, the middle portion of the IL2 molecule was replaced with systematically altered hydrophobic segments, leucine, alanine, or leucine/alanine mixed clusters. Furthermore, charged amino acid residues were introduced just downstream of the hydrophobic segments. These modified IL2 peptides were synthesized with wheat germ cell-free system in the presence of rough microsomes and the topology of the peptides in the microsomes was assessed by post-translational digestion with proteinase K. We obtained the following results. (i) Each modified protein was processed to the mature form but the extent of stop-translocation varied widely. The ratio of the stopped to the translocated products increased as the length and hydrophobicity of the inserted segment increased. (ii) Shorter hydrophobic segments than naturally occurring native transmembrane segment promoted stop-translocation. (iii) Proteins with hydrophobic segments followed by positive charges were more efficiently stop-translocated than those having negative charges. (iv) If the hydrophobicity of the segment was sufficiently high, the positive charges after the segment were not essential for stop-translocation. We also suggest that the stop-transfer process includes protein-protein interaction between the hydrophobic segment and translocation channel.
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Masahiro Abe, Katsuhito Takahashi, Kunio Hiwada
1990 Volume 108 Issue 5 Pages
835-838
Published: 1990
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Calponin inhibited the actin-activated myosin MgATPase activity in a dose-dependent manner without affecting the phosphorylation level of myosin light chain. This inhibition was Ca
2+-independent. The decrease in enzymatic activity of myosin was correlated with binding of calponin to actin-tropomyosin filaments. Caldesmon showed a further inhibition of the calponin-induced inhibition of MgATPase activity of the thiophosphorylated myosin. Calponin-induced inhibition of the myosin MgATPase activity was reversed by the addition of calmodulin only in the presence of Ca
2+. These results suggest that calponin acts as an inhibitory component of smooth muscle thin filaments.
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Kazunori Hirokawa, Nobuo Aoki
1990 Volume 108 Issue 5 Pages
839-845
Published: 1990
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Previous studies have shown that thrombomodulin (TM) on endothelial cells is down-regulated by endotoxin, interleukin-1β (IL-1β), and tumor necrosis factor (TNF). This loss of anti-coagulant potential is thought to be related to the hypercoagulable state in sepsis, inflammation, and cancer. The current studies describe up-regulation of TM in human umbilical vein endothelial cells (HUVECs) by several compounds as judged by increased surface cofactor activity, surface TM antigen, and TM mRNA levels. Surface TM activity was increased by active phorbol esters (10
-8M, 24-48h), analogs of cAMP (1-10mM, 4h), and forskolin (10
-5M, 24-48h). Up-regulation of TM in HUVECs by 4β-phorbol 12-myristate 13-acetate (PMA) and dibutyryl cAMP (dBcAMP) was due to
de novo synthesis of TM protein resulting from increased TM mRNA levels. The results suggest that protein kinase C and protein kinase A may be involved in cellular regulatory mechanisms for TM expression. In addition, PMA effects on surface TM activity are biphasic, with an initial reduction followed by a significant enhacement. Hence, we propose that compounds capable of increasing intracellular cAMP concentrations in HUVECs may be useful in preventing thrombosis by increasing the anti-thrombotic properties of endothelial cells.
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Takemitsu Mizunaga, Yoshio Katakura, Tadashi Miura, Yoshiharu Maruyama
1990 Volume 108 Issue 5 Pages
846-851
Published: 1990
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Protein disulfide-isomerase (PDI), which reactivates inactive scrambled RNase, was purified from
Saccharomyces cerevisiae. The enzyme was purified 1, 850-fold to apparent homogeneity by five purification steps: 30-70% ammonium sulfate fractionation, DEAE Toyopearl-650S and Butyl Toyopearl-650S chromatographies, and differential Phenyl-5PW HPLC with or without cysteine. The native enzyme had an apparent
Mr of 140, 000 on gel filtration chromatography, and its NH
2-terminal was blocked. The
Mr of its subunits were estimated to be 70, 000 by sodium dodecyl sulfate polyacrylamide gel electrophoresis, indicating that the enzyme is probably composed of two identical subunits. The
Mr of the subunits changed to 60, 000 on endoglucosaminidase H treatment, indicating that the enzyme is transported into the endoplasmic reticulum. The enzyme has a pH optimum of 8.5, and pI of 4.02. Its enzymic properties were compared with those of purified bovine liver PDI. The
Km values of yeast and bovine PDIs for scrambled RNase were 1×10
-5 and 2×10
-5 M, and their
Vmax values were 6 and 7 units/mg protein, respectively. The two enzymes showed no significant differences in
Km or
Vmax values with respect to thiol compounds. Bacitracin inhibited both PDIs in the same fashion. These results indicate that this yeast PDI corresponds to mammalian PDI.
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Ri-ichiroh Manabe, Naoko Manabe, Hiroshi Ochiai
1990 Volume 108 Issue 5 Pages
852-858
Published: 1990
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Monoclonal antibodies were prepared against a putative cell-cell adhesion glycoprotein, with an apparent molecular mass of 64, 000 (gp64), of the cellular slime mold,
Polysphondylium pallidum. Five monoclonal antibodies obtained by means of an enzyme-linked immunoadsorbent assay did not bind to the antigens which were subjected to gel electrophoresis and blotting method in the presence of a reducing agent, but they did bind specifically to the antigens prepared in unreducing conditions of samples and then processed by the same blotting method. To solubilize gp64 in a sodium dodecyl sulfate (SDS)-sample buffer without mercaptoethanol (heated) or SDS-sample buffer with 2-mercaptoethanol (nonheated) was critical for the antibody binding onto gp64 on a membrane. Hence the antibodies seem to bind to a surface portion(s) of the localized protein structure folded up by disulfide cross-linkages. One of the antibodies obtained blocked cell-cell adhesion by about 20%.
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Hiroko Murakami, Yoshiyasu Yabusaki, Toshiyuki Sakaki, Megumi Shibata, ...
1990 Volume 108 Issue 5 Pages
859-865
Published: 1990
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The NADPH-cytochrome P450 reductase gene isolated from the yeast
Saccharomyces cerevisiae [Yabusaki
et al., J. Biochem. 103, 1004-1010 (1988)] was expressed on a multi-copy plasmid in the yeast. The transformed yeast cells with the recombinant plasmid carrying the reductase gene with a length of 3kb produced the corresponding mRNA read from the original transcription initiation site under the control of its own promoter with a maximum length of 300bp. The reductase content in the transformed cells was 25 times higher than that of the endogenous reductase. When the coding region for the reductase was placed between the alcohol dehydrogenase I gene promoter and the terminator of the expression vector pAAH5, the expression level was 32 times higher than at the endogenous level. These recombinant yeast strains showed enhanced cytochrome
c reductase activity with increased cellular reductase levels. A simultaneous expression of yeast P450 reductase with rat P450c or bovine P450
17a resulted in 25 times or a 5 times increase in the corresponding P450-dependent monooxygenase activity of the recombinant yeast strains, respectively, as compared with that of the yeast cells expressing the corresponding P450 species. These results suggested that the overproduction of yeast P450 reductase with a simultaneous expression of the mammalian P450 species enhanced the P450c- and P450
17a-dependent monooxygenase activities in the recombinant yeast strains, probably due to the increased frequency of the interaction between yeast P450 reductase and P450c or P450
17a in the yeast microsomes.
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Morio Ishizuka, Kunihiro Machida, Shinichi Shimada, Ayumi Mogi, Toru T ...
1990 Volume 108 Issue 5 Pages
866-873
Published: 1990
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The gene coding for four subunits of cytochrome
aa3-type oxidase was isolated from a genomic DNA library of the thermophilic bacterium PS3 and sequenced. The N-terminus of each subunit was also sequenced to verify the initiation site of the reading frame. The deduced amino acid sequences contained 615 amino acid residues for subunit I (CO1/
caaB product), 333 residues for subunit II (CO2/
caaA product), 207 residues for subunit III (CO3/
caaC product), and 109 residues for subunit IV (CO4/
caaD product) after processing. Re-examination of the sequencing of
caa revealed a longer open reading frame for C01, which contains 14 transmembrane segments instead of 12 [ Sone
et al. (1988)
J. Biochem. 103, 606-610], although the main portions of the sequences constituting cytochrome
a (Fe
A), cytochrome
a3 (Fe
B), and Cu
B are correct. PS3 CO2 has an additional sequence for cytochrome
c after the Cu
A binding protein portion with 2 transmembrane segments, which is homologous to the mitochondrial counterpart. PS3 CO3 has DCCD-binding glutamyl residues but contains only 5 transmembrane segments, unlike the mitochondria' counterpart, which has 7 segments. The subunits of PS3 cytochrome oxidase (aa3-type) show clear similarity in amino acid sequences with those of cytochrome
bo-type oxidase from
Escherichia coli as well, in spite of the difference of hemes. PS3 CO3 and C04 are much more similar to
E. coli CO3 and C04 than to mitochondrial CO3 and C04, respectively.
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Tatsuya Hayashi, Koji Suzuki
1990 Volume 108 Issue 5 Pages
874-878
Published: 1990
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Monoclonal antibodies for human thrombomodulin, a cofactor for thrombin-catalyzed activation of protein C, were prepared and their epitopes characterized. All six antibodies (MFTM-1-MFTM-6) bound to an elastase-digested active fragment of thrombomodulin, which contains six consecutive EGF domains. Binding of thrombomodulin to these antibodies did not depend on Ca
2+ concentration. MFTM-4, MFTM-5, and MFTM-6 strongly inhibited protein C activation by thrombin and thrombomodulin. MFTM-4 and MFTM-5 inhibited thrombin binding to fixed thrombomodulin and bound to a recombinant mutant EGF456 protein, which contained the fourth, fifth, and sixth EGF domains of thrombomodulin. However, MFTM-6 did not inhibit thrombin binding to thrombomodulin and did not bind to EGF456 protein. Binding of thrombomodulin to fixed MFTM-4 or MFTM-5 was competitively inhibited by a recombinant mutant EGF45 protein which contained the fourth and fifth EGF domains and also by a mutant EGF56 protein which contained the fifth and sixth EGF-domains. These results suggest that epitopes of MFTM-4 and MFTM-5 are located in the fifth EGF domain of thrombomodulin. Thus, the binding site for thrombin is located in the fifth EGF domain. These results also suggest that an epitope for MFTM-6 is located at a region near the binding site for γ-carboxyglutamic acid residues of protein C
via Ca
2+ on thrombomodulin.
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Peng Wang, Jun Nishihata, Fusao Makishima, Kohji Moriishi, Bunei Syuto ...
1990 Volume 108 Issue 5 Pages
879-885
Published: 1990
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ADP-ribosyltransferase from
Clostridium botulinum type C strain was found to induce an increase of inositol phosphates (IPs) formation in murine thymocytes membranes. Incubation of electropermeabilized murine thymocytes with the enzyme also caused an increase of IPs formation in the cells. This increase of IPs formation in the enzyme-treated membranes and electropermeabilized cells was dependent on the amount of both NAD and the enzyme, suggesting that the stimulation of phosphoinositide-specific phospholipase C (PLC) was related to ADP-ribosylation of membrane proteins by the enzyme. On the other hand, in calf and murine thymocytes two proteins with the same molecular weight of 21, 000 were found to be ADP-ribosylated by the botulinum ADP-ribosyltransferase. A minor ADP-ribosyla-tion substrate was shown by two-dimensional polyacrylamide gel electrophoresis to be G21k, a low-molecular-weight GTP-binding protein (G protein) suggested previously by us to be involved in PLC regulation [Wang, P.
et al. (1987)
J. Biochem. 102, 1275-1287; (1988) 103, 137-142; and (1989) 105, 461-466], and the other major ADP-ribosylation substrate was identified as a rho A protein. Under the experimental conditions of the IPs formation study, ADP-ribosylation of both G21k and rho A proteins by botulinum ADP-ribosyltransferase in membranes and permeabilized cells was observed. These results suggest that botulinum ADP-ribosyltransferase-induced PLC stimulation in thymocytes is closely correlated with ADP-ribosylation of the low-molecular-weight G proteins.
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Tadashi Ueda, Hidenori Yamada, Hidenori Aoki, Taiji Imoto
1990 Volume 108 Issue 5 Pages
886-892
Published: 1990
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The effects of chemical modifications of Trp62 and Trp108 on the folding of hen egg-white lysozyme from the reduced form were investigated by means of the sulfhydryl-disulfide interchange reaction at pH 8 and 40°C. The folding of reduced lysozyme was monitored by following the recovery of the original activity. Under the conditions employed, the apparent first-order rate constant for the folding of reduced lysozyme was not changed by the modifications of both Trp62 and Trp108 and the folding was completed within 30 min. However, the extent of the correct folding was changed by the modification of Trp62 but not by that of Trp108. Native and oxindolealanine108 lysozymes recovered 80 and 81% of their original activities after 30-min refolding, respectively, but Trp62-modified lysozymes recovered their activities to a lesser extent than native and oxindolealanine108 lysozymes. The recovered activities of Trp62-modified lysozymes after 30-min refolding were 63% for oxindolealanine62 lysozyme, 65% for δ1 -carboxamidomethylthiotryptophan62 lysozyme, and 52% for δ1 -carboxymethylthiotryptophan62 lysozyme. These results suggest that Trp62 is important for preventing the misfolding of reduced lysozyme, but that neither Trp62 nor Trp108 is involved in the rate-determining step (the slowest step) in the folding pathway. A decrease in the hydrophobic nature of Trp62 seems to increase the misfolding and thus to decrease the extent of the correct folding of reduced lysozyme. A mechanism for the involvement of Trp62 in the folding pathway of reduced lysozyme is proposed.
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