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Reiko Nitta, Naoko Katayama, Yukie Okabe, Masanori Iwama, Hideaki Wata ...
1989 Volume 106 Issue 5 Pages
729-735
Published: 1989
Released on J-STAGE: January 25, 2011
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A pyrimidine base-specific ribonuclease was purified from bullfrog (
Rana catesbeiana) liver by means of CM-cellulose column chromatography and affinity chromatography on heparin-Sepharose CL-6B, which gave single band on SDS-slab electrophoresis. The primary structure of the bullfrog liver RNase was determined. It consisted of 111 amino acid residues, including 8 half-cystine residues. From the sequence, it was concluded that three disulfide bridges in RNase A were conserved in the bullfrog RNase, that a disulfide bridge in RNase A [Cys
65-Cys
126 (RNase A numbering)] was deleted, and that a new disulfide bridge was created in the C-terminal part of the enzyme. In this frog RNase, the amino acid residues thought to be essential for catalysis in bovine pancreatic RNase A were conserved except for Asp
121 (RNase A numbering). The sequence homology of the bullfrog liver RNase with bovine pancreatic RNase A was 30.6%. The sequence of bullfrog liver RNase was very similar to those of lectins obtained from bullfrog egg by Titani
et al.[
Biochemistry (1988) 26, 2189-2194] and
R. japonica egg by Kamiya
et al.[
Seikagaku (in Japanese)(1989) 60, 733; and personal communication from Kamiya, Y., Oyama, F., Oyama, R., Sakakibara, F., Nitta, K., Kawauchi, H., and Titani, K.]. The sequence homology between the bullfrog liver RNase and the two lectins was 70.2 and 64.8%, respectively.
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Nobuhiro Suzuki, Hirokazu Matsumoto, Chieko Kitada, Sadao Kimura, Masa ...
1989 Volume 106 Issue 5 Pages
736-741
Published: 1989
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Immunoreactive endothelin (ET) and big-endothelin (big-ET) in conditioned media of endothelial and of non-endothelial cells were studied using sandwich-type enzyme immunoassays. Immunoreactivities of both ETs were detected in the media of all four endothelial cells tested. Among non-endothelial cells, tumor cell lines with epithelial-like morphology also produced immunoreactive ET and/or big-ET, although the total amount of ETs was one or two orders of magnitude less than that produced by PAE (porcine aortic endothelial cells). Immunoreactive ETs produced by HepG-2 (human hepatocellular carcinoma) and some other cells were characterized by gel-filtration HPLC and reversephase HPLC. These studies revealed the production of ET-1 and human big-ET-1 by these cells, although the immunoreactive ETs produced by the tumor cells were more heterogeneous than those produced by endothelial cells. The regulatory effects of thrombin and transforming growth factor-β(TGF-β) on the production of ETs were investigated. TGF-β markedly stimulated the production of both ETs in HepG-2 and slightly decreased the big-ET level in A549 (human lung carcinoma). HPLC analysis showed that the major immunoreactive ETs induced by TGF-β in HepG-2 were identical to ET-1 and human big-ET-1. These results demonstrated that production of ET-1 and big-ET-1 was not restricted to endothelial cells and was induced by TGF-β in HepG-2 at the same levels as those produced by PAE.
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Motoshi Suzuki, Eishi Takagi, Kiyohide Kojima, Shunji Izuta, Shonen Yo ...
1989 Volume 106 Issue 5 Pages
742-744
Published: 1989
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We have developed a rapid purification method for DNA topoisomerase I from Raji cells, a human Burkitt lymphoma cell line, using ammonium sulfate fractionation followed by chromatography on a Mono S column (FPLC, Pharmacia). By this method, the enzyme could be purified to near homogeneity within one day. Electrophoresis on sodium dodecyl sulfate polyacrylamide gel revealed that the final preparation is mainly composed of a 100-kDa protein. The major enzyme activity sedimented through a glycerol density gradient at 5.7S, accompanied with a minor peak at 8.7S. The former may correspond to the monomer of the 100-kDa polypeptide, and the latter, to its dimeric form. The gel filtration study of the crude extract revealed an active molecular species of 200 kDa, in addition to 100 kDa, and lower molecular weight forms. These results suggest that DNA topoisomerase I is largely in monomeric form, but also has a minor population of the dimeric form.
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Joan Estelrich, Maria Teresa Montero
1989 Volume 106 Issue 5 Pages
745-750
Published: 1989
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A human erythrocyte glycoprotein was isolated and purified fromblood of group A
+1 by a procedure involving chloroform-methanol extraction and affinity chromatography on
Helix pomatia lectin-Sepharose 6MB, and some of its physicochemical properties were determined. The resulting preparation was homogeneous as indicated by polyacrylamide gel electrophoresis. The glycoprotein contained nearly 60% carbohydrate and 40% protein. It was water-soluble and inhibited the agglutination of A-erythrocytes. Its molecular weight was 41, 900 (amino acid analysis) or 55, 200 (light scattering), whereas electrophoresis revealed two bands of 43, 000 and 76, 000 Da. The ORD spectrum was consistent with 30%α-helix, 20%β-sheet, and 50% random coil. Intrinsic viscosity was 14.61 ml·g
-1, partial specific volume was roughly 0.66, isoelectric and isoionic points were 6.90 and 6.95, respectively. The glycoprotein differs from glycophorin and appears to be one of the minor glycoproteins of the human erythrocyte membrane.
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Eisaku Katayama
1989 Volume 106 Issue 5 Pages
751-770
Published: 1989
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Stereo electron microscopy of negatively stained images showed that myosin heads in acto-subfragment-1 (S1) covalently cross-linked with 1-ethyl-3-[3-(dimethylamino) propyl] carbodiimide were predominantly short and round when ATP was added, in contrast to their uniform tilted appearance in the rigor state. As an attempt to exclude molecules which were actually dissociated but still tethered to actin by artificial cross-links, quickfreeze deep-etch electron microscopy was counled with the mica flake method to observe uncross-linked native acto-S1 in the presence of ATP. To maintain the low affinity S1 associated to actin in the presence of ATP, a high concentration of acto-S1 was applied to mica flakes whose absorption had been chemically modified. The image of acto-S1 with added ATP agreed well with the expected time-course of reversible dissociation and reassociation, confirming the applicability of this approach to examination of the structural changes of acto-S1. S1 molecules attached to F-actin under rigor conditions or in the presence of ADP were elongated, with the long axis tilted to F-actin. Actin-attached S1 became short and round upon addition of ATP or ADP-inorganic vanadate. Adenyl-5'-ylimidodiphosphate and inorganic pyrophosphate each partially dissociated S1 from actin, as expected.
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Shigeko Kijimoto-Ochiai, Taiko Hatae, Yohko U. Katagiri, Harue Okuyama
1989 Volume 106 Issue 5 Pages
771-777
Published: 1989
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The β chain of human histocompatibility complex class II antigen, HLA-DR, showed 4 to 5 microheterogeneous spots on a gel obtained by two-dimensional polyacrylamide gel electrophoresis. The types of oligosaccharide chains on the β chains were analyzed by the lectin-nitrocellulose sheet method for each microheterogeneous spot with 3 cell lines of two haplotypes (HLA-DR 4, 4, and 3, 3). Two kinds of oligosaccharide chains were observed and were essentially the same in the microheterogeneous spots from all three cell lines. One, the oligosaccharide chain on the most basic spot (β1), was stained with peroxidase-coupled concanavalin A (Con A-P.O.) but not with peroxidase-coupled wheat germ agglutinin and was sensitive to endo-β-N-acetylglucosaminidase H (endo H), indicating that it was a high-mannose type. The oligosaccharide chains on other spots that were not stained with Con A-P.O. but were stained with peroxidase-coupled
Ricinus communis agglutinin were resistant to endo H.β2 and β3 were stained with E-PHA. Thus, they probably had bisected biantennary and others probably had multiantennary complex-type oligosaccharides. Sialidase experiments showed that the charge heterogeneity was due to post-translational sialylation of the oligosaccharide chains. In pulse-chase experiments, the most basic spot of β chain (β1) was labeled first, β2 and β3 were labeled next, and β4 was labeled last. These labeling characters accorded well with the results on the oligosaccharide types mentioned above.
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Takashi Takagi, Michio Yazawa, Tomohisa Ueno, Satoru Suzuki, Koichi Ya ...
1989 Volume 106 Issue 5 Pages
778-783
Published: 1989
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Three major calmodulin-binding eyanogen bromide peptides (fragments A, B, and D) were isolated from chicken gizzard muscle caldesmon and their amino acid sequences were determined. The molecular masses of fragments A, B, and D were estimated to 16, 12, and 9kDa, respectively, by SDS-urea polyacrylamide gel electrophoresis. Fragment A was composed of 102 amino acid residues and contained homoserine at the C terminus. The amino acid sequence from the 37th residue of fragment A corresponds to the N-terminal sequence of the 15kDa peptide which was obtained by thrombin dige8tion [Mornet, D., Audemard, E., & Derancourt, J.(1988)
Biochem. Biophys. Res. Commun. 154, 564-571]. Thrombin 15kDa peptide binds to F-actin but does not bind to calmodulin. Thus the N-terminal 36 residues and the C-terminal part from the 37th residue of fragment A are supposed to bind to calmodulin and F-actin, respectively. The sequences of fragments B and D were identical, but fragment D was composed of 64 amino acid residues and ended with tryptophan, whereas fragment B was of 98 or 99 amino acid residues and ended with proline. Both fragments B and D are supposed to be the C-terminal peptides of chicken caldesmon. Fragment B had heterogeneous sequence8 at the C-terminal region. These results can explain the reported heterogeneity of chicken caldesmon in charge and molecular mass.
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Kiyoshi Nakazawa, Noriko Takeuchi, Shuzo Iwata
1989 Volume 106 Issue 5 Pages
784-793
Published: 1989
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Chick lens epithelial cells were cultured on plastic and type IV collagen substrata, and the confluent cultures were labeled continuously with [
35S] sulfate for 20h. Intact lenses were also labeled in the same way.
35S-Proteoglycans isolated from those cultures were compared for their molecular sizes and glycosaminoglycan compositions. The results have shown that: 1) Proteoglycans synthesized by cells on type IV collagen were significantly smaller than those by cells on plastic. 2) Proteoglycans of intact lens showed a broad distribution of molecular size and contained a high proportion of chondroitin sulfate in the medium fraction compared to those of the two cell cultures. In order to explain such differences between proteoglycans from cultures, label-chase experiments with [
35S] sulfate were done for proteoglycans synthesized.
35S-Proteoglycans isolated at each chase time (0, 2.5, and 17 h) were compared and the following results were found: 1) The cell layers of both “plastic” and “type IV collagen” cultures contained glycosaminoglycan species predominantly at each chase time rather than proteoglycans. 2) Changes in the glycosaminoglycan compositions of medium fractions of cell cultures were observed during the chase period; in medium of the “plastic” culture, proteoheparan sulfate increased with chase time, whereas in medium of the “type IV collagen” culture, chondroitin sulfate glycosaminoglycan (not proteoglycan) increased with chase time. 3) In intact lens culture, lens capsule fraction at every chase time contained a proteoglycan unique in molecular size, which was not found in cell culture fractions. 4) All fractions from intact lens cultures contained a higher content of chondroitin sulfate at every chase time than the respective fractions from cell cultures. These results suggest that adhesion of the cells to type IV collagen or lens capsule influences the degradation and secretion of proteoglycans. In addition, they can account partially for the above-described differences in molecular sizes and glycosaminoglycan compositions between
35S-proteoglycans from various cultures continuously labeled with [
35S] sulfate.
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II. Accumulation of Phospholipids during Myeloid Body Formation and Histological Studies on Myeloid Bodies Using Twelve Aminoglycoside Antibiotics
Mieko Oshima, Mitsuko Hashiguchi, Miyuki Nakasuji, Noboru Shindo, Seii ...
1989 Volume 106 Issue 5 Pages
794-797
Published: 1989
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Cultured human skin fibroblasts take up aminoglycoside antibiotics into lysosomes to form myeloid bodies. Gentamicin (GM), one such antibiotic, was taken up until the cellular concentration reached an estimated 64mM on the 3rd day when cells were incubated with 2mM gentamicin. The rate of release of intracellular GM was high on the first day of incubation and gradually slowed down over the next 4d. About 50% of the GM remained in the cells even on longer incubation in GM-free medium, suggesting it may irrversibly bind to cellular components. With myeloid body formation, the cellular phospholipid content increased 1.5 times. Bis (monoacyl-glyceryl) phosphate, which is known as a marker of lysosomal phospholipid, phosphatidylcholine and phosphatidylserine showed 250, 162, and 153% increases, respectively. Sphingomyelin was not accumulated, while lysosomal sphingomyelinase was dramatically inhibited. Of 12 different aminoglycoside antibiotics, paromomycin is the most prominent myeloid body-forming antibiotic. The myeloid body-formation is not directly correlated to human nephrotoxicity. On the other hand, the number of myeloid bodies is well correlated to the affinity to the brush border membrane, suggesting that such aminoglycoside antibiotics are taken up easily through cellular endocytosis. The cytotoxic effects of aminoglycoside antibiotics may be due to by their binding to cellular organelles other than lysosomes.
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Hiroyuki Hori, Mineo Saneyoshi, Izumi Kumagai, Kin-ichiro Miura, Kimit ...
1989 Volume 106 Issue 5 Pages
798-802
Published: 1989
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tRNA (guanosine-2'-)-methyltransferases (Gm-methylases) isolated from extreme thermophiles,
Thermus thermophilus strains HB 27 and HB 8, methylate the 2'-OH of the G
18 ribose of the GG sequence in the D loop of tRNA, by recognizing the D “loop-stem” structure as a minimal requirement. To examine the role of the consensus uridine residue at position 8 (U
8) adjacent to the D “loop-stem” region in the recognition of Gm-methylase, 4-thiouridine at this position (s
4U
8) in Escherichia coli tRNA
Metf was modified reversibly with
Sbenzylthioisothiourea (sBTIU) or irreversibly by UV light. The initial velocities of the methylation reaction for the sBTIU-modified and the UV-induced cross-linked tRNAs were decreased to 40 and 30%, respectively, of that of the intact tRNA, but the sBTIU-modified tRNA regained almost full activity on reduction with β-mercaptoethanol. Although both of the modified tRNA
Metfs showed larger Km (although to different extents) and slightly smaller V
max than the intact tRNA
Metf, they retained full activities of methylation with tRNA (adenine-1-)-methyltransferase (m
1A-methylase) and of aminoacylation with aminoacyl-tRNA synthetase (ARS) fraction as well, both of which were prepared from
T. thermophilus strain HB 27. The 5'-half fragments derived from the sBTIU-modified and cross-linked tRNA
Metfs showed methylation efficiency (V
max/K
m) not appreciably different from that of the unmodified 5'-half fragment. These results suggest that the conformation of s
4U
8 residue of tRNA is deeply involved in the recognition of tRNA by Gm-methylase.
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Masashi Hamai, Kunihiko Konno
1989 Volume 106 Issue 5 Pages
803-807
Published: 1989
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The stabilizing effect of nucleotides against thermal denaturation of subfragment-1 (S-1) was studied quantitatively. We showed that ATP-(ADP·P
i) and AMPPNP-bound species are very stable, while ADP- and P
i-bound species are much less so (after ligand affinities are accounted for); these two stability classes correspond to the two CD and UV spectral classes. We further showed that the result of P
i- and ADP-binding is a weakening of the affinity between S-1 heavy chain and light chains, resulting in turbidity due to formation of aggregates of naked S-1 heavy chain. We also showed that while P
i- and ADP-binding decrease the inactivation rate of S-1 ATPase by protecting the 50-kDa fragment, they actually increase the denaturation rate of the remaining moieties (27 and 20 kDa); the rate of decrease of the 27- and 20-kDa tryptic fragments is much slower than that of the 50-kDa fragment with ligand-free S-1; however, the rate of decrease of these two fragments is fast and equal to that of the 50-kDa fragment with ADP- and P
i-bound S-1. Therefore, nucleotide binding to S-1 strongly affects the structure of the light chain binding site on the heavy chain, and it seems that the domain structure of S-1 is maintained by light chain binding.
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Isolation and Charaeterization
Hideko Atoda, Takashi Morita
1989 Volume 106 Issue 5 Pages
808-813
Published: 1989
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Using affinity chromatography on a column of factor X-Cellulofine, we have isolated a novelblood coagulation factor X-binding protein with anticoagulant activity from the venom of
Trimeresurus flavoviridis (Habu snake). This anticoagulant protein was also purified bychromatography on Sephadex G-75 and S-Sepharose Fast Flow. The yield of the purifiedprotein was approximately 16mg from 400mg of crude venom. The purified protein gave asingle band on both analytical alkaline disc-gel electrophoresis and SDS-PAGE. Thisprotein had a relative molecular weight (
Mr) after SDS-PAGE of 27, 000 before reduction of disulfide bonds and 14, 000 after reduction of disulfide bonds. The protein prolonged theclotting time induced by kaolin or factor Xa. In the presence of Ca
2+, it formed a complexwith factor X, the molar ratio being 1 to 1. Similar complex formation was observed withfactor Xa and factor IX/factor IXa, but not with other vitamin K-dependent coagulationfactors,
i. e., prothrombin, factor VII, protein C, protein S, and protein Z. The interaction of this anticoagulant protein with factor IX/factor X was dependent on γ-carboxyglutamicacid (Gla) domains, since Gla-domainless derivatives of factor X and factor IXaβ' did notinteract with this anticoagulant protein.
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Description of HPLC Gels in Terms of Porous Fractals
Marc Le Maire, Alexandre Ghazi, Michel Martin, Francoise Brochard
1989 Volume 106 Issue 5 Pages
814-817
Published: 1989
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We have tested the proposition that porous media used in protein size exclusion chromatography are surface fractals. The data obtained in the calibration of classical gels (Sephacryland Sepharose) and of HPLC gels (TSK SW and PW) using a wide range of protein sizes, havebeen analyzed within the framework of this theory. While the model does not apply toclassical gels, it seems that HPLC gels can be described as fractals in the range of proteinsizes. This finding has interesting implications for the calibration procedure.
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Makoto Nakanishi, Akihiko Moriyama, Yasuko Narita, Makoto Sasaki
1989 Volume 106 Issue 5 Pages
818-825
Published: 1989
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Aminopeptidase M [EC 3.4.11.2] was purified 772-fold to homogeneity from the microsomalfraction of human liver, with a yield of 18.9%, by a combination of solubilization with 0.5% Triton X-100 and then 1M urea and chromatography on columns of DEAE-cellulose, hydroxylapatite, Butyl-Toyopearl, and Sephacryl S-300. The purified enzyme had amolecular weight of 140, 000 by SDS-polyacrylamide gel electrophoresis and of 280, 000 bygel filtration on a column of TSK gel 2000 SW. It was reconstituted into proteoliposomeswith asolectin, showing its amphiphilic nature. The aminopeptidase M from liver wasfound to be efficiently inhibited by bile acids. The enzyme was almost completely inhibitedby chenodeoxycholic acid and 70-90% inhibited by cholic acid at a concentration of 6mM.The extent of inhibition by conjugated and unconjugated bile acids was in the order: unconjugated>glycoconjugated>tauroconjugated bile acid, independent of the nature ofthe substrates used. The inhibition by the various bile acids was totally reversible.Further, it was immunochemically revealed that a considerable amount of liver aminopep-tidase M was released into the bile duct. The role of the aminopeptidase M on the bilecanalicular membrane and of the enzyme released in the bile duct is discussed in relationto the effects of bile acids.
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Makoto Nakanishi, Akihiko Moriyama, Yasuko Narita, Makoto Sasaki
1989 Volume 106 Issue 5 Pages
826-830
Published: 1989
Released on J-STAGE: January 25, 2011
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The mechanism of inhibition of aminopeptidase M by bile acids was analyzed by application of the specific velocity plot that was introduced by Baici [
Eur. J. Biochem. 119, 9-14 (1981)]. Kinetic studies with three bile acids (cholic acid, deoxycholic acid, and chenodeoxycholic acid) and three substrates (Leu-Met, Leu-Gly, and Leu-
pNA) showed that the inhibition constants
Ki for the bile acids were appreciably different from each other, but that the
Ki for each was not affected by the substrates used, being 0.89-1.03mM for cholic acid, 0.42-0.66mM for deoxycholic acid, and 0.24-0.31mM for chenodeoxycholic acid. The values of the kinetic coefficient α[(apparent
Ks in the presence of inhibitor)/
Ks] for cholic acid with Leu-Met and Leu-Gly were 9.0 and 2.5, respectively. These values were very similar to those for chenodeoxycholic acid (7.0 and 2.7) but smaller than those for deoxycholic acid (21 and 11). The values of the other kinetic coefficient β[(apparent
kp in the presence of inhibitor)/
kp] were 0 except in the case of the combinations of Leu-Gly with cholic acid (0.33) and Leu-Gly with chenodeoxycholic acid (0.13). On the basis of these kinetic parameters, the inhibitions by bile acids were classified into 4 types: competitivenoncompetitive linear mixed type (1<α<∞, β=0), noncompetitivencompetitive linear mixed type (0<α<∞, β=0), pure noncompetitive type (α=1, β=0), and hyperbolic mixed type (1<α<∞, 0<β<1). In addition, studies using cholic acid and a series of dipeptides with side chains of different lengths showed that the values of α for Leu-Y peptides increased from 2.5 for Leu-Gly to 9.3 for Leu-Met, with increase in the length of the side chain of the Y residue from 0.033 to 0.60nm. From all these values, the mutual interactions of aminopeptidase M, substrates, and bile acids were analyzed and a model which shows the relative positions of the dipeptide substrate and bile acid on the enzyme molecule was proposed.
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Hideo Koga, Eiichi Yamaguchi, Kiyoshi Matsunaga, Hironori Aramaki, Tad ...
1989 Volume 106 Issue 5 Pages
831-836
Published: 1989
Released on J-STAGE: January 25, 2011
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Pseudomonas putida PpGl, which carries the CAM plasmid encoding enzymes involved in the degradation pathway of D-camphor, can utilize D-camphor as a sole carbon source. Cytochrome P-450cam and related enzymes participate in the early oxidation steps of D-camphor degradation metabolism. We cloned from a
HindIII DNA library of PpGl a 2.9 kbp CAM segment which carries the major part of
camA gene encoding NADH-putidaredoxin reductase and the entire
cam>B< gene encoding putidaredoxin. The 2.9kbp CAM segment was adjacent to the 4.27kbp
HindIII CAM segment which has been previously cloned (Koga
et al.(1986)
J. Bacterial. 166, 1089-1095). Thus, the total 7.17kbp
HindIII CAM directed all the genes responsible for early steps of D-camphor degradation,
i.e. 5-exo-hydroxycamphor dehydrogenase (
camD gene), cytochrome P-450cam (
camC), NADH-putidaredoxin reductase (
camA), and putidaredoxin (
camB). These
cam genes form an operon,
camDCAB, and are under negative control by the gene
camR located immediately upstream from the
camD gene. The total number of amino acids deduced from the nucleotide sequence is 422 for putidaredoxin reductase, and 106 for putidaredoxin.
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Naoto Yamaguchi, Takeo Takahashi, Nobuyuki Harada, Kiyoshi Takatsu
1989 Volume 106 Issue 5 Pages
837-843
Published: 1989
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Interleukin 5 (IL5), a lymphokine produced by T cells, induces differentiation of B cell chronic leukemia BCL
1-B20 cells into IgM-producing cells accompanied with growth arrest. To elucidate the intracellular mechanisms, the roles of Ca
2+ mobilization and protein phosphorylation in the activation of the cells were examined. F (ab') 2 fragment of anti-immunoglobulin (anti-Ig), which cross-links membrane-bound Ig, and calciumionophore A23187 caused a rapid increase in the intracellular free calcium concentration ([Ca
2+]
1), whereas these stimulants did not give rise to differentiation of the cells. In contrast, treatment with IL5 did not affect either [Ca
2+]
1 or the rates of Ca
2+ uptake from the outside and release from the inside of the cells. Analysis by two-dimensional gel electrophoresis revealed that the
in vitro phosphorylation of acidic 80-, 60-, and 45-kDa proteins was induced upon stimulation with IL5. Treatment with IL5 also caused a marked decrease in the
in vitro phosphorylation of an acidic 100-kDa protein which was highly phosphorylatedin the unstimulated state. Addition of phorbol 12-myristate 13-acetate (PMA) to theculture inhibited IL5-mediated differentiative responses. Therefore, these results suggest that Ca
2+ mobilization is not involved but activities of stimulatory and inhibitory kinases may be involved in the IL5-mediated differentiation process.
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Yoshihide Ohe, Hiroaki Hayashi, Koichi Iwai
1989 Volume 106 Issue 5 Pages
844-857
Published: 1989
Released on J-STAGE: June 07, 2011
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Following the previous determination of the main variant H1b of human spleen histone H1, we have determined the complete amino acid sequence of another variant, H1d. Limited chymotryptic digestion of H1d produced four fragments, I to IV, and one partial fragment I-II, as in the case of H1b. These fragments were aligned with two overlapping peptides, produced by another enzyme from the intact H1d. We also confirmed the C-terminalsequence of H1d by carboxypeptidase digestion. This H1d has an acetylated N-terminal serine, equimolar alanine or valine residue at 17, and is composed of 212 residues. The molecular weight was 21, 233 for the alanine variant and 21, 261 for the valine variant in the unmodified form. We also deduced the total sequences of H1a and H1c in a similar way, considering the maximum homology with H1b and H1d. Each N-terminal serine residue is acetylated, too. H1a consists of 222 amino acid residues and has a molecular weight of 22, 178 in its unmodified form; the H1c consists of 220 residues and has a molecular weight of 22, 218 in that form. The human spleen H1 sequences varied to about the same extent in the N-terminal 40 and C-terminal 110 residues. However, the sequences of the about 70 internal residues are well conserved between the variants. The extent of differences among the human H1 variants is similar to, or rather smaller than, those among the mammalian somatic H1 species. The implications of these differences in the sequence for H1 function are discussed from the evolutionary viewpoint.
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Hideo Oe, Nobuynki Takabashi, Etsushiro Doi, Masaaki Hirose
1989 Volume 106 Issue 5 Pages
858-863
Published: 1989
Released on J-STAGE: January 25, 2011
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A previous paper (Harris (1985)
Biochemistry 24, 7412-7418) reported the occurrence of two classes of anion binding sites in transferrin. To evaluate the locations of the two anion binding sites in relation to the two major domains of transferrin we determined the binding constants of whole ovotransferrin and its two half-molecules by means of the difference UV spectroscopic technique. Anions induced strong negative absorbance at 245nm in the order: citrate>phosphate>bicarbonate for whole ovotransferrin and the N-terminal half-molecule; and: phosphate>citrate>bicarbonate for the C-terminal half-molecule.The anion dissociation constants of the N-terminal half-molecule were consistent with lower dissociation constants, and those of the C-terminal half-molecule, with higher dissociation constants of whole ovotransferrin, indicating that the two classes of anion binding sites correspond to the binding sites in individual structural domains. Anion binding markedly protected the N-terminal half-molecule, but not the C-terminal halfmolecule from digestion with trypsin and disulfide reduction with dithiothreitol. As to the far and near ultraviolet CD spectra data, however, there was no significant difference between in the presence and absence of an anion. Therefore, the binding of an anion would induce some conformational changes which were not reflected by the CD spectrum.
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Yasuo Ogawa, Hikaru Harafuji
1989 Volume 106 Issue 5 Pages
864-867
Published: 1989
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To clarify the biological role of phosphoinositides including inositol trisphosphate (IP
3) in the skeletal muscle, we examined the Ca-releasing action on the heavy fraction of sarcoplasmic reticulum (HFSR) from bullfrog skeletal muscle of IP
3, phosphatidylinositol monophosphate (PIP), phosphatidylinositol 4, 5-bisphosphate (PIP
2), and glycerophospho-inositol 4, 5-bisphosphate (GPIP
2). Only PIP
2 caused dose-dependent Ca release. IP3 (up to 55 μM), PIP (up to 37μM), and GPIP
2(up to 33μM) were ineffective. The PIP
2-induced Ca release is due to the direct action of PIP
2, but not its metabolite (s). The properties of the PIP
2-induced Ca release are unique and cannot be accounted for by the Ca releasemechanisms already reported, such as Ca
2+-induced, ionic substitution-induced, or IP
3induced Ca release. The rate of the PIP
2-induced Ca release, however, is so slow that it may have no physiological relevance unless stimulating factors or agents exist.
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Masayuki Takahashi, Fumi Morita
1989 Volume 106 Issue 5 Pages
868-871
Published: 1989
Released on J-STAGE: January 25, 2011
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Myosin (opaque myosin) isolated from the opaque portion of scallop smooth muscle, a catch muscle, was subjected to limited digestion by trypsin during the steady-state ATPase reaction. The 200-kDa heavy chain of opaque myosin was cleaved into 125- and 74-kDa fragments. The proteolytic rate in the absence of Ca
2+ was lower than that in the presence of Ca
2+, and was similar to that in the presence of ADP and absence of Ca
2+. The results suggest that the steady-state intermediate of opaque myosin ATPase in the absence of Ca
2+ is EADP, which is consistent with the previous results based on the difference UVabsorption spectrum (Takahashi, M., Sohma, H., & Morita, F.(1988)
J. Biochem. 104, 102-107). In the presence of F-actin, the proteolytic rates were decreased, but the digestive patterns by trypsin were similar to those of myosin alone. Even in the presence of F-actin, the proteolytic rate during the ATPase reaction in the absence of Ca
2+ was lower than that in the presence of Ca
2+, and was similar to that in the presence of ADP and absence of Ca
2+.In addition, there was another trypsin-susceptible site which is probably located at 18 kDa from the N-terminal of the heavy chain. The site in the absence of Ca
2+ was hardly cleaved when ATP or ADP was present. Similar tendencies were observed even in the presence of F-actin. These findings suggest that the intermediate of opaque myosin ATPase at the steady state in the absence of Ca
2+ is EADP even in the presence of F-actin. Opaque myosin may remain as EADP
in vivo during the catch contraction which is attained after the Ca
2+ concentration decreases.
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Toshiyuki Takasago, Toshiaki Imagawa, Munekazu Shigekawa
1989 Volume 106 Issue 5 Pages
872-877
Published: 1989
Released on J-STAGE: January 25, 2011
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The phosphorylation of canine cardiac and skeletal muscle ryanodine receptors by thecatalytic subunit of cAMP-dependent protein kinase has been studied. A high-molecular-weight protein (
Mr 400, 000) in cardiac microsomes was phosphorylated by the catalyticsubunit of cAMP-dependent protein kinase. A monoclonal antibody against the cardiacryanodine receptor immunoprecipitated this phosphoprotein. In contrast, high-molecular-weight proteins (
Mr 400, 000-450, 000) in canine skeletal microsomes isolatedfrom extensor carpi radialis (fast) or superficial digitalis flexor (slow) muscle fibers werenot significantly phosphorylated. In agreement with these findings, the ryanodine receptorpurified from cardiac microsomes was also phosphorylated by cAMP-dependent protetnkinase. Phosphorylation of the cardiac ryanodine receptor in microsomal and purifiedpreparations occurred at the ratio of about one mol per mol of ryanodine-binding site. Uponphosphorylation of the cardiac ryanodine receptor, the levels of [
3H] ryanodine binding atsaturating concentrations of this ligand increased by up to 30% in the presence of Ca
2+ concentrations above 1μM in both cardiac microsomes and the purified cardiac ryanodinereceptor preparation. In contrast, the Ca
2+ concentration dependence of [
3H] ryanodinebinding did not change significantly. These results suggest that phosphorylation of theryanodine receptor by cAMP-dependent protein kinase may be an important regulatorymechanism for the calcium release channel function in the cardiac sarcoplasmic reticulum.
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Ken-ichi Nagai, David D. Roberts, Toshihiko Toida, Hajime Matsumoto, Y ...
1989 Volume 106 Issue 5 Pages
878-886
Published: 1989
Released on J-STAGE: January 25, 2011
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A novel sulfated glycosphingolipid that belongs to “globo-series” was isolated from human kidney. This lipid was purified from a pooled kidney preparation by chloroform-methanol extraction, mild alkaline treatment, DEAE-Sephadex and silicic acid column chromatographies, and preparative TLC. The structure and the properties were studied by IR spectroscopy, proton NMR spectroscopy, negative secondary ion-mass spectrometry, solvolysis, periodate oxidation, compositional and methylation analyses, monoclonal antibodies, and a sulfatide-binding protein. From the results of the above analyses, the structure of this glycolipid was proposed to be HSO
3-3GalNAc β 1-3Gal α 1-4Gal β 1-4Glc β 1-1ceramide. This sulfated lipid reacted with a monoclonal anti-SSEA-3 (stage-specific embryonic antigen-3)(MC-631)(Kannagi, R., Cochran, N. A., Ishigami, F., Hakomori, S., Andrews, P. W., Knowles, B. B., & Solter, D.(1983)
EMBO J. 2, 2355-2361), whose epitope is R-3GalNAc β 1-3Gal α 1-4Gal β 1-R', on TLC and solid-phase radioimmunoassay. This lipid also bound to the
125I-labeled sulfatide-binding protein, thrombospondin. The yield of this sulfated glycolipid was 34pmol/g of tissue, which was about 0.028, 0.16, and 18mol% of galactosyl- and lactosylceramide sulfates, and globopentaosylceramide sulfate (Nagai, K.-i., Roberts, D. D., Toida, T., Matsumoto, H., Kushi, Y., Handa, S., & Ishizuka, I.(1989)
J. Biol. Chem. 264, in press), respectively, in human kidney.
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Tohru Nakano, Saichi Matsumoto, Kohji Hanasaki, Hitoshi Arita
1989 Volume 106 Issue 5 Pages
887-893
Published: 1989
Released on J-STAGE: January 25, 2011
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Only tetraprenol (
n=4), among the (
n)-polyprenols studied, induced activation of rabbitplatelets. Tetraprenol-induced responses, including platelet aggregation, Ca
2+ mobiliza-tion, inositol phosphate formation, and arachidonic acid release, were greatly inhibited bya thromboxane A
2 (TXA
2) receptor antagonist and a cyclooxygenase inhibitor, indicating anessential role for endogenously produced TXA
2. The TXA
2-mimetic agonist U46619 inducedplatelet aggregation, Ca
2+ mobilization and phospholipase C action but did not inducearachidonic acid release. These results suggest that arachidonic acid is not released
via phospholipase C but by phospholipase A
2, and this is also supported by the finding thatphospholipase C action was inhibited by depletion of extracellular Ca
2+, while arachidonicacid release was not. Full arachidonic acid release was found to be induced by thesynergistic action of U46619 and tetraprenol. Therefore, the initial, most essential responseinduced by tetraprenol is a small arachidonic acid release by phospholipase A
2, whichresults in initial TXA
2 formation. Further action of phospholipase C as well as Ca
2+ mobilization and aggregation were induced by the initially formed TXA
2 while furtheractivation of phospholipase A
2 required the synergistic action of tetraprenol and TXA
2.
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Akira Yoshida, Haruhiko Takisawa, Takao Nakamura
1989 Volume 106 Issue 5 Pages
894-899
Published: 1989
Released on J-STAGE: January 25, 2011
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We studied the regulation of dephosphorylation of cAMP-dependent phosphorylatedproteins of isolated, permeabilized (skinned) myocardial cells from adult rat. Staurospo-rine, a potent inhibitor of protein kinase, inhibited cAMP-dependent phosphorylation ofphospholamban and troponin-I, the key proteins in the control of contraction and relaxa-tion of the myocardial cells. Staurosporine antagonized the stimulatory action of cAMP onthe spontaneous beating of the myocytes accompanied by dephosphorylation of phos-pholamban but not of troponin-I at pCa 7-8. In cold ATP dilution experiments withapparent stoppage of protein phosphorylation, dephosphorylation of phospholamban wasaccelerated both by Ca
2+ and staurosporine but that of troponin-I took place only in thepresence of Ca
2+ ion (pCa<6.5). These phenomena suggest a bi-directional regulation of dephosphorylation of the key proteins by the intracellular messengers cAMP and Ca
2+.
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Akira Hara, Makoto Nakagawa, Hiroyuki Taniguchi, Hideo Sawada
1989 Volume 106 Issue 5 Pages
900-903
Published: 1989
Released on J-STAGE: January 25, 2011
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Homogeneous indanol dehydrogenase from monkey liver catalyzed the reversible conver-sion of 3α- or 20α-hydroxy groups of several bile acids and 5β-pregnanes to the corre-sponding 3- or 20-ketosteroids. The
kcat values for the steroids determined at pH7.4 werelow, but the
kcat/
Km values for the 3-ketosteroids were comparable to or exceeded those for1-indanol and xenobiotic carbonyl substrates. The enzyme transferred the 4-pro-
R-hydrogen atom of NADPH to the 3β- or 20β-face of the ketosteroid substrate. Competitiveinhibition of the hydroxysteroid dehydrogenase activity of the enzyme by medroxyproges-terone acetate, hexestrol, and 1, 10-phenanthroline suggests that both 1-indanol andhydroxysteroid are oxidized at the same active site on the enzyme. The specific inhibitor ofthe enzyme, 1, 10-phenanthroline, suppressed the 3α-hydroxysteroid dehydrogenase activ-ity in the crude extract of monkey liver by 50%. The results strongly suggest that indanoldehydrogenase acts as a 3 (20)α-hydroxysteroid dehydrogenase in the metabolism of certainsteroid hormones and bile acids.
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Nobuaki Okumura, Kouichi Takimoto, Masato Okada, Hachiro Nakagawa
1989 Volume 106 Issue 5 Pages
904-909
Published: 1989
Released on J-STAGE: January 25, 2011
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With purified preparations of basic fibroblast growth factor (bFGF), we studied the effect of its growth-promoting activity on C6 glioma cells. We also examined with its antibody whether the cultured glioma cells could produce it. It was shown that bFGF stimulated the DNA synthesis and proliferation of C6 glioma cells in serum-free medium, and that the activity was potentiated by heparin, the bFGF concentrations for half-maximal stimulation being 0.2 and 5ng/ml in the presence and absence of heparin, respectively. This effect of heparin was dose-dependent and was half-maximal at 0.5μg/ml. Next, we raised the antiserum against bFGF and detected a single immunoreactive band from extracts of C6 glioma cells by immunoblot analysis. The immunoreactive substance was partially purified on a heparin-Sepharose column and was shown to stimulate the DNA synthesis of C6 glioma cells. On the basis of its immunoreactivity, molecular weight, affinity for heparin, and growth-promoting activity, this substance was identified as bFGF. The content of bFGF in the cells was elevated as the cell density increased, but no immunoreactivity was detected in the conditioned medium of the cells. These results suggest that C6 glioma cells produce and store bFGF which is potent in stimulating their own growth.
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Inhibition by Sulfated Glycosaminoglycans and Activation by Polyamines and Basic Peptides Including a Polylysine-Containing Segment of the c-Ki-ras 2 Protein
Kazuyuki Sugahara, Mayumi Nakamura, Junko Nagisa, Masao Masuda, Youich ...
1989 Volume 106 Issue 5 Pages
910-919
Published: 1989
Released on J-STAGE: January 25, 2011
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The regulatory mechanisms for the glycosaminoglycan sulfotransferases in fetal calf serum were investigated. The enzymes examined were those which transfer sulfate from 3'-phosphoadenosine 5'-phosphosulfate to 1) position 6 of the internal
N-acetylgalactosamine units of chondroitin, 2) position 6 of galactose units of keratan sulfate, and 3) position 2 (an amino group) of glucosamine units of heparan sulfate. The former two enzymes were activated by spermidine, spermine, protamine, and poly L-lysine. All the enzymes were strongly inhibited by heparin and dextran sulfate, whereas only the chondroitin 6-
O-sulfotransferase was inhibited by sulfated galactosaminoglycans. The inhibition of this enzyme by the sulfated glycosaminoglycans was abolished by polylysine, indicating that the activation by polylysine is partly due to the neutralization of endogenous acidic inhibitors, including sulfated glycosaminoglycans. Affinity chromatographic studies demonstrated that heparin specifically binds to the three enzymes, which have anionic isoelectric points, and that chondroitin 6-sulfate, spermine, and polylysine bind to the former two enzymes under physiological conditions. Thus, the activation by spermine and polylysine as well as the inhibition by sulfated glycosaminoglycans also appears to occur through their binding to the enzymes. Studies with synthetic lysine oligomers and an affinity-purified (approximately 700-fold) fraction containing the former two enzymes indicated that the pentamer is the minimum unit required for the activation. A synthetic peptide, containing six consecutive lysines at the carboxy terminus of the human c-Ki-ras 2 protein, also regulated the two enzyme activities at micromolar concentrations. The possible physiological implications of the oberved effects of these regulatory substances on the glycosaminoglycan sulfotransferases are discussed in relation to glycosaminoglycan synthesis during the proliferation, differentiation, and transformation of cells. The possibility of sulfated glycosaminoglycans being enzyme regulators is also discussed.
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Senarath B. P. Athauda, Masao Tanji, Takashi Kageyama, Kenji Takahashi
1989 Volume 106 Issue 5 Pages
920-927
Published: 1989
Released on J-STAGE: January 25, 2011
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Six pepsinogen isozymogens, including five forms of pepsinogen A (PGA) and an apparently single form of pepsinogen C (PGC), were isolated simultaneously from the purified total pepsinogen fraction of human gastric mucosa by fast protein liquid chromatography on a Mono Q column, and their NH
2-terminal amino acid sequences and some other properties were compared. Upon activation at pH 2.0, all the isozymogens were converted to the corresponding pepsins in a stepwise manner through intermediate forms. The activation rates and the cleavage sites in the activation peptide segment to generate intermediate forms were significantly different among the isozymogens. The NH
2-terminal 85-residue amino acid sequences of these isozymogens were determined, including the sequences of the activation peptide segments and the NH
2-terminal regions of the corresponding pepsins. Differences in amino acid sequence were found at positions 43 and 77 among the pepsinogen A isozymogens; the residue at position 43 was Lys in PGA-5, PGA-4, and PGA-3a, and Glu in PGA-3 and PGA-2, and the residue at position 77 was Leu in PGA-5 and PGA-4 and Val in PGA-3 and PGA-2. Phosphate was not found in any of the isozymogens. The corresponding pepsins also showed significant variations in properties such as specific activities toward synthetic and protein substrates, pH dependence of activity, susceptibility to various inhibitors, and thermal and alkaline stabilities.
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Tadayoshi Imai
1989 Volume 106 Issue 5 Pages
928-937
Published: 1989
Released on J-STAGE: January 25, 2011
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A particulate NMN glycohydrolase of rabbit spleen was solubilized with Triton X100 and purified approximately 100-fold. The enzyme was shown to have a pH maximum of 6.5, a
Km of 0.25 mM, a
Vmax of 5.3 μmol/min/mg protein, an activation energy of 7.9kcal/mol, and a molecular weight of approximately 400, 000. Both of the purified and the particulate enzymes exhibited identical catalytic properties with respect to substrate specificity, activation energy, pH profile and exchange reaction with nicotinic acid, except that the purified enzyme was highly activated with Triton X100 as compared with the particulate enzyme; it appears that the purified enzyme possesses the same catalytic properties as the enzyme present in the tissue and that solubilization does not significantly alter the native protein. In addition to catalytic activity with NMN, the rabbit spleen enzyme catalyzed an irreversible hydrolysis with NAD and NADP, exhibiting catalyzing activity ratios of NMN: NAD: NADP=1.00: 1.45: 0.44 and
Vmax/
Km ratios of 1.00: 1.7: 2.3, respectively. These ratios of activity remained constant throughout purification of the enzyme and no separation of these activities was detected. Mutually competitive inhibition of the enzyme with
K1 values similar to
Km, and identical rates of thermal denaturation of the enzyme and activity-pH profiles with NMN or NAD indicated the hydrolysis of the C-N glycosidic linkage of the pyridine nucleotides to be catalyzed by the same enzyme. The enzyme was less specific for the purine structure of the substrate dinucleotides but was stereospecific for the glycosidic linkage cleaved. Nicotinamide riboside, the nicotinic acid analogs and the reduced forms were not hydrolyzed. A linear noncompetitive inhibition of NMN hydrolysis with nicotinamide indicated an ordered Uni-Bi mechanism in which nicotinamide was the first product released from the enzyme. A property that the rabbit spleen enzyme appears to share with other NAD glycohydrolases is the transglycosidation reaction. The ratio of transglycosidation reaction vs. hydrolysis catalyzed by the enzyme in the presence of NMN and nicotinic acid indicated that the enzyme could function as a primary transglycosidase rather than a hydrolytic enzyme
in vivo.
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Tadayoshi Imai
1989 Volume 106 Issue 5 Pages
938-948
Published: 1989
Released on J-STAGE: January 25, 2011
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Properties of the transglycosidation reaction catalyzed by rabbit spleen pyridine nucleotide glycohydrolase were characterized using a modified cyanide addition method by which initial velocities of the transglycosidation (νT) and hydrolysis (νH) of pyridine nucleotides could be monitored simultaneously.(1) The νT was routinely determined with NMN and nicotinic acid used as substrates and was observed to be maximal at pH 6. Arrhenius plots of νT and νH indicated that the activation energies for transglycosidation and hydrolysis were 8.7 and 10.7 kcal/mol, respectively.(2) The enzyme showed a broad spectrum of substrate specificity with respect to both pyridine nucleotides and bases. Of the compounds tested, NMN and nicotinic acid were shown to be the best substrates when compared on the basis of
Vmax/
Km values. Kinetic constants for the enzyme-catalyzed transglycosidation reaction were as follows;
Km (NMN) =0.53mM,
Km (nicotinic acid) , as acid form=15mM, apparent
Vmax=7.8μmol/min/mg protein, in the presence of 0.2M nicotinic acid.(3) The ratio of νT/νH was sbown to be dependent on botb pH and nicotinic acid concentration. However, transglycosidation versus hydrolysis partition at a fixed pH was constant regardless of the nicotinic acid concentration employed and approximated to be 1.2×10
4 at the maximal pH.(4) Nicotinamide, one of the most potent inhibitors for the enzymecatalyzed hydrolysis, was shown to function as an antagonist for the transglycosidation reaction with NMN and nicotinic acid used as substrates. The inhilbition mechanism with nicotinamide was purely noncompetitive with respect to nicotinic acid; on the other hand, the double reciprocal plot of the transglycosidation velocity against NMN concentration at a fixed concentration of nicotinamide was concave downwards.(5) The equilibrium constant of the reaction, NMN +3-acetylpyridine-3-acetylpyridine mononucleotide+nicotinamide, was 0.61, whereas the conversion of NMN with nicotinic acid to nicotinic acid mononucleotide was essentially irreversible. These enzymatic properties of rabbit spleen pyridine nucleotide glycohydrolase suggested that the enzyme should not function as a glycohydrolase but as a transglycosidase and could serve in an important mechanism for an alternative biosynthetic pathway of nicotinic acid mononucleotide, one of the precursors for NAD synthesis, when nicotinic acid is supplied.
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