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Volume 114 (1993)
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Volume 114, Issue 3
Displaying 1-25 of 25 articles from this issue
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Takeo Ohichi, Tetsu Hozumi, Sugie Higashi-Fujime1993 Volume 114 Issue 3 Pages 299-302
Published: 1993
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe in vitro motility of enzymatically cleaved S-1 was investigated with a novel procedure for the coating of a glass surface with poly-L-lysine. On a lysine-coated surface, HMM and S-1 (mixture of 95 and 91 kDa fragments) moved F-actin filaments at average velocities of 4.41 and 1.54 fzm/s, respectively, which are similar to those, 5.96 and 1.47 pm/s, on a collodion-coated surface, respectively. Myosin S-1 composed of the 91 kDa polypeptide could move F-actin at 1.15 μm/s. Tryptically cleaved chymotryptic S-1 could move F-actin filaments at an average velocity of 2.8 μm/s: about 2 times that in the case of S-1. This activation of the sliding movement on cleaved S-1 arises partly from reduction of the drag force due to the low affinity of cleaved S-1 for F-actin. The motility of S-1 cleaved at other sites was also examined on a lysine-coated surface.View full abstractDownload PDF (2416K) -
Kimiko Asano, Masahiko Nakamura, Tsuneko Sato, Hisashi Tauchi, Akira A ...1993 Volume 114 Issue 3 Pages 303-306
Published: 1993
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSMitochondria) DNA contents were measured using non-radioactive DNA probes for specific mtDNA sequences. The nuclear repetitive DNA sequence, LINE, was used as an internal standard marker. Age-dependent decreases in the mtDNA/nucDNA ratio in rat hepatic cells were confirmed with this method and, furthermore, a similar decrease was found in cardiac muscles. On the other hand, in thigh skeletal muscles such a decrease was not observed, slight increases being rather found in 18- to 24-month-old rats. The possible relationship of the decrease in mtDNA and the mechanism underlying animal senescence was discussed.View full abstractDownload PDF (392K) -
Takeshi Shigenaga, Yoko Takayenoki, Shoko Kawasaki, Noriaki Seki, Tats ...1993 Volume 114 Issue 3 Pages 307-316
Published: 1993
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWe designed a method for separating two types of granules, a smaller (S) but dense and a larger (L) but less dense granule from hemocytes of the horseshoe crab (Tachypleus tridentatus), using continuous sucrose density gradient centrifugation. The isolated L-granules contained at least three clotting factors plus a clottable protein, coagulogen, as the major component. The known anti-lipopolysaccharide factor and 7 additional unknown protein components were also present in the L-granules. Two known natural substrates, Pro-rich protein and 8.6 kDa protein, for limulus transglutaminase [Tokunaga, F., Yamada, M., Miyata, T., Ding, Y. -L., Hiranaga-Kawabata, M., Muta, T., Iwanaga, S., Ichinose, A., & Davie, E. W. (1993) J. Biol. Chem. 268, 252-261] were present in the L-granules. On the other hand, the isolated S-granules contained antimicrobial tachy-plesins I and II (17 amino acids in length) as the major component, in addition to 6 unidentified proteins with molecular masses of less than 30 kDa. The structural analyses of tachyplesin analogs indicated that all these peptides of mature form are stored in the S-granules, together with a processing intermediate containing the COOH-terminal GlyLys sequence. We also found an Arg-rich protein of 22 kDa and a Leu-rich protein of 30 kDa in S-granules. Based on these observations, we speculate that protein components in L-granules, which probably contain all the factors essential for the limulus clotting system, participate in immobilization of invading microbes and that factors in the S-granules containing tachyplesins contribute to a self-defense system against invaders.View full abstractDownload PDF (5396K) -
Masao Yukawa, Masato Sakon, Jun-ichi Kambayashi, Ei-ichi Shiba, Tomio ...1993 Volume 114 Issue 3 Pages 317-323
Published: 1993
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSAn endogenous activator of 20 S proteasome was purified from human platelets and its effect on three peptidase activities of proteasome was studied. This activator had a molecular weight of 170 kDa, and was composed of 32 kDa polypeptides as determined by SDS-PAGE. It was highly labile upon heat treatment (56°C, 20 s) and proteinase (pronase CB) digestion. Suc-LLVY-MCA degrading activity of the platelet proteasome showed positive cooper-ativity between two or more catalytic sites because the coefficient was 1.54 when analyzed by use of the Hill plot. The endogenous activator increased Vmax and caused a loss of cooperativity. The plot of reaction velocity as a function of activator concentration yielded a saturation curve, implying the binding of the activator to proteasome. Boc-LTR-MCA degrading activity followed Michaelis-Menten kinetics. The activator enhanced the activity by increasing Vmax, and decreasing Km. In contrast, CBz-LLE-2NA degrading activity could not be analyzed according to any kinetic scheme reported so far. The activator stimulated this activity at lower substrate concentrations (below 200 μM), while it inhibited the activity at higher substrate concentrations (400-800μM). It is concluded from these findings that the endogenous protein activator may regulate the intracellular proteasome activity by functioning as a positive allosteric effector.View full abstractDownload PDF (1577K) -
Torn Hisabori, Gunther Kothen, Heinrich Strotmann1993 Volume 114 Issue 3 Pages 324-328
Published: 1993
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSIrradiation of isolated chloroplast thylakoids with TNP-ADP results in non-covalent binding and covalent incorporation of a reaction product of TNP-ADP formed by photo-synthetic reduction into the so-called “tight” nucleotide binding site of CF1 [Ponse et al. (1992) Z. Naturforsch. 47 c, 264-274]. CF1 extracted from thus-loaded thylakoid membranes yielded maximal incorporation of 1 mol/mol of CF1. Almost half had the covalent bond with CF1. In experiments with TNP-[14C] ADP, radioactivity was detected almost equivalently on α and β subunits, suggesting that the binding site may be at the interface between a and β subunits. Enzyme activities of the thylakoid membrane-bound enzyme after covalent labeling were measured. Inhibition, ranging from 20 to 25%, was less than expected from the percentage of CF1 molecules labeled (40-50%). It is suggested that only half of the labeled enzymes, probably those with the nucleotide analog linked to the β subunit, are inactive.View full abstractDownload PDF (1149K) -
Toyoki Amano, Takayoushi Wakagi, Tairo Oshima1993 Volume 114 Issue 3 Pages 329-333
Published: 1993
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSMembranes of Sulfolobus acidocaldarius, a thermoacidophilic archaebacterium, show novel enzymatic activities to hydrolyze PPi, ATP, and ADP at an optimal pH of 3, equal to the growth optimum. The activity increased by about 2-fold on addition of PPi and/or P1 to the growth medium, when yeast extract and casamino acids were removed. The enzyme which hydrolyzes PPi at pH 3 was solubilized and purified by successive chromatogra-phies. The final preparation showed a 26 kDa single band on SDS-PAGE, and a molecular mass of 35 kDa on gel permeation chromatography. The Km and Vmax for PPi were 0.16mM and 33μmol Pi released/min/mg at 55°C. ATP and ADP were also good substrates. Divalent cations were not essential for activity. Substrate inhibition at more than 5mM PPi, ATP or ADP was observed. AMP, glucose-6-phosphate, and p-nitrophenyl phosphate were not hydrolyzed at all. The activity was 4-fold stimulated by addition of the lipid fraction extracted from the organism.View full abstractDownload PDF (1395K) -
Hiroyuki Shimizu, Kou Ochiai, Kazuhiro Ikenaka, Katsuhiko Mikoshiba, S ...1993 Volume 114 Issue 3 Pages 334-338
Published: 1993
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSSugar chains were liberated from mouse tissues (liver, heart, spleen, kidney, thymus, cerebrum, cerebellum, and brain stem) by hydrazinolysis. After acetylation of the free amino groups, the sugar chains released were pyridylaminated. Pyridylamino (PA-) derivatives of the sugar chains from each tissue were fractionated by anion-exchange high-performance liquid chromatography (HPLC) according to their negative charges. Fractions containing PA-neutral sugar chains thus obtained were separated successively by size-fractionation HPLC and reversed-phase HPLC. Two sugar chains that were more abundant in neural tissues than in other tissues were purified. The structures of the two sugar chains were determined by sugar composition analysis, sequential exoglycosidase digestion, and methylation analysis. The proposed structures are shown below. A structure with n=1 was found in the cerebrum, cerebellum, and brain stem. A structure with n=0 was abundant in the cerebrum and brain stem, but less so in the cerebellum.View full abstractDownload PDF (450K) -
Masahiro Iwakura, Tomokuni Kokubu1993 Volume 114 Issue 3 Pages 339-343
Published: 1993
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA cysteine residue with a short amino acid chain spacer was attached to the C-terminal end of mutant dihydrofolate reductase [DHFR(C152E)] by a recombinant DNA technique. By using 5, 5'-dithiobis (2-nitrobenzoic acid) (DTNB) as a SH reagent, it was determined that the reactivity of the SH group of the introduced C-terminal cysteine was much higher than that of cysteines with the wild-type DHFR (C 85 and C 152), probably due to an increase of the solvent accessible surface area of the SH group. By utilizing the increased reactivity of the SH group of the C-terminal cysteine, the engineered DHFR was effectively immobilized to a thiopropyl-Sepharose gel. The amount of immobilized DHFR was estimated to be ca. 6mg/g of dried gel, and the activity of the bound DHFR was comparable to that of the free enzyme. Thus, the attachment of the cysteine residue to the C-terminal was extremely useful for immobilization of the enzyme.View full abstractDownload PDF (544K) -
Masaru Nakamoto, Masayuki Ozawa, Sonia D. Jacinto, Tatsuhiko Furukawa, ...1993 Volume 114 Issue 3 Pages 344-349
Published: 1993
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSHeparin binding protein-44 (HBP-44) is a heparin binding protein of 44 kDa, found by cDNA cloning using antibodies against teratocarcinoma glycoproteins [Furukawa, T. et al. (1990) J. Biochem. 108, 297-302]. The N-terminal sequence analysis reported in this publication establishes the structure of its mature form. Immunohistochemical staining revealed that HBP-44 was located in the tubular brush border of the kidney. HBP-44 formed a complex with brushin, a high molecular weight (450 kDa) glycoprotein antigen common to the kidney and teratocarcinoma, but not with OR8 antigen, another antigen (350 kDa) of the same category. Brushin was shown to be the mouse counterpart of rat Heymann nephritis antigen, called gp330. The association between HBP-44 and brushin was revealed not only by co-precipitation upon indirect immunoprecipitation, but also by ligand blotting with HBP-44-maltose binding protein fusion protein. Calcium ion stabilized the association. Disulfide bonds in brushin seemed to be necessary for the complex formation, since reductive cleavage of the bonds resulted in failure of the protein to associate with HBP-44 in a ligand blotting experiment. Association of HBP-44 with brushin occurred both in teratocarcinoma cells, in which these molecules are mainly located in extraembryonic endoderm cells, and in the kidney, suggesting that the complex has an unknown common function in the renal tubular brush border and the extraembryonic endoderm.View full abstractDownload PDF (2579K) -
Shinobu Nagasawa, Kazuya Ishige, Takeshi Mizuno1993 Volume 114 Issue 3 Pages 350-357
Published: 1993
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA variety of adaptive response systems in prokaryotes often involve two families (two components) of signal transduction proteins, namely, sensory kinases and response-regulators. To extend the list of such sensor/regulator genes for Escherichia coli, we adopted a random screening method in this study. In particular, we isolated a series of recombinant plasmids that are able phenotypically to suppress mutational lesions of both the envZ and phoR/creC genes, each of which encodes a well-characterized sensory-kinase. Among the recombinant plasmids thus isolated, two clones (named pSN11 and pSN25) were subjected to characterization in detail. These analyses allowed us to identify the genetic loci specifying novel members of the sensor/regulator families. One (pSN11) is located around 45min on the E. coli genetic map, that contains two adjacent coding-sequences (baeS and baeR). The other (pSN25) is located around 93min of the genetic map, that also comprises two adjacent coding-sequences (basS and basR). These two pairs of gene-products, thus newly identified, were revealed to belong to typical members of the sensor/regulator families. Furthermore, they were demonstrated to exhibit the in vitro phosphotransfer reaction in the presence of ATP, that is also a characteristic of the sensory-kinase and response-regulator proteins.View full abstractDownload PDF (1754K) -
Masatoshi Muraoka, Toshihiko Hayashi1993 Volume 114 Issue 3 Pages 358-362
Published: 1993
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSStudies of intact type IV collagen from deposits of cultured cells or from tissues in culture, or more recently, isolated from EHS tumor have suggested that type IV collagen molecule is composed of two procollagen-like polypeptides (Mr=185k and 170 k). We show that the major components of intact type IV collagen in bovine lens capsule are three polypeptides, two with sizes (Mr=180k and 175 k) comparable to those of the procollagen-like polypep-tides and one with a smaller size (Mr=160k). Both CNBr peptide mapping and electropho-retic analysis by utilizing a gel containing urea showed that the 180 k polypeptide and the 160 k polypeptide are chemically and genetically very similar to each other, but that the 175 k polypeptide is chemically distinct from the other two polypeptides. It is unlikely that the 160 k polypeptide resulted from cleavage of the 180 k polypeptide during experimental manipulation, since a change of the acidic pH of extraction buffer to neutral pH, storage of the acid extract in acid for a prolonged time or incubation of the acid extract at 80°C after neutralization gave rise to essentially no change in relative amount or size of the three polypeptides.View full abstractDownload PDF (2474K) -
Tomohide Uno, Yoshio Imai, Masahiko Nakamura, Noriaki Okamoto, Tomohik ...1993 Volume 114 Issue 3 Pages 363-369
Published: 1993
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSProline-480 and proline-481 are highly conserved in the P450 2 family. When these successive prolines of P450 2C2 were replaced by Ala-Thr, its activities of laurate (ω-1)-hydroxylation and benzphetamine N-demethylation were lost. On the other hand, the mutated P 450 which retained one of the proline residues was 60-100% active in the (ω-1)-hydroxylation and 100-120% active in the N-demethylation. Pro-Pro (480, 481) to Ala-Thr mutated P 450 2C14 was also inactive in testosterone 16α-hydroxylation and benzphetamine N-demethylation, the activities of the wild-type P 450 2C14. In the carboxy-terminal region of the P 450 2C subfamily, the sequence of P 450 2C2 from residues 469-473 is noticeably different from those of the other members of this subfamily. The Ser-473 to Val mutation of P 450 2C2 caused a decrease in the (ω-1)-hydroxylase activity to one-fifth but the N-demethylase activity was not much affected. When the sequence of P 450 2C2 from residues 469-473 was replaced by that of P 450 2C14 (mutation at four residues), the 16 α-hydroxylase and N-demethylase activities were increased by seven- and three-fold, respectively, and the (ω-1)-hydroxylase activity was decreased to one-third. The mutated P 450 2C2, in which the carboxy-terminal four residue sequence or the proline cluster adjacent to the amino-terminal membrane-anchor signal sequences was deleted, was not accumulated in yeast cells transformed with plasmids directing the synthesis of such P 450 s. The results obtained here suggest that the successive prolines play an important role in forming the local structure which is essential to the catalytic activity, that residues 469-473 participate in the contacts between the substrate and protein molecules, and that the carboxy-terminal few residues and the amino-terminal proline cluster contribute to stabilization of the conformation of P 450 2C2 protein.View full abstractDownload PDF (1664K) -
Hiroshi Kawaguchi, Kenji Inagaki, Yasuyuki Kuwata, Hidehiko Tanaka, Ta ...1993 Volume 114 Issue 3 Pages 370-377
Published: 1993
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESS3-Isopropylmalate dehydrogenase encoded by the Thiobacillus ferrooxidans leuB gene was purified to homogeneity from Escherichia coli cells harboring a recombinant plasmid containing the leuB gene. The native enzyme molecule is a dimer of molecular weight 38, 000. The Km value for 3-isopropylmalate was estimated to be 26 μM and that for NAD+ 0.8mM. The presence of K+ or NH4+ is essential for the enzyme reaction. The enzyme is activated about 4-fold by the addition of 1.0mM Mg2+ or Co2+. The optimum pH and temperature for the activity are 9.0 and 60°C, respectively. The properties of the enzyme are similar to those of the Salmonella typhimurium and Thermus thermophilus enzymes, except for substrate specificity. T. ferrooxidans 3-isopropylmalate dehydrogenase is able to utilize D- and L-malate as substrates in addition to 3-isopropylmalate. Sequencing of subcloned DNA revealed that the leuB gene consists of a 1, 074 by open reading frame and encodes 358 amino acid residues corresponding to the subunit (38, 462 Da). The amino acid sequence of 3-isopropylmalate dehydrogenase from T. ferrooxidans and those of some heterotrophic microorganisms have high homology.View full abstractDownload PDF (1540K) -
Yutaka Inaguma, Sachiyo Goto, Haruo Shinohara, Kaori Hasegawa, Kunihir ...1993 Volume 114 Issue 3 Pages 378-384
Published: 1993
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe two small stress proteins, HSP 27 and αB crystallin, are expressed widely in normal rat tissues and abundantly in skeletal muscle. In order to clarify the physiological significance of these stress proteins, the changes in their levels were determined immunochemically, in the slow-twitch soleus muscle and fast-twitch extensor digitorum longus muscle or rectus femoris muscle of growing rats, and in those of adult rats during denervation and tenotomy. HSP27 was quantitated by specific immunoassay, similar to that for all crystallin, with antibodies raised in rabbits against purified rat HSP27. In adult rats, HSP27 was present at high levels in tissues composed of striated muscle, and it was present at much higher levels in the soleus muscle than in the rectus femoris or extensor digitorum longus muscle, as is αB crystallin. However, in rats of perinatal age (from prenatal day 2 to postnatal day 3), levels of HSP27 in the rectus femoris muscle were enhanced like those in the soleus muscle, reaching the maximum levels at postnatal day 3. Thereafter HSP27 in the fast-twitch muscle showed a steep decrease. The increase in αB crystallin in the hindlimb muscles was also observed in the perinatal period. However, αB crystallin concentrations in the soleus muscle of perinatal rats were as low as those in rectus femoris muscle. The transection of the sciatic nerve resulted in decreases in the levels of HSP27 and αB crystallin in the soleus muscle of adult rats, together with increases in the levels of the two proteins in the extensor digitorum longus muscle. The decrease in HSP27 and αB crystallin in the soleus muscle was also observed after tenotomy. However, there was no increase in the concentrations of the two proteins in the tenotomized extensor digitorum longus muscle. These results suggest that the two small stress proteins in the hindlimb muscle, in particular HSP27, are induced by events associated with birth, and that the expression of HSP27 and αB crystallin in the skeletal muscles is controlled similarly in adult rats.View full abstractDownload PDF (1948K) -
Munehiko Asayama, Yasuo Kobayashi1993 Volume 114 Issue 3 Pages 385-388
Published: 1993
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSSpoOA is both a positive and a negative transcriptional regulator which plays a very important role in sporulation initiation in Bacillus subtilis. Its N-terminal amino acid sequence is homologous to that of regulator proteins of the two-component regulatory systems involved in signal transduction in bacteria. Phosphorylation of SpoOA through phosphorelay has been reported by Burbulys et al. (1991). In this study, we found that (i) SpoOA is phosphorylated effectively with phospho-EnvZ* (N-terminal truncated EnvZ), which is a heterologous osmotic sensor protein in Escherichia coli, and (ii) a phosphorylation deficient mutant of SpoOA protein is completely defective in initiating sporulation. These results suggest that SpoOA phosphorylation may be an essential event in signal transduction of sporulation in B. subtilis and the signal transduction mechanism has a common feature in Gram-positive and Gram-negative bacteria.View full abstractDownload PDF (1157K) -
Akira Tachibana, Toshio Tanaka, Makoto Taniguchi, Susumu Oi1993 Volume 114 Issue 3 Pages 389-392
Published: 1993
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe catalytic properties of geranylgeranyl diphosphate (GGPP) synthase [EC 2. 5. 1. 29] purified from Methanobacterium thermoformicicum SF-4 were studied by kinetic procedures. The plots of 1/v versus 1/[S] and inhibition patterns by enzyme reaction products, PPi and GGPP, showed that the GGPP synthase reaction mechanism is an ordered-sequential Bi Bi one. Monovalent cations at low concentration (0.05M) enhanced the enzyme activity, but at high concentration (0.4M) they were inhibitory, except for K+. The K+ ion was found to be a modifier forming a parallel reaction pathway and accelerated the binding of substrates to the enzyme, especially the binding of isopentenyl diphosphate (IPP). When substrate concentrations are near the Km values, the rate-limiting step of the GGPP synthase reaction may be the substrate-binding step, probably the IPP-binding step, rather than the conversion step of the enzyme-farnesyl diphosphate-IPP complex to the enzyme-PPi-GGPP complex.View full abstractDownload PDF (343K) -
Sachiko Murase, Yasushi Kawata, Noboru Yumoto1993 Volume 114 Issue 3 Pages 393-397
Published: 1993
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe guanidine-HCl (Gu-HCl)-induced denaturation of a tetrameric enzyme, aspartase from Escherichia coli has been studied by size-exclusion chromatography, and circular dichroism and fluorescence spectroscopies. The size-exclusion analysis showed that in the presence of 0.4M Gu-HCl, the enzyme has a dimeric structure with 45% of the native activity. The fluorescence and CD studies showed that only a small change occurred in the secondary and tertiary structures in 0.4M Gu-HCl. In the range of 0.4 to 1M Gu-HCl, decrease in the activity was observed as the secondary and tertiary structures were disrupted, whereas the dimeric enzyme did not dissociate into inactive monomer until 1M Gu-HCl. When the enzyme was denatured in less than 1M Gu-HCl, more than 90% of the original activity was recovered from the renaturation reaction, indicating that the dissociation process from tetramer to dimer is reversible. In contrast, the renaturation yield was 43% when the enzyme was diluted from more than 1M Gu-HCl, indicating that the process of dissociation into monomer is not reversible. Thus, we identified an active dimeric form as a denaturation intermediate in this study, although the intermediates (including dimer) that were detected in renaturation experiments at low temperature were inactive, as reported previously [Imaishi, H., Yumoto, N., & Tokushige, M. (1989) Physiol. Chem. Phys. Med. NMR 21, 221-228].View full abstractDownload PDF (467K) -
Hidenori Yamada, Tadashi Ueda, Taiji Imoto1993 Volume 114 Issue 3 Pages 398-403
Published: 1993
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSFor the stabilization of a protein against irreversible denaturation caused by rapid reaction of the unfolded form of the protein (kinetic stabilization), the free energy change of activation for unfolding should be increased. First, we demonstrated that this strategy was effective to stabilize a protein against protease digestion. For kinetic stabilization, it is important to stabilize a protein at a site where the local structures are largely unfolded in the transition state for unfolding. We developed a method to find such sites by comparison of the thermodynamic stabilities and the unfolding rate constants between unmodified and modified proteins. Application of this method to analyze the transition state of hen-egg lysozyme using some chemically modified derivatives is also described. Moreover, it was confirmed that the protease digestion method is superior to the relaxation method for estimation of the unfolding rate constant. Namely, the protease digestion method may be useful in analyzing the transition state of protein unfolding.View full abstractDownload PDF (676K) -
Francisco J. G. Muriana, Valentina Ruiz-Gutierrez, José Bolufer1993 Volume 114 Issue 3 Pages 404-407
Published: 1993
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe hepatopancreas and muscle of the prawn, Penaeus japonicus, were analyzed as to the phospholipid fatty acid composition. The most important lipids in the hepatopancreas are triglycerides and phospholipids, while the lipids in muscle are principally phospholipids and free cholesterol. Fatty acids such as palmitic (16:0), stearic (18:0), oleic (18:1 n-9), eicosapentaenoic (20:5 n-3), and docosahexaenoic (22:6 n-3) were prominent in the total phospholipid fatty acid composition. However, phosphatidylethanolamine appeared to be particularly rich in n-3 polyunsaturated fatty acids, sphingomyelin in saturated fatty acids (mainly 18:0), and phosphatidylinositol in monounsaturated fatty acids (mainly 18:1 n-9). Our data indicated that each phospholipid class is characterized by a specific paraffin chain composition.View full abstractDownload PDF (455K) -
Yoshiyuki Horio, Yasutake Mori, Ichiro Higuchi, Katsumi Fujimoto, Seij ...1993 Volume 114 Issue 3 Pages 408-414
Published: 1993
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe histamine H1 receptor gene was isolated from a guinea-pig gene library. The gene contains no introns and encodes a protein of 488 amino acid residues. The structure of the guinea-pig histamine H1 receptor is predicted to contain seven putative transmembrane regions, which are similar to those of receptors coupling with GTP binding proteins. Although the third intracellular domain, the predicted binding site for the GTP binding protein, showed only 50% identity with those of the bovine and rat H1 receptors, the expressed guinea-pig H1 receptor was fully able to bind with [3H] mepyramine. Northern blot analysis indicated that the cerebrum, cerebellum, lung, adrenal, intestine, and heart expressed 3.3 kb guinea-pig H1 receptor mRNA. Expression of histamine H1 mRNA of guinea-pig peripheral organs was greater than that of rat organs, suggesting the high sensitivity of guinea-pig organs as to histamine is due to the contents of histamine H1 receptor mRNA. In addition, the lung, adrenal, intestine, and heart expressed 3.9 kb mRNA, In situ hybridization showed that the hippocampus, cerebral cortex, thalamus, and granular layer of the cerebellum each contained a large amount of histamine H1 receptors. Southern blot analysis showed that there was another gene quite similar to the cloned histamine H1 receptor gene.View full abstractDownload PDF (3034K) -
Kathleen M. Botham, Maria José, Begpña Ochoa1993 Volume 114 Issue 3 Pages 415-420
Published: 1993
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe activities of a lysosomal acid and two neutral cholesteryl ester hydrolases (microsomal and cytosolic) found in the mammary gland were studied in subcellular fractions prepared from tissue from rats at various stages of pregnancy, lactation, and after weaning the pups. The relationship between the activities of these enzymes and that of acyl coenzyme A: cholesterol acyl transferase (ACAT) was also investigated. Acid cholesteryl ester hydrolase activity was increased considerably in glands from lactating as compared to pregnant animals, and was sharply decreased 2 days after weaning. The microsomal neutral cholesteryl ester hydrolase activity followed a similar pattern, but took longer to return to pre-lactating values on weaning. In contrast, the activity of the cytosolic neutral cholesteryl ester hydrolase increased throughout pregnancy, then remained relatively constant in lactation and for 2 days after weaning. At 8 days post-weaning, however, activity was markedly decreased. No correlation between the changes in the activities of any the cholesteryl ester hydrolases and ACAT during mammary gland development was detected. The microsomal neutral cholesteryl ester hydrolase activity showed a strong positive linear correlation with the unesterified and esterified cholesterol content of the microsomal fraction. No relationship, however, was found between the cytosolic neutral cholesteryl ester hydrolase and the level of either form of cholesterol in the cytosol. In addition, there was no correlation between ACAT activity and microsomal cholesterol concentrations. These results provide evidence for an important role for the enzymes responsible for cholesteryl ester synthesis and hydrolysis in the mammary gland in the regulation of cholesterol metabolism and the provision of cholesterol for secretion into milk.View full abstractDownload PDF (783K) -
Jean-Marc Lancelin, Mariana Stein, Jean-Pierre Jacquot1993 Volume 114 Issue 3 Pages 421-431
Published: 1993
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe recombinant form of the chloroplastic thioredoxin Ch2 from the green alga Chlamydomonas reinhardtii [Jacquot et al. (1992) Nucleic Acids Res. 20, 617] that preferen-tially activates the NADP dependent malate dehydrogenase [EC 1. 1. 1. 82] (m-type thiore-doxin) through a light promoted reductive system, has been subjected to an extensive two-dimensional 1H NMR analysis. A complete 1H NMR assignment of the resonance lines in both the oxidized and the reduced states at pH 5.8 has been obtained allowing the recognition of the secondary structure patterns and the global protein folding. The single polypeptide chain, made of 106 residues plus one additional Met located at the N-terminal position (11.6 kDa) due to the protein expression system, folds into a pattern characteristic of the open twisted α/β structures already found for Escherichia coli and human thiore-doxins for which the protein shares 46 and 20% of sequence identity, respectively. The open α/β structure is made of 5 β-sheets associated in a parallel (β1 to β3) and anti parallel manner (β3 to β5) and surrounded by 4 helices. This represents the first structural exploratory study of the ubiquitous oxido-reductase thioredoxins in a photosynthetic living system.View full abstractDownload PDF (1021K) -
Rika Nanbu, Takeo Kubo, Takashi Hashimoto, Shunji Natori1993 Volume 114 Issue 3 Pages 432-437
Published: 1993
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA protein that binds to the AU-rich sequence in the 3'-untranslated region of sarcotoxin IIA mRNA was purified from a Sarcophaga pupal extract to near homogeneity. The molecular mass of this protein was estimated to be 39 kDa by SDS-polyacrylamide gel electropho-resis. The partial amino acid sequences of two peptides obtained from the 39 kDa protein showed striking similarities to partial amino acid sequences of rat and yeast 3-oxoacyl-CoA thiolase, suggesting that this protein is a Sarcophaga thiolase. In fact, the purified 39 kDa protein was found to have thiolase activity. Moreover, rat mitochondrial 3-oxoacyl-CoA thiolase showed affinity to the AU-rich RNA. These results suggest that the RNA binding activity is an intrinsic character of thiolase.View full abstractDownload PDF (4681K) -
Setsuko Fujita-Becker, Lan Kluwe, Andrea Miegel, Kayo Maeda, Yuichiro ...1993 Volume 114 Issue 3 Pages 438-444
Published: 1993
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThree subunits of rabbit skeletal muscle troponin were expressed in and purified from Escherichia coli. The procedures were optimized, and the reconstituted troponin complex is highly homogeneous, stable, and obtainable in large quantities, allowing us to conduct crystallization studies of the troponin complex. The three subunits expressed and purified are β-TnT (N'-208), TnI (C64A, C133S), and the wild type TnC. β-TnT (N'-208) is a 25 kDa fragment of β-troponin T, which consists of 208 amino acids and lacks 58 residues in the N-terminal variable region. TnI (C64A, C133S) is a mutant troponin I, in which Cys-64 and Cys-133 are replaced by Ala and Ser, respectively. Each subunit was separately expressed in E. coli, purified by column chromatography including HPLC, and reassembled to form troponin complex. The reconstituted troponin complex was not distinguishable from authentic troponin prepared from rabbit skeletal muscle; the acto-S 1 ATPase rate, as well as the superprecipitation, was calcium-sensitive. Small flat crystals up to 0.2mm long have been reproducibly obtained in preliminary crystallization trials.View full abstractDownload PDF (2530K) -
Masayuki Komori, Atsuko Higami, Yoshio Imai, Susumu Imaoka, Yoshihiko ...1993 Volume 114 Issue 3 Pages 445-448
Published: 1993
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA form of P 450 [termed P 450(h-1)] was purified from the liver microsomes of a male horse to electrophoretic homogeneity. The specific content of the final P 450 (h-1) preparation was 14.8 nmol/mg of protein and the recovery was 0.38% of the microsomal P 450. The apparent molecular weight of P 450 (h-1) was 52, 000 Da. The absorption spectra of P 450 (h-1) indicated that P 450 (h-1) was a low- and high-spin mixed type P 450 in the oxidized form. The reconstituted system containing P 450 (h-1) could catalyze benzphetamine N-demethylation, 7-ethoxycoumarin O-deethylation, and testosterone 16α-hydroxylation. In the horse hepatic microsomes, aniline p-hydroxylation and testosterone 6β-hydroxylation, in addition to the above reactions, were detected. The N-terminal amino acid sequence of P 450 (h-1) was highly homologous to that of rat P 450 2C11. Western blot analysis using anti-P 450 (h-1) antibody revealed that this antibody most strongly recognized P 450 2C13 among ten rat P 450 s belonging to eight different subfamilies involved in hepatic drug metabolism. This anti-P 450 (h-1) antibody inhibited the testosterone 16α-hydroxylase activity in horse liver microsomes. These results suggest that P 450 (h-1) belongs to the P 450 2 C subfamily and contributes to the testosterone 16α-hydroxylation in horse liver microsomes.View full abstractDownload PDF (1304K)
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