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Volume 94 (1983)
- Issue 6 Pages 1723-
- Issue 5 Pages 1353-
- Issue 4 Pages 1029-
- Issue 3 Pages 623-
- Issue 2 Pages 331-
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Volume 94, Issue 4
Displaying 1-37 of 37 articles from this issue
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Toshikazu NAKAMURA, Yumiko TOMITA, Akira ICHIHARA1983 Volume 94 Issue 4 Pages 1029-1035
Published: October 01, 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSAdult rat hepatocytes in primary culture, which show various liver functions, did not show any mitosis at confluent cell density, although they entered the S phase and remained in the G2 phase, judging by cytofluorometry, when insulin and epidermal growth factor (EGF) were added to 2-day cultures (Tomita, Y., Nakamura, T., & Ichihara, A. (1981) Exp. Cell Res. 135, 363-371). However, when the cell density was decreased by half or one third, the number of nuclei and cell number increased to 1.5-2.0 times that after culture for 35 h with insulin and EGF. Moreover, at these lower densities, DNA synthesis started much earlier, although at the usual high density DNA synthesis with these two hormones did not start until the hepatocytes had been cultured for over 40 h. These results suggest that proliferation of mature rat hepatocytes is regulated by the cell density. First, cells in Go enter the G1 phase density-dependently; then cells in the G1 phase seem to be stimulated to enter the S phase by insulin and EGF, and a low cell density may permit cells after DNA synthesis to enter the M phase. DNA synthesis of rat hepatocyte cultures at low cell density was strongly inhibited by co-culture with a dense culture. Therefore, the density-dependent mechanism of hepatocyte proliferation seems to involve regulation by a soluble inhibitor (s) secreted by the hepatocytes into the culture medium.View full abstractDownload PDF (1823K) -
Hiroshi TSUCHIHASHI, Toshiro YADOMAE, Toshio MIYAZAKI1983 Volume 94 Issue 4 Pages 1037-1044
Published: October 01, 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWhen the cold water-extract of Kobayasia nipponica containing a glycuronan, protuberic acid (PA), was allowed to stand at room temperature, PA was hydrolyzed. The optimum conditions for this PA-hydrolysis were 37°C and pH 4-5 in 0.1M acetate buffer. Characterization of the hydrolytic products was performed by chemical analysis, and by 1H- and 13C-NMR spectroscopy. They were identified as D-GIcUA and O-(α-L-IdUAp)-(1-4)-D-GIcUA. These results suggest that PAhydrolytic enzyme (s) include at least endo-β-D-glucuronidase.View full abstractDownload PDF (485K) -
Shintaro KIKUCHI, Takashi KUSAKA1983 Volume 94 Issue 4 Pages 1045-1051
Published: October 01, 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESS3-Oxoacyl-CoA synthase, that condenses malonyl-CoA to other acyl-CoAs and takes part in the malonyl-CoA-dependent, acyl carrier protein (ACP)-non-requring fatty acid elongation system (“fatty acid elongation system II or elongation system II” (Kikuchi, S. & Kusaka, T. (1982) J. Biochem. 92, 839-844)), was purified to homogeneity for the first time from the crude extract of Mycobacterium smegmatis by columnchromatographies. The molecular weight of this enzyme was estimated to be around 64, 000 by Sephacryl S-300 gel filtration and 59, 000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzymic product from malonyl-CoA and stearoyl-CoA was identified as 3-oxoeicosanoyl-CoA by mass-spectrometry. Km values of the enzyme for malonyl-CoA and stearoyl-CoA were 41.7 μM and 52.6 μM, respectively. The enzyme was more active toward acyl-CoAs having acyl-carbon-numbers of 18 or more, either saturated or monounsaturated, than those with below 18. Cerulenin, a specific inhibitor of 3-oxoacyl-ACP synthase [EC 2. 3. 1. 41], had no effect on this enzyme but iodoacetamide and N-ethylmaleimide (NEM) showed inhibitory effects.View full abstractDownload PDF (440K) -
Satoshi SEKIGUCHI, Sachiko SEKI, Makoto ISHIMOTO1983 Volume 94 Issue 4 Pages 1053-1059
Published: October 01, 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSNitrite reductase from Clostridium perfringens was purified by chromatographies on DEAE-cellulose, DEAE-Sephadex, Sephadex G-150, and hydroxylapatite and by isoelectric focussing to a homogeneous state, showing essentially a single protein band in disc gel electrophoresis and a single immuno-precipitation line in double diffusion against antiserum obtained from immunized rabbits. The reductase was induced in the presence of nitrate. It had a molecular weight of 54, 000 and showed no absorption peak in the visible region. The pH optimum was 6.2 and Km for nitrite was 5mM. Ferredoxin, as well as viologen dyes, was found to be an electron donor. The product of nitrite reduction was hydroxylamine. This reductase was inhibited by o-phenanthroline and azide but not by cyanide or diethyldithiocarbamate.View full abstractDownload PDF (1039K) -
Kunihiko KONNO, Ken-ichi ARAI, Shizuo WATANABE1983 Volume 94 Issue 4 Pages 1061-1066
Published: October 01, 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESS1. Dissociation and association of regulatory light chains of scallop myosin were found to be accompanied by changes in the fluorescence intensity and in the UV absorption spectrum.
2. The changes in the two optical properties of scallop myosin and the dissociation and association of regulatory light chains were studied as a function of the magnesium and calcium concentrations. The results thus obtained suggested that there are two different types of attachment between regulatory light chains and “desensitized” myosin; one type is a calcium-specific attachment, and the other type of attachment can be mediated by either calcium or magnesium ions.
3. These changes in the optical properties of scallop myosin were distinguishable from those induced by Mg-ATP; for example, with “desensitized” scallop myosin, the former changes were not observed but the latter were.View full abstractDownload PDF (457K) -
Hideyoshi YOKOSAWA, Misako MIYATA, Hitoshi SAWADA, Shin-ichi ISHII1983 Volume 94 Issue 4 Pages 1067-1076
Published: October 01, 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA post-proline cleaving enzyme and its endogenous inhibitor have been demonstrated to be present in sperm of the ascidian, Halocynthia roretzi. The enzyme was extracted with artificial sea water from frozen and thawed sperm and isolated from accompanying acrosin-like and chymotrypsin-like enzymes by DEAE-cellulose chromatography. It was then separated from the endogenous inhibitor by ammonium sulfate fractionation and DEAE-Sephacel chromatography. Three subsequent chromatographic operations using hydroxylapatite, Sephadex G-150 and Z-Gly-Pro-Leu-Gly-aminohexyl-Sepharose yielded the highly purified enzyme. The molecular weight and isoelectric point of the enzyme were estimated to be 66, 000 and 5.5, respectively. The pH optimum of the activity was 7.0. The enzyme was inactivated with diisopropylphosphorofluoridate, phenylmethylsulfonyl fluoride, Z-Gly-Pro-chloromethyl ketone and sulfhydryl-directed reagents; these inhibitor suscep-tibilities were similar to those reported for the enzymes of mammalian origins. The ascidian enzyme hydrolyzed oxytocin, angiotensin II, luteinizing hormone releasing hormone and neurotensin at the carboxyl side of proline residues. The endogenous inhibitor was heat stable. The molecular weight of its main component was estimated to be about 8, 000. The presence of salt at high concentrations weakened the enzyme-inhibitor interaction. Z-Gly-Pro-chloromethyl ketone inhibited fertilization of the ascidian, suggesting possible involvement of the post-proline cleaving enzyme in fertilization.View full abstractDownload PDF (728K) -
Hiroshi OHMIZU, Takuji SASAKI, Norihiro TSUKAGOSHI, Shigezo UDAKA, Nor ...1983 Volume 94 Issue 4 Pages 1077-1084
Published: October 01, 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSB. brevis 47 secretes a vast amount of protein consisting mainly of two kinds with approximate molecular weights of 130, 000 and 150, 000. The two major extracellular proteins were indistinguishable from those of cell wall protein by SDS-polyacrylamide gel electrophoresis. Based on the results of analysis of amino acid composition, limited proteolysis followed by electrophoresis, and the cross-reactivity of antisera, we conclude that the 130K and 150K extracellular proteins are derived from the respective cell wall proteins. Furthermore, the NH2-terminal amino acid analysis suggests that the two major extracellular proteins are released from the cell wall without any modification of the NH2-terminal portion.View full abstractDownload PDF (2614K) -
Hajime MATSUE, Mitsuo MAJIMA1983 Volume 94 Issue 4 Pages 1085-1094
Published: October 01, 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSαFuc-Ser, αFuc-Thr, αFuc-Ser, and αFuc-Thr were synthesized stereoselectively in good yields, and their properties were investigated. β-Fucosylserine (and -threonine) was synthesized by Koenigs-Knorr reaction of per-O-acetylfucopyranosyl bromide with N-carbobenzoxy-L-serine (and -threonine) benzyl ester using mercuric cyanide, followed by hydrogenolysis and deacetylation. α-Fucosylserine (and -threonine) was synthesized by the halide-ion catalyzed reaction of per-O-benzylfucopyranosyl bromide with the serine (and threonine) derivative described above, followed by hydrogenolysis. These substances each gave a single peak on an amino acid analyzer. The anomeric configurations of these compounds were determined by 1H-NMR spectroscopy, optical rotation measurement and enzymatic degradations of these compounds.
On acid hydrolysis, αFuc-Ser and αFuc-Thr were more rapidly hydrolyzed than the corresponding β-series compounds. Under mild alkaline conditions in the presence of sodium borohydride, per-protected βFuc-Thr released about 50% of fucose but free βFuc-Thr did not. However, free βFuc-Thr and βFuc-Thr were cleaved to give acetylated fucose during acetylation with acetic anhydride-pyridine. α-L-Fucosidases, which were purified from abalone and rabbit livers by affinity chromatography, acted very well on aFuc-Ser and aFuc-Thr, but not on the β-series compounds.View full abstractDownload PDF (745K) -
Mizuho KOMATSU-TAKAKI1983 Volume 94 Issue 4 Pages 1095-1100
Published: October 01, 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSADP binding brought about by inorganic phosphate addition (P1-dependent ADP binding) on membrane-bound chloroplast coupling factor was studied and the following results were obtained.
1. Under energization by illumination or by acid-base transition, P1 brought about the binding of ADP with an apparent Km value of 0.22mM. This effect of P1 was lost rapidly after turning the light off or after acid to base transition, concomitant with the loss of ATP synthesizing activity.
2. P1-dependent ADP binding was inhibited by phlorizin to nearly the same extent as was ATP synthesis. The inhibitory effects of phlorizin on both the PP1-dependent ADP binding and ATP synthesis increased with the decrease of Pi concentration.
These results suggest that the P1-dependent ADP binding reaction participates in the ATP synthesis reaction and that phlorizin inhibits the P1 binding process.View full abstractDownload PDF (457K) -
Kazuo NAGASAKI, Michiki KASAI1983 Volume 94 Issue 4 Pages 1101-1109
Published: October 01, 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSRapid Ca2+ release rate from sarcoplasmic reticulum vesicles was determined by the stopped flow method in terms of chlortetracycline fluorescence. Intensity of chlortetracycline fluorescence was proportional to the intravesicular free Ca2+ concentration. Ca2+ efflux was activated by extravesicular Ca2+ with an apparent dissociation constant of 25 μm and was inhibited with an inhibition constant of 120 μm in the absence of Mg2+. Caffeine enhanced the Ca2+ release rate by increasing only the affinity of Ca2+ for the activation site. Mg2+ reduced the Ca2+ release rate by competitive binding to the activation site. ATP increased the Ca2+ release rate very much without changing the affinities of Ca2+ for the activation and inhibition sites, i.e., ATP seems to increase the pore radius or number of the Ca2+ channels without affecting the gating mechanism of the channel. These results are consistent with those reported in skinned muscle sarcoplasmic reticulum. The maximum rate of Ca2+ release in the presence of ATP reached 80 s-1. This value is considered to be sufficient to cause muscular contraction.View full abstractDownload PDF (696K) -
Ca2+-Induced Ca2+ Release from Fragmented Sarcoplasmic Reticulum: Ca2+-Dependent Passive Ca2+ EffluxYutaka KIRINO, Masanori OSAKABE, Hiroshi SHIMIZU1983 Volume 94 Issue 4 Pages 1111-1118
Published: October 01, 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSCharacterization of the putative Ca2+-gated Ca2+ channel of sarcoplasmic reticulum, which is thought to mediate Ca2+-induced Ca2+ release, was carried out in order to elucidate the mechanism of Ca2+-induced Ca2+ release. Heavy and light fractions of fragmented sarcoplasmic reticulum isolated from rabbit skeletal muscle were loaded passively with Ca2+, and then passive Ca2+ efflux was measured under various conditions. The fast phase of the Ca2+ efflux depended on the extravesicular free Ca2+ concentration and was assigned to the Ca2+ efflux through the Ca2+-gated Ca2+ channel. Vesicles with the Ca2+-gated Ca2+ channels comprised about 85% of the heavy fraction and about 40% of the light fraction. The amount of Ca2+ loaded in FSR was found to be much larger than that estimated on the basis of vesicle inner volume and the equilibration of intravesicular with extravesicular Ca2+, indicating Ca2+ binding inside FSR. Taking this fact into account, the Ca2+ efflux curve was quantitatively analyzed and the dependence of the Ca2+ efflux rate constant on the extravesicular free Ca2+ concentration was determined. The Ca2+ efflux was maximal, with the rate constant of 0.75 s-1, when the extravesicular free Ca2+ was at 3 μM. Caffeine increased the affinity for Ca2+ of Ca2+-binding sites for opening the channel with only a slight change in the maximum rate of Ca2+ efflux. Mg2+ inhibited the Ca2+ binding to the sites for opening the channel while procaine seemed to inhibit the Ca2+ efflux by blocking the ionophore moiety of the channel.View full abstractDownload PDF (631K) -
Hideaki TSUNEMATSU, Yoshihiro HATANAKA, Yuji SUGAHARA, Satoru MAKISUMI1983 Volume 94 Issue 4 Pages 1119-1125
Published: October 01, 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe phenylthiazolone (PTA) of p-guanidinophenylalanine (GPA) was synthesized and the susceptibility of this compound to trypsin and related enzymes was compared with that of the PTA of arginine (Arg). Both PTA-GPA and PTA-Arg were almost completely and rapidly hydrolyzed by trypsin and pronase. PTA-Arg was hydrolyzed rapidly by thrombin, whereas PTA-GPA was less susceptible to this enzyme. The rates of hydrolysis of the two PTAs by α-chymotrypsin and papain were fairly slow.
The specificity constant (kcat/Km) for the hydrolysis of PTA-GPA by trypsin was about 4 times larger than that of PTA-Arg. Thus, PTA-GPA, as well as PTA-Arg, behaves as a specific internal thioester substrate for trypsin and is the best one in the series of PTA substrates examined so far. However, PTA derivatives of GPA and Arg were hydrolyzed with kcat/Km values smaller than those of Nα-benzoyl ethyl esters of L-GPA and L-Arg by factors of 4 and 17, respectively.View full abstractDownload PDF (465K) -
Tatsuji HANEJI, Noriyoshi KURIHARA, Katsumi IKEDA, Masayoshi KUMEGAWA1983 Volume 94 Issue 4 Pages 1127-1132
Published: October 01, 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSIn order to study the effects of vitamin D metabolites on bone metabolism, clone MC3T3-E1 cells, which have retained osteoblastic activity, were cultured with various concentrations of the hormone, lα, 25-dihydroxyvitamin D3 [1α, 25 (OH)2D3]. A physiological concentration of 1α, 25 (OH)2D3 stimulated alkaline phosphatase (ALP) activity in the cells. Other metabolites-1α, 24-dihydroxyvitamin D3 [1α, 24 (OH)2D3], 1α-hydroxyvitamin D3 [1α (OH)D3], and 24R, 25-dihydroxyvitamin D3 [24R, 25 (OH)2D3] -also induced increases in ALP activity in a dose-dependent fashion. However, their effective concentrations were 100 or 1, 000 times greater than that of 1α, 25 (OH)2D3. Hormone-induced and native ALP activities in the cells were of the same type as that found in newborn mouse calvaria; that is, they were heat-labile, L-homoarginine- and levamisole-sensitive, and L-phenylalanine-insensitive (liver-bone-kidney type). These results show that vitamin D metabolites stimulate bone formation in vitro and that they may be involved in bone formation in vivo as well.View full abstractDownload PDF (428K) -
Daisaburo FUJIMOTO, Masanori SUZUKI, Akiyoshi UCHIYAMA, Shigehito MIYA ...1983 Volume 94 Issue 4 Pages 1133-1136
Published: October 01, 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSPyridinoline, a cross-linking compound of collagen fibers, was found in human urine. A significant portion of urinary pyridinoline was in free form. The ratio of total pyridinoline to creatinine changed with age. It was high in children and decreased with growth. It was low and constant in adults, and increased slightly in old age. It was increased significantly in patients with certain bone and joint diseases. Urinary pyridinoline may serve as a useful marker for the breakdown of collagen fibers of skeletal tissues.View full abstractDownload PDF (297K) -
Energy Transfer between Fluorescent Dyes Attached to Ca2+, Mg2+-ATPase in the Sarcoplasmic ReticulumRobert E. YANTORNO, Taibo YAMAMOTO, Yuji TONOMURA1983 Volume 94 Issue 4 Pages 1137-1145
Published: October 01, 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSSarcoplasmic reticulum (SR) isolated from rabbit skeletal muscle was solubilized with a nonionic detergent, dodecyl octaethyleneglycol monoether (C12E8), at a weight ratio of detergent to protein of greater than 10, so that the Ca2+, Mg2+ dependent ATPase existed mainly in a monomeric form (7). The solubilized ATPase was reacted with 10 μM N-1-P or 5 μM DACM in the presence of 5 mcvi CaCl2, 0.4M KCl, 20% glycerol and 50mM TES at pH 7.5 and 20°C. Under these conditions, about 1 mol of N-1-P was incorporated into 105g SR protein on 10min incubation and 1 mol of DACM was incorporated into the same amount of SR on 5min incubation. Analysis of the tryptic digest of the N-1-P- or DACM-labeled ATPase on SDS polyacrylamide gel revealed that almost all the fluorescence was associated with the 30 Km. w. subfragment of the ATPase protein. Even when the amount of the probe incorporated into SR-ATPase was increased from 1 to 3 mol per 105g SR protein, all was incorporated into the 30 K subfragment. Both the activities of formation and decomposition of the phosphorylated intermediate (EP) were unaffected by these modifications. When the separately labeled ATPases were mixed together in the presence of C12E8 and the detergent was removed by incubation with Bio-Beads SM-2, a significant amount of fluorescence energy transfer was observed between N-1-P and DACM. However, energy transfer did not occur when the labeled ATPases were mixed after removal of C12E8. The amount of energy transfer increased as a function of incubation time with Bio-Beads. This time course corresponded well to that of the restoration of the Mg2+ sensitivity of EP decomposition, indicating that ATPase-ATPase interaction is essential for the enzymatic reaction and associated Ca2+ transport across the SR membrane.View full abstractDownload PDF (587K) -
Hirofumi ONISHI, Takeyuki WAKABAYASHI, Toshio KAMATA, Shizuo WATANABE1983 Volume 94 Issue 4 Pages 1147-1154
Published: October 01, 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe conformation of thiophosphorylated myosin molecules of chicken gizzard muscle was studied by electron microscopy with the rotary shadowing technique and by the light scattering method.
1. In the absence of ATP, the radius of gyration (RG) of gizzard thiophosphorylated myosin was 478 Å, and was essentially equal to that (474 Å) of unthiophosphorylated myosin. In the presence of ATP, it was 355 Å, and was much larger than that (146 Å) of unthiophosphorylated myosin.
2. In the presence of ATP, 84 percent of the unthiophosphorylated myosin molecules had intramolecular loops at their tails, but only 23 percent of the thiophosphorylated myosin molecules had them.
3. There were two flexible regions in the unthiophosphorylated myosin tail. The considerable flexibility at both regions remained even when the light chain was thiophosphorylated.
4. The two globular heads of the unthiophosphorylated myosin molecules had a tendency to bend back towards the tail in the presence of ATP, but this tendency was reduced by the light chain thiophosphorylation.
5. The myosin molecules with “looped” tails were mostly, if not all, in one of two mirror-image forms, and the mirror-image asymmetry was independent of the thiophosphory lation.View full abstractDownload PDF (3226K) -
Masato HIRATA, Takafumi HAMACHI, Toshihiko HASHIMOTO, Eiichi SUEMATSU, ...1983 Volume 94 Issue 4 Pages 1155-1163
Published: October 01, 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSPassive permeability of the endoplasmic reticulum of saponin-treated macrophages to Ca2+ was studied by the filtration method using 45Ca. The Ca2+ release from the endoplasmic reticulum of macrophages was enhanced by the presence of submicromolar concentrations of Ca2+ in the medium. The Ca2+ release was enhanced by caffeine, and suppressed by MgCl2. These phenomena are similar to the Ca2+-induced Ca2+ release reported for the sarcoplasmic reticulum of skeletal muscle. On the other hand, adenine suppressed the Ca2+ release from the endoplasmic reticulum, while it reportedly enhanced the Ca2+-induced Ca2+ release of the skeletal muscle. The threshold concentration of Ca2+ for the Ca2+-induced Ca2+ release was approximately 10-8 M in the presence of 0.95mM MgCl2 in macrophages. The spontaneous spreading of macrophages and spontaneous migration of macrophages were inhibited by adenine, and also by caffeine in spite of the enhancement of the Ca2+-induced Ca2+ release.View full abstractDownload PDF (1861K) -
Tsunehiro KITAGAWA, Kunio FUJIWARA, Shoichi TOMONOH, Kazuyo TAKAHASHI, ...1983 Volume 94 Issue 4 Pages 1165-1172
Published: October 01, 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSAn antiserum against kanamycin (anti-KM) was elicited in rabbits immunized with a kanamycin immunogen prepared by a three-step procedure using N- (m-maleimido-benzoyloxy) succinimide as a cross-linker. KM and tobramycin (TOB) were labeled with β-D-galactosidase utilizing another cross-linker, N- (gamma-maleimidobutyr-yloxy) succinimid. The labeled KM showed very strong affinity to anti-KM antiserum and that of TOB had an adequate affinity to anti-KM. Increases in the assay sensitivities at the B/B0 value of 50% of KM and dibekacin were 183- and 191, 000-times, respectively, on changing the enzyme label from KM to TOB. The optimal conditions for highly sensitive enzyme immunoassay (EIA) of KM using anti-KM and the enzyme labeled with TOB with satisfactory accuracy and precision were determined. Highly sensitive EIAs of four KM analogs with measurement ranges of 1 to 100 ng/tube were also developed using the labeled TOB and anti-KM as common reagents. Various commonly used drugs were found to have little reactivity in this immunoassay, indicating that the EIA is specific to KM and its analogs. The reasoning and the selection of TOB as the label are also discussed.View full abstractDownload PDF (549K) -
Kazushige YOKOTA, Fumiaki SHONO, Shozo YAMAMOTO, Eiki KOMINAMI, Nobuhi ...1983 Volume 94 Issue 4 Pages 1173-1178
Published: October 01, 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSAmong several intracellular protease tested, cathepsin H transformed leukotriene D4 to E4 with a release of glycine in a stoichiometric quantity. Under the optimal conditions the rate of leukotriene D4 transformation by cathepsin H was about 3% of the hydrolysis rate of α-N-benzoyl-DL-arginine-2-naphthylamide which is commonly utilized as a very efficient substrate to test the peptidase activity of the enzyme. Leukotriene C4 was not transformed to leukotriene D4, by cathepsin H. Neither cathepsin B nor C was active with leukotrienes C4 and D4.View full abstractDownload PDF (415K) -
Tadashi YOSHIMOTO, Toshiharu NISHIMURA, Taizo KITA, Daisuke TSURU1983 Volume 94 Issue 4 Pages 1179-1190
Published: October 01, 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA post-proline cleaving enzyme [prolyl endopeptidase, EC 3. 4. 21. 26] was purified about 3, 700-fold from an extract of bovine brain by a series of column chromatog-raphies on DEAE-Sephadex, hydroxyapatite and PCMB-T-Sepharose, and gel filtration on Sephadex G-200 using N-carbobenzoxy-Gly-Pro-β-naphthylamide (Z-Gly-Pro-2-NNap), thyrotropin releasing hormone (TRH) and oxytocin as substrates. The purified enzyme appeared homogeneous as judged by disc gel and SDS gel electrophoreses. The enzyme was most active at pH 7.5 and 7.2 with Z-Gly-Pro-2-NNap and TRH, respectively, and hydrolyzed peptide bonds involving Pro-X (X=amino acid, peptide, ester and amide) bonds of synthetic substrates, oxytocin, vasopressin, neurotensin, substance P, tuftsin, bradykinin, and insulin B chain. However, the enzyme was inert toward collagen, gelatin, and casein. The enzyme was completely inactivated by diisopropylphosphorofluoridate (DFP), Z-Gly-Pro-chloromethyl ketone and p-chloromercuribenzoate (PCMB), while it was not inhibited by phenylmethane sulfonylfluoride (PMSF) or metal chelators. Determination of the amino acid composition revealed that the enzyme contained 25 half-cystines. Modification of three cysteine residues of the enzyme by PCMB led to complete inactivation. The isoelectric point of the enzyme was 4.8, and the molecular weight was estimated to be 76, 000 by ultracentrifugal analysis and 75, 000-74, 000 by both gel filtration and sodium dodecyl sulfate (SDS) gel electrophoresis, suggesting that the enzyme is present as a monomer. These results indicate that the post-proline cleaving enzyme from bovine brain is very similar to those previously purified from lamb brain and kidney in its enzymatic properties, substrate specificity and physicochemical properties, in sharp contrast with the results obtained by Tate, who reported that the bovine brain prolyl endopeptidase was inert toward oxytocin, vasopressin and bradykinin.View full abstractDownload PDF (861K) -
Kazuo ASAOKA, Kenji TAKAHASHI1983 Volume 94 Issue 4 Pages 1191-1199
Published: October 01, 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSGlutathione S-transferase was purified from porcine brain. Four enzyme fractions were obtained by column chromatography on CM-cellulose and the main enzyme fraction was purified to apparent homogeneity as judged by disc gel electrophoresis. The action spectra of these enzyme fractions toward some typical substrates were roughly similar. The optimum pH range of the purified enzyme was from 6.5 to 7.5 with o-dinitrobenzene as a substrate. The main enzyme showed a molecular weight of about 43, 000 on Sephadex G-150 chromatography, and about 22, 000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. It was composed of two subunits of apparently identical molecular weight bound to each other noncovalently. The amino acid composition showed characteristic high contents of leucine and glutamic acid residues. A notable difference was observed in the lysine content as compared with that of the liver enzyme. The brain enzyme bound bilirubin less strongly than the monkey liver enzyme and human albumin. In addition, the regional and subcellular distributions of the enzyme in porcine brain was investigated with o-dinitrobenzene as a substrate. The enzyme activity was found to be distributed fairly evenly in various regions of the brain, and was especially abundant in the cytosol fraction. However, the enzyme activity was also detected considerably in the microsomal and mitochondrial fractions but not in the synaptosomal fraction.View full abstractDownload PDF (1235K) -
Bernard Yukihiro HIRAOKA, Katsuhiko FUKASAWA, Minoru HARADA1983 Volume 94 Issue 4 Pages 1201-1208
Published: October 01, 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSTwo novel aminopeptidases (I and II) which have specificity for amino-terminal arginine residues and strong sensitivity to divalent cations were purified from Streptococcus mitis ATCC 9811 by a procedure that involved treatment with a lytic enzyme for bacterial cell walls, followed by a series of chromatographies. Enzyme I was obtained as a homogeneous protein as judged by polyacrylamide gel electrophoresis and had a specific activity of 484.8 units per mg protein using L-arginine-2-naphthylamide as substrate; its Km value was 2.6×10-5M. The molecular weight was estimated to be 62, 000, and its isoelectric point was pH 4.4. Enzyme 11 was purified to a specific activity of 128.0 units per mg protein and had a Km value of 3.8×10-5M. The molecular weight was estimated to be 360, 000, and its isoelectric point was pH 5.7. The pH optima of enzymes I and II were 8.6 and 7.6, respectively. Both enzymes were inactivated by sulfhydryl reagents and metal ions but were markedly activated by EDTA. The chloride ion had an inhibitory rather than a stimulatory effect on the activity of both enzymes. Substrate specificity studies indicated that both the enzymes specifically hydrolyze N-terminal arginine residues from α-aminoacyl 2-naphthylamides and peptides, but they could not attack the L-arginyl-L-prolyl-peptide.View full abstractDownload PDF (1165K) -
Masaharu ISHIKAWA, Hiromu MUROFUSHI, Hikoichi SAKAI1983 Volume 94 Issue 4 Pages 1209-1217
Published: October 01, 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSFodrin is a spectrin-like protein present in the cortical cytoplasm of neurons and binds to F-actin to induce gelation of actin. We found that fodrin purified from porcine brains co-sedimented with microtubules which were assembled from phosphocellulose-purified tubulin prepared from porcine brains. This indicates that fodrin bound to microtubules. An unusual enhancement of turbidity at 350 nm was observed when microtubules were assembled in the presence of fodrin. Microscopic observations showed that fodrin bundled the microtubules. The interaction between fodrin and microtubules was decreased by microtubule-associated proteins (MAPS), indicating that fodrin and MAPS interacted with microtubules competitively. These data raise the possibility that microtubules are involved in the submembranous cytoskeleton of neurons with fodrin.View full abstractDownload PDF (3906K) -
Hirofumi SHOUN, Yoshiaki SUDO, Yasuo SETO, Teruhiko BEPPU1983 Volume 94 Issue 4 Pages 1219-1229
Published: October 01, 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA cytochrome P-450 was isolated from a fungus, Fusarium oxysporum, which grew on a medium containing soybean oil as a sole carbon source. It was found as a heme protein that possesses lipoxygenase activity, and seemed to exist in the soluble fraction of cell-free extracts. The cytochrome revealed multiplicity and could be separated into at least 3 fractions (A, B, and C). Two of them, termed Fusarium P-450A and -B, were highly purified. The complex of the ferrous Fusarium P-450 with carbon monoxide showed a Soret peak at 447 nm. The properties of the cytochromes (P-450A and -B) were closely similar to each other, the only detectable difference being in the pl (isoelectric point) value (5.2 and 5.0, respectively). The pI and molecular weight (48, 000) values together with amino acid composition of Fusarium P-450 were similar to those of other cytochromes P-450 from various sources. Some other spectral properties as well as interactions with various ligands were also studied. Peroxidase or chloroperoxidase activity was not detected with Fusarium P-450.View full abstractDownload PDF (1136K) -
Yousuke SEYAMA, Kazumasa OHASHI, Toshiharu IMAMURA, Takeshi KASAMA, Hi ...1983 Volume 94 Issue 4 Pages 1231-1239
Published: October 01, 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSFatty acid compositions of phosphatidyl ethanolamine and phosphatidyl choline extracted from the guinea pig Harderian gland were examined by gas chromatography and gas chromatography-mass spectrometry. Oleic acid was the major component of both phospholipids, but a large amount of saturated branched chain fatty acids was found: 34.5% in phosphatidyl ethanolamine, and 42.9% in phosphatidyl choline. On the other hand, linolenic and arachidonic acids were not found in these phospholipids. At the 1-position of phosphatidyl choline and phosphatidyl ethanolamine, 54.3% and 40.9% of fatty acids, respectively, had methyl branches. These methyl branches were located at the even-numbered carbon atoms. Branched chain fatty acids were also found at the 2-position of both lipids: 36.2% in phosphatidyl choline and 31.2% in phosphatidyl ethanolamine. The fatty acids of phosphatidyl ethanolamine and phosphatidyl choline from liver, cerebrum, cerebellum, erythrocytes, and plasma of the same animal were also analyzed. Saturated and unsaturated fatty acids, including arachidonic acid, were the major components. Branched chain fatty acids were also found in these lipids, but in very small amounts.View full abstractDownload PDF (2006K) -
Hiroyuki ABURATANI, Tatsuhiko KODAMA, Atsushi IKAI, Hiroshige ITAKURA, ...1983 Volume 94 Issue 4 Pages 1241-1245
Published: October 01, 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSApolipoproteins B100 and B48 in human and rat plasma were studied by using sodium dodecyl sulfate (SDS) polyacrylamide gradient gel electrophoresis. On SDS gradient gel electrophoresis, human and rat apoprotein B100 co-migrated and had an apparent Mr=258, 000±12, 000. Human and rat apoprotein B48 had an apparent Mr=189, 000±6, 000. The molecular weight of human apoprotein B100 determined by sedimentation equilibrium analysis was 270, 000±20, 000, which was similar to the value determined by SDS gradient gel electrophoresis. However, on SDS polyacrylamide gel electrophoresis at constant concentration, the relative migration value of human apoprotein B100 was not constant when the concentration of polyacrylamide was changed. These results indicate that SDS gradient gel electrophoresis is more suitable for the analysis of apolipoprotein B's than ordinary SDS polyacrylamide gel electrophoresis.View full abstractDownload PDF (1678K) -
Hubert E. BLUM, Bruno DEUS, Wolfgang GEROK1983 Volume 94 Issue 4 Pages 1247-1257
Published: October 01, 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSAn isoenzyme of creatine kinase [ATP: creatine N-phosphotransferase, EC 2. 7. 3. 2] was isolated in pure, crystalline form from mitochondria of human heart muscle. The enzyme has a molecular weight of 84, 000 and consists of two subunits with identical molecular weight, each containing two reactive sulfhydryl groups. The enzyme has a specific activity of 15±2 U/mg in the direction of creatine phosphate synthesis at optimum pH of 8.7 and of 45±5 U/mg in the direction of ATP synthesis at optimum pH of 6.7. Mitochondrial creatine kinase has Michaelis constants of 1.70mM for MgATP2-, 8.00mM for creatine, 0.15mM for MgADP-, and 3.00mM for creatine phosphate. The mitochondrial enzyme differs from the other creatine kinase isoenzymes, i.e. from muscle (CK-MM), brain (CK-BB), and their hybrid form (CK-MB) in (I) its amino acid composition and its amino terminal amino acid sequence, (II) its electrophoretic mobility, and (III) its immunological properties. It thus constitutes a fourth isoenzyme of creatine kinase. By analogy to CK-MM, CK-BB, and CK-MB, the mitochondrial isoenzyme was designated as CK-MiMi.View full abstractDownload PDF (2603K) -
Shyh-Horng CHIOU1983 Volume 94 Issue 4 Pages 1259-1267
Published: October 01, 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSL-Ascorbic acid, when combined with either copper (II) ion or a copper(II) -tripeptide complex, extensively cleaved several viral DNAs and proteins under in vitro conditions. Neither ascorbate nor copper tripeptide (Cu2+-diglycyl-L-histidine) alone caused any apparent changes on these molecules. Various transition metal ions and reducing agents were examined under comparable conditions to determine the basic requirements for both DNA degradation and protein scission activities. Copper and iron are the two most effective transition metal ions examined that exhibit these activities in the presence of ascorbate. The addition of catalase, but not superoxide dismutase, can partially inhibit the scission of DNA in vitro, suggesting that H2O2 may be involved in these activities. Among the various reducing agents tested, ascorbate was most effective in causing DNA scission and protein cleavage, corroborating the possible role of H2O2 in the cleavage reactions. One of the products of the reactions of copper/ascorbate is probably the hydroxyl radical generated from H2O2, which can be formed from the oxidation of ascorbate.View full abstractDownload PDF (2072K) -
Yasuhiro NOMATA, Takehiko WATANABE, Hiroshi WADA1983 Volume 94 Issue 4 Pages 1269-1278
Published: October 01, 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSIn a continuation of previous work [Nomata et al. (1983) J. Biochem. 93, 825-831], this paper reports the purification and properties of the two other acidic proteins from human brain, and the immunochemical comparison of the three proteins. The protein described previously was named Glu-50 protein, because 50%. of its amino acid residues were glutamic acid. By analogy, the proteins reported here were named Glu-20 and Glu-35 proteins on the basis of their glutamic acid contents. Both proteins were purified to a homogenous state from human brain by ammonium sulfate fractionation, DEAE-Sephadex A-50 column chromatography and gel filtration on Sephadex G-75 and G-100, like Glu-50 protein. The molecular weights of the Glu-20 and Glu-35 proteins in the native state were 61, 000 and 48, 000 daltons and the values in SDS medium were 60, 000 and 16, 000, respectively, suggesting that Glu-20 protein is a monomer, and Glu-35 a trimer. The isoelectric points of Glu-20 and Glu-35 proteins were pH 4.2 and 3.6, respectively. Glu-20 protein contained 20.5%. Glu, 18.4%. Asp and 10.5% Lys, and Glu-35 protein contained 33.9% Glu and 17.2% Asp. The N-terminal amino acids of both proteins were Gly, whereas the C-termini of Glu-20 and Glu-35 proteins were Leu and Lys, respectively. Glu-20 protein gave a slightly positive periodic acid-Schiff reaction, suggesting that it is a glycoprotein, but Glu-35 protein gave a negative reaction.
The three acidic proteins, Glu-50, Glu-35, and Glu-20 proteins, were compared immunochemically. Antibodies could be produced in rabbits against Glu-50 and Glu-20 proteins only after immunization with the performate-oxidized proteins, but no antibody could be produced against Glu-35 by similar immunization. Anti Glu-50 antibody reacted only with Glu-50 protein, not with Glu-35 or Glu-20 protein, calmodulin, or S-100 protein. Similarly, anti Glu-20 antibody was specific for Glu-20 protein and did not cross-react with Glu-35 or Glu-50 protein, calmodulin, or S-100. Anti S-100 antibody did not react with Glu-50, Glu-35, or Glu-20 protein. The cross-reactivities of these antibodies were tested with extracts of various tissues of a goat. Anti Glu-50 antibody reacted only with an extract of the brain, not with extracts of other tissues, whereas anti Glu-20 antibody reacted with all the tissue extracts examined.View full abstractDownload PDF (2279K) -
Fumio SHISHIKURA, Shin NAKAMURA, Kenji TAKAHASHI, Koichi SEKIGUCHI1983 Volume 94 Issue 4 Pages 1279-1287
Published: October 01, 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSCoagulogens from four kinds of horseshoe crabs (Limulus polyphemus (LP), Tachypleus tridentatus (TT), Tachypleus gigas (TG), and Carcinoscorpius rotundicauda (CR)) were compared in biochemical and immunochemical properties.
On SDS-polyacrylamide gel electrophoresis, the reduced coagulogens had a common apparent molecular weight of 20, 000, whereas the molecular weights of unreduced samples were 24, 000 for the LP protein and 21, 000 for the proteins of the three Asian species (TT, TG, and CR). Gel-filtration yielded apparent molecular weights of 19, 500 for the LP protein and 15, 500 for the other proteins.
All coagulogens consisted of 173-175 amino acid residues and their compositions were very similar to one another, e. g., no methionine, and high proportions of basic amino acids, cystine, valine, and phenylalanine. The amino-terminal 14-residue sequence of the LP coagulogen was Gly-Asp-Pro-Asn-Val-Pro-Thr-Cys-Leu-Cys-Glu-Glu-Pro-Thr. Those of the other coagulogens, Ala-Asp-Thr-Asn-Ala-Pro-Ile-Cys-Leu-Cys-Asp-Glu-Pro-Gly, were identical to one another except for the amino-terminal aspartic acid residue of the TG protein and leucine7 of the CR protein. The carboxyl-terminal residues of the coagulogens were identified as serine (LP), phenylalanine (TT and TG), and tyrosine (CR), respectively.
An antiserum to the LP coagulogen produced a single precipitin line against the LP protein but none against the Asian coagulogens. On the other hand, each of the antiserums to TT, TG, and CR coagulogens yielded a precipitin line against the Asian proteins but gave no line against the LP protein.
These results indicate that the LP coagulogen differs markedly in biochemical and immunochemical properties from TT, TG, and CR coagulogens. Hence a phylogenetic separation between LP (Limulinae) and TT, TG, or CR (Tachypleinae) and a close relationship among TT, TG, and CR were suggested.View full abstractDownload PDF (2416K) -
Kazuo HORIE, Hiroyuki HAGIHARA, Akira WADA, Hideo FUKUTOME1983 Volume 94 Issue 4 Pages 1289-1299
Published: October 01, 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSE. coli ribosomes and rRNA's released 20 to 50 protons upon jump of magnesium ion concentration from 1mM to 20mM. The Mg2+-induced proton release was measured separately for 16S rRNA, 23S rRNA, 30S subunit, and 50S subunit by a new spectrophotometric method that had a much better sensitivity than the pH-stat method. The proton release from the subunits and rRNA's were similar in the number of protons, the pH dependence that had a minimum at neutral pH, and the upward concaveness of the Scatchard plot. From these results, the main source of protons in ribosomal subunits was assigned to nucleotide bases of rRNA's that showed a downward pKa shift upon Mg2+-ion binding.
The subunits and rRNA's, however, differed in the proton release. 16S rRNA released protons somewhat more effectively than 23S rRNA, while 30S subunit released protons 2 to 5 times more effectively than 50S subunit. The marked difference between the two subunits suggests that ionizable bases in 16S and 23S rRNA's are covered and their pKa values are shifted by ribosomal proteins to different extents.
The association of 30S and 50S subunits induced little proton release, showing that few ionizable groups with pKa near neutral pH are involved in the association. E. coli tRNA and poly U also showed Mg2+-induced proton release. The amounts of protons released from rRNA's, tRNA, and poly U were roughly proportional to the amount of bases not hydrogen bonded. The Mg2+-induced proton release from the natural and synthetic RNA's can be explained by the electrostatic field effect of polyphosphate backbones on bases not hydrogen bonded, as proposed in a previous paper. It also reflects the conformational structure of each RNA molecule.View full abstractDownload PDF (809K) -
Dongchon KANG, Hideki NARABAYASHI, Takeyoshi SATA, Koichiro TAKESHIGE1983 Volume 94 Issue 4 Pages 1301-1306
Published: October 01, 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSFormation of superoxide anions (O2-) by bovine heart NADH-dehydrogenase preparation (Complex I) supported by an NADH- or NADPH-generating system was studied kinetically. Both NADH- and NADPH-dependent superoxide-forming activities of Complex I were biphasic in double reciprocal plots. The NADH-dependent reaction had two sets of kinetic parameters: One Km value for NADH was 10 times higher than the other one, and the Vmax of the reaction with high Km was about 4 times higher than that of the reaction with low Km. Similar Vinax values were obtained for the NADPH-dependent reactions but the Km values were a thousand times higher than those of the NADH-dependent reactions. The plots of the NADH-dependent activity of rotenone-treated submitochondrial particles were also biphasic. The double reciprocal plots of NADH- and NADPH-dependent 2, 6-dichlorophenolindophenol (DCIP) reductase activities of Complex I were linear and the plots of the superoxide-forming activities against the DCIP reductase activities were biphasic. These results indicate that the biphasic double reciprocal plots of the superoxide-forming activities against reduced coenzymes are not caused by interaction between reduced coenzymes and the NADH-dehydrogenase but are due to the presence of at least two superoxide-forming sites in the respiratory chain NADH-dehydrogenase.View full abstractDownload PDF (455K) -
Soichi TSUJI1983 Volume 94 Issue 4 Pages 1307-1315
Published: October 01, 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSOrnithine transcarbamylase [EC 2. 1. 3. 3] has been purified from chick kidney to homogeneity. The molecular weight is 110, 000 as determined by gel filtration. Sodium dodecylsulfate polyacrylamide gel electrophoresis of the enzyme showed that the enzyme exists as a trimer of identical subunits of 36, 000 daltons like other mammalian species ornithine transcarbamylases. In 0.1M triethanolamine/HCl, the apparent optimum pH of the purified enzyme was 7.5 in the presence of 5mM ornithine. The curve shifted toward a more alkaline region with a decrease in ornithine concentration. The specific activity of the purified enzyme was 77 units at pH 7.5. The Km for carbamyl phosphate was 0.11mM and the Km for ornithine was 1.21mM. With an increase in pH, a decrease in Km values for ornithine and an increase in the extent of inhibition by ornithine were observed. On using antibody against bovine liver ornithine transcarbamylase, the precipitin lines for the chick and bovine enzymes showed a spur pattern. Even when excess amounts of the antibody were added, the chick enzyme did not lose the activity while the bovine enzyme activity was inhibited completely.View full abstractDownload PDF (1738K) -
Norio KANEDA, Fumio TANAKA, Kunio YAGI1983 Volume 94 Issue 4 Pages 1317-1328
Published: October 01, 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSChange in aggregation state of insulin upon conjugation with 5-dimethylamino-naphthalene-1-sulfonyl (DNS) group was investigated at neutral pH. DNS group was introduced exclusively into Bl phenylalanine, the N-terminus of the B-chain of insulin. The association state of insulin shifted toward a more highly aggregated one upon conjugation, depending on the mole fraction (d) of DNS group to insulin monomer; at d=0.3 the equilibrium between dimer and hexamer was dominant over the range of 1-600 μM, while at d=1.0-1.5 DNS-insulin formed a larger aggregate (dodecamer) which is stable over the range of 67-600 μm. The dissociation constant of dimer-hexamer equilibrium at d=0.3 was evaluated to be 2.5×10-10-10M2 from the fluorescence anisotropy of the DNS group, which was about one order of magnitude smaller than that of the dimer-hexamer equilibrium in native insulin.
Spectroscopic data and fluorescence decay analyses indicated that there exist at least two different environments surrounding the dye bound to Bl phenylalanine and that they are both relatively hydrophilic. It is considered that the major part of DNS group has excitation and emission maxima at longer wavelengths with relatively low quantum yield, while the minor part has excitation and emission maxima at shorter wavelengths with relatively high quantum yield. The fluorescence lifetime of the dye was modified by the change in quaternary structure of DNS-insulin.
Remarkable depolarization of DNS fluorescence was observed at d=1.0 and d=1.5 due to energy transfer between DNS groups conjugated to Bl phenylalanine in the hexamer or the dodecamer. Critical transfer distance for inter-DNS energy transfer was evaluated to be 15 Å. From the molecular model of the insulin crystal, this energy transfer is ascribed to the close proximity, within about 15 Å, between DNS groups in dimer units of the hexamer or the dodecamer.View full abstractDownload PDF (1433K) -
Shohei MAEKAWA, Sachiko ENDO, Hikoichi SAKAI, Hikoichi SAKAI1983 Volume 94 Issue 4 Pages 1329-1337
Published: October 01, 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA new protein capable of bundling actin filaments was purified from porcine brain by ammonium sulfate fractionation and Sephacryl S-300, hydroxyapatite and Whatman DE 52 column chromatographies. Co-sedimentability of this protein with actin filaments on low speed centrifugation was used as an index in the purification process. This protein had a molecular weight of 53, 000 as estimated by SDS-polyacrylamide gel electrophoresis. The Stokes' radius of the protein was determined to be 3.3 nm by the gel filtration method, which indicates a monomeric form of the protein in solution. On isoelectric focusing, it showed a single protein band having a pI of 5.62. This protein caused bundling of actin filaments as seen on electron microscopy, thereby lowering the specific viscosity of the actin solution in a concentration-dependent fashion. The bundling activity of the 53 K protein was modulated by changes in ionic strength and pH of the medium as well as by ATP and Mg ions. Low shear falling ball viscometry showed the formation of a gelling structure on mixing of actin filaments with this protein.View full abstractDownload PDF (2561K) -
Keiji TANAKA, Akira ICHIHARA1983 Volume 94 Issue 4 Pages 1339-1348
Published: October 01, 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSPrimary cultured rat hepatocytes synthesize various proteins and secrete half of them into the medium. When the cells were cultured in medium deficient in amino acids for 30 h, they maintained their normal level of intracellular protein synthesis, but their synthesis of extracellular proteins decreased by half. This reduced production of extracellular proteins could be restored by addition of amino acid mixture to the medium. Since secretion of protein was not inhibited and no albumin accumulated in the cells on amino acid deprivation, the reduced secretion of extracellular protein was due to inhibition of its synthesis rather than inhibition of its secretion.
Addition of the protease inhibitor leupeptin to cells cultured in medium supplemented with amino acids inhibited protein degradation 60%, but it did not change the rate of protein synthesis. Addition of this inhibitor to cells in amino acid deficient medium strongly inhibited the syntheses of both intra- and extracellular proteins.
Electron microscopic examination of the cells showed that amino acid deprivation markedly decreased the amount of rough endoplasmic reticulum and caused the appearance of autophagic vacuoles.
These results show that extracellular amino acids control the syntheses of both intra- and extracellular proteins, but have a more direct effect on the latter, the intracellular proteins being synthesized by reutilization of amino acids liberated on protein degradation. Moreover, protein degradation seems to be accelerated by formation of phagosomes, when the medium is deficient in amino acids. When this protein degradation is inhibited by a protease inhibitor, the synthesis of intracellular proteins decreases. Therefore, there seem to be two compartments of amino acids in hepatocytes, one supplied by lysosomal degradation of proteins and used mainly for synthesis of intracellular protein, and the other supplied from exogenous amino acids and used for syntheses of both intra- and extracellular proteins.View full abstractDownload PDF (2320K) -
Issei MABUCHI1983 Volume 94 Issue 4 Pages 1349-1352
Published: October 01, 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSG-actin freed from exogenous ATP was added to the pieces of isolated acrosomal actin bundles from horseshoe crab sperm to form filaments as reported earlier (Tilney, L. G., Bonder, E. M., & DeRosier, D. J. (1981) J. Cell Biol. 90, 485-494). The growth of a filament was far more rapid at one end (the preferred end) than the other end. These ends were shown to correspond to the barbed and pointed ends, respectively, by decoration of the filaments with myosin subfragment 1. Cytochalasin B inhibited the monomer addition at the preferred end. This technique is useful in determining the ends to which actin filament end-binding proteins from nonmuscle cells bind, which are considered to regulate the actin polymerization in the cells.View full abstractDownload PDF (1433K)
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