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Volume 94 (1983)
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- Issue 5 Pages 1353-
- Issue 4 Pages 1029-
- Issue 3 Pages 623-
- Issue 2 Pages 331-
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Volume 94, Issue 2
Displaying 1-36 of 36 articles from this issue
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Takeshi MIWA, Eiko AKABOSHI, Kenichi MATSUBARA1983 Volume 94 Issue 2 Pages 331-338
Published: 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSλdv plasmids having an amber mutation in an initiator gene, O or P, were constructed from mutant λ phages by recombinant DNA techniques and several properties of such derivatives were investigated. These plasmids are perpetuated in suppressorplus (amber-permissive) cells, but not in non-suppressor cells. The plasmid copy number in the suppressor-plus cells was low as compared to that of the plasmid without the amber mutation. In cells carrying a thermosensitive suppressor 2, raising the temperature is expected to block new production of amber proteins, but should not affect conservation of the protein made prior to heating. It was observed, however, that replication of the plasmids carrying an amber mutation in the O or P gene was abolished soon after raising the temperature, suggesting that neither of the initiator proteins can continue functioning unless replenished. Pulsechase experiments demonstrated that O protein decays with a half-life of 8min. Several lines of evidence suggest that this degradation occurs independently of the protein function. On the other hand, P protein was not degraded under the same experimental conditions. These observations are discussed in connection with functional instability of the initiator molecules. It appears that they do not work catalytically.View full abstractDownload PDF (591K) -
Motoaki ANAI, Atsuko MUTA, Morio UMENO, Mieko SASAKI1983 Volume 94 Issue 2 Pages 339-344
Published: 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSAn acid deoxyribonuclease has been purified from rat small intestinal mucosa by a procedure including ammonium sulfate fractionation, chromatographies on DEAE-cellulose, CM-cellulose and SE-Sephadex and finally isoelectric focusing. Polyacrylamide gel electrophoresis of the purified enzyme preparation showed one major and two minor bands, and the enzyme activity corresponded to one of the minor bands. The enzyme preparation was free of contaminating DNase I, DNase III, alkaline RNase, acid and alkaline phosphatases and nonspecific phosphodiesterase, but slight activities of DNase IV and acid RNase were detected. The enzyme did not require divalent cations for activity, had a pH optimum of 4.5 in 0.33M sodium acetate buffer, and had an optimum temperature of 50 to 60°C when assayed for 30min. The rate of hydrolysis of native DNA was about 2.5-fold faster than that observed with denatured DNA. Its molecular weight was obtained as 36, 000 by sucrose density gradient centrifugation. Its isoelectric point was found to be 9.0±0.1. The enzyme catalyzes the endonucleolytic cleavage of native and denatured DNA, yielding oligonucleotides which have an average chain length of about 7, and which contain 3'-phosphoryl termini. The mode of action of the enzyme is doublestrand scission.View full abstractDownload PDF (1118K) -
Inhibitory Effect of Bovine Serum Albumin on Acid Deoxyribonuclease from Rat Small Intestinal MucosaNatsuko ESHIMA, Atsuko MUTA, Motoaki ANAI1983 Volume 94 Issue 2 Pages 345-352
Published: 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSBovine serum albumin was found to have an inhibitory effect on acid DNase from rat small intestinal mucosa. The inhibitory activity showed pH-dependency. Thus, the highest inhibition was observed at about pH 4.3 but conversely the enzyme was activated at about pH 4.7. The inhibitory effect was heat-inactivated most strongly at about pH 5, but at more acidic or alkaline pHs, no inactivation was observed. Inhibitory activities of serum albumin of various species were comparable with that of bovine serum albumin. Acid DNases from guinea pig kidney and small intestinal mucosa and from rat spleen and kidney were similarly inhibited by the albumin. The acid DNase displays typical Michaelis-Menten kinetics but the kinetics became sigmoidal in the presence of the inhibitor. With increasing inhibitor concentration, the sigmoidal shape became more pronounced, and at high concentration, the DNA was able to compete with the inhibitor and to reverse its action. Among the cyanogen bromide-cleaved fragments of bovine serum albumin, fragment C (derived from the carboxyl-terminal two-thirds of the albumin) had an inhibitory effect comparable to that of intact bovine albumin, but fragment N (derived from the amino-terminal one-third of the albumin) had no activity. Reduced fragment C showed a markedly decreased effect and lost the activity completely after separation into its three component peptides. Acetylation of bovine serum albumin completely destroyed its inhibitory activity.View full abstractDownload PDF (529K) -
Koichiro KISHI, Haruhiko NODA1983 Volume 94 Issue 2 Pages 353-359
Published: 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe Raman spectra of purified myosin, C-protein and myosin-C-protein complex in aqueous solution, as well as that of pelleted filaments, were analyzed. The analyses confirmed that C-protein adopts a predominantly non-α-helical structure, while myosin adopts a predominantly α-helical structure. Neither 10mM MgCl2 nor filament formation induced large conformational changes of purified myosin. Thermal denaturation of purified myosin, however, induced large changes in the Raman spectrum owing to conformational changes. It is likely that the binding of myosin and C-protein is not accompanied by large conformational changes of the proteins.View full abstractDownload PDF (1044K) -
Takuji SUZAKI, Takashi IYANAGI, Shigeki KOBAYASHI1983 Volume 94 Issue 2 Pages 361-371
Published: 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe effect of 2, 4-dinitrophenol (2, 4-DNP) on the oxidative metabolism of hexobarbital by cytochrome P-450 was investigated in perfused rat liver. In the livers from fed, phenobarbital (PB)-treated rats, 2, 4-DNP (50 μm) had no effect on the redox state of cytochrome P-450 or on oxygen uptake during mixed-function oxidation of hexobarbital. In the livers from fasted, PB-treated rats, 2, 4-DNP (50 μM) significantly decreased the amount of reduced (oxygenated) cytochrome P-450 and the drug-induced oxygen uptake by about 50%. 2, 4-DNP caused a decrease of metabolites of hexobarbital in perfusate, in the fasted but not in the fed state. These results suggest that in fed, PB-treated rats NADPH for mixed-function oxidation of hexobarbital can be predominantly supplied from an extramitochondrial source (most probably via the cytosolic pentose phosphate shunt), but in fasted, PB-treated rats, about 50% of the NADPH required for the mixed-function oxidation is supplied from an intramitochondrial source.
In the livers from PB-treated rats, infusion of sorbitol (4mM), a glycogenic substrate in fasted rats, stimulated the rate of drug-induced oxygen uptake and the steady-state level of reduced (oxygenated) cytochrome P-450 increased during mixedfunction oxidation of hexobarbital. These effects of sorbitol were almost completely abolished in the presence of 2, 4-DNP. Complete inhibition of gluconeogenesis was also observed in the livers from fasted, PB-treated rats in the presence of 2, 4-DNP (50 μM). The amount of metabolites of hexobarbital in the perfusate was increased by the addition of sorbitol in the fasted but not in the fed state. The effect of sorbitol on drug metabolism was inhibited by 2, 4-DNP. These data may be explained by assuming that ATP is required for the conversion of sorbitol to metabolites (e. g. glucose-6-phosphate) which can produce NADPH in the cytosol.View full abstractDownload PDF (979K) -
Tamiko SUZUKI, Keizo INOUE, Shoshichi NOJIMA, Herbert WIEGANDT1983 Volume 94 Issue 2 Pages 373-377
Published: 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSUsing a synthetic glycolipid derived from maltotetraose and a spin-labeled fatty acid, the lateral distribution and molecular motion of the spin-labeled glycolipid on phosphatidylcholine liposomes in the presence and absence of concanavalin A were examined. When the spin-labeled glycolipid was added to preformed egg yolk phosphatidylcholine-dicetyl phosphate (molar ratio, 10:1) liposomes, most of the spin-labeled glycolipid molecules could be incorporated into liposomes as shown by their concanavalin A-induced agglutination. Concanavalin A also caused a change in line width of the ESR signal of liposome-bound spin-labeled glycolipid, whereas the overall splitting value 2A// did not change significantly. It is suggested that the binding of glycolipid molecules to concanavalin A increased the interactions among the radicals of the probe but that the mobility of the acyl chain of glycolipids was not affected. These signal changes were also observed with succinyl-concanavalin A. However, in contrast to concanavalin A, no appreciable agglutination of liposomes could be induced by the latter concanavalin A derivative. Both the agglutination of liposomes and the change in line width of the ESR signal were completely inhibited by α-methyl-D-mannoside.View full abstractDownload PDF (386K) -
Takeo YAMAGUCHI, Yoshinori FUJITA, Sumio KUROKI, Koji OHTSUKA, Eiji KI ...1983 Volume 94 Issue 2 Pages 379-386
Published: 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSMembrane fluidity of human erythrocytes treated with H2O2 (1-20mM) was studied using three kinds of fatty acid spin labels. A strongly immobilized signal appeared on exposure of erythrocytes to H2O2 but was not observed in either H2O2-or Fenton's reagent-treated ghosts or lipid vesicles prepared from H2O2-treated erythrocytes, indicating that the appearance of this signal necessitates the reaction of hemoglobin with H2O2 and is not due to lipid peroxidation.
The ESR spectrum of maleimide-prelabeled erythrocytes showed an isotropic signal and the rotational correlation time (τe) increased as the concentration of H2O2 was increased. Furthermore, maleimide labeling of H2O2-pretreated erythrocytes showed a strongly immobilized component, in addition to a weakly immobilized component. From the relative ratio of the signal intensity of hemoglobin and membrane proteins, it was found that label molecules bound predominantly to hemoglobin. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of H2O2-treated erythrocytes demonstrated globin aggregation. Therefore, the changes in the ESR signal observed on H2O2 treatment may be due to some change in hemoglobin, such as globin aggregation or its binding to the membranes.
The ESR spectrum of H2O2-treated erythrocytes at -196°C is characterized by signals of nonheme ferric iron type (g=4.3), low spin ferric iron, and free radical type at g=2.00. At higher H2O2 concentrations, the ESR lines due to low spin ferric iron became broad and their peak heights decreased, compared with that at g=2.00 or 4.3.
These results indicate that oxidative stress such as decrease of membrane fluidity, lipid peroxidation, and globin aggregation in H2O2-treated erythrocytes is dependent on the reaction of hemoglobin with H2O2.View full abstractDownload PDF (1219K) -
Keishiro WADA, Toshiaki TAMURA, Hiroshi MATSUBARA, Keiun KODO1983 Volume 94 Issue 2 Pages 387-393
Published: 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe preparation procedure for Spirulina ferredoxin-NADP+ reductase (ferredoxin: NADP+ oxidoreductase, EC 1. 18. 1. 2, FNR) was improved by adding protease inhibitors, phenylmethylsulfonylfluoride (PMSF) and EDTA, through the whole process of preparation and by introducing an affinity chromatography step on Blue Sepharose CL-6B.
The addition of the inhibitors largely prevented the formation of the minor component (FNR I), and the affinity gel chromatography simplified the preparation process, shortening the exposure period of FNR to proteolysis. However, complete removal of the heterogeneity of FNR found at the amino (N)-terminal region was not achieved even by applying the new method. The affinity chromatography on the Blue Sepharose gel was also effective in purifying spinach FNR. The affinity of this gel for Spirulina FNR was compared with that for the enzyme derived from spinach leaves. The spinach enzyme had a higher affinity than the Spirulina one. Both enzymes showed the highest affinities to Blue Sepharose at 20-30mM NaCl concentration.
The N-terminal sequence analysis revealed that there were 4 forms, which were probably modifications produced by exopeptidase action during the preparation, or even in the living cells. The longest component gave the N-terminal sequence Ala-Lys-Thr-Asp-Ile-Pro-Val-Asn-Ile-Tyr-. The others lacked amino acids successively one by one from the N-terminus. In contrast, the carboxyl (C)-terminal residues of all 4 FNR forms were tyrosine. The probable C-terminal sequence was predicted to be-Trp-His-Val-Gln-Thr-Tyr based on a study of a cyanogen bromide peptide.View full abstractDownload PDF (561K) -
Tetsuya ASAKAWA, Naomi AZUMA1983 Volume 94 Issue 2 Pages 395-401
Published: 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe experimental conditions for release of the regulatory light chain (RLC) of scallop myosin at 30°C were studied. Substantially all RLC was released from myosin by incubation for 5min in medium containing buffer and KCl. This release of RLC was inhibited strongly by Ca2+ while the effect of Mg2+ was about 10, 000 times weaker than that of Ca2+. Even in the absence of Ca2+, MgATP and MgADP inhibited the release of RLC, while the protective effect of AMPPNP was negligible. Other Mg nucleotides also showed some protective effect, though appreciably less than MgATP.
The incubation of scallop myosin with abalone regulatory light chain (LC2) at 30°C for 5 min produced a hybrid myosin. In the presence of 5mM MgCl2, 1 of the 2 mol of RLC per mol of scallop myosin was exchanged with 1 mol of LC2. In the presence of Ca2+ or MgATP, myosin bound 1 extra mole of LC2 besides the 2 mol each of SH-LC and RLC.View full abstractDownload PDF (1116K) -
Yasuyuki OGURA, Isao YAMAZAKI1983 Volume 94 Issue 2 Pages 403-408
Published: 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSUnder carefully controlled experimental conditions, the Michaelis constant for H2O2 was measured to be 1.39 and 1.29M in the reactions of beef erythrocyte and liver catalases, respectively. These values remained unchanged at temperatures between 1 and 26°C. The turnover number of the Michaelis complex was about 2.25×107 s-1 for either enzyme at 26°C. The cyanide inhibition in the catalase reaction has been reported to be noncompetitive in spite of the fact that cyanide and H2O2 compete for the same site on the catalase molecule. At high concentrations of H2O2, however, the inhibition became clearly competitive. The existence of the Michaelis complex and the anomalous features of cyanide inhibition were clearly accounted for on the basis of simple kinetic models.
At H2O2 concentrations below 100mM, the catalase reaction obeyed first order kinetics with respect to H2O2 and its apparent second order rate constant was measured to be 7.6×166 and 7.9×106M-1•s-1 for erythrocyte and liver catalases, respectively.View full abstractDownload PDF (363K) -
XII. Reaction Catalyzed by 2, 4-Dienoyl-CoA Reductase of Escherichia coliMichinao MIZUGAKI, Chiharu KIMURA, Tomoko NISHIMAKI, Akihiko KAWAGUCHI ...1983 Volume 94 Issue 2 Pages 409-413
Published: 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSIncorporation of deuterium atoms from deuterium-labeled NADPH and 2H2O during the reaction catalyzed by 2, 4-dienoyl-CoA reductase of Escherichia coli (E. coli) was investigated. When traps-2, cis-4-decadienoyl-CoA was incubated with 4R- or 4S-[4-2H1]NADPH in the presence of purified 2, 4-dienoyl-CoA reductase, no deuterium was detected in the reaction product by gas chromatography-mass spectrometry after derivatization to its pyrrolidine amide. On the other hand, when the dienoyl-CoA was incubated in the presence of NADPH and the reductase in 2H2O two deuterium atoms were incorporated: One deuterium atom was located at the C-4 position of trans-2-decenoate, and the other at the C-5 position. The UV and shorter wavelengths of the visible spectrum of the reductase solution revealed that the reductase contained flavin as a prosthetic group. Therefore it is considered that a hydrogen atom of NADPH was first transferred to the flavin moiety of the reductase, and then the hydrogen atom was rapidly exchanged for one in the medium before its direct transfer to the substrate.View full abstractDownload PDF (363K) -
Masayuki YOKOKAWA, Akiko FUJIWARA, Hiraku SHIMADA, Ikuo YASUMASU1983 Volume 94 Issue 2 Pages 415-420
Published: 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSRNA polymerase from Escherichia coli was inhibited by long chain fatty acyl CoAs, such as myristoyl CoA (K1=17.2 μM), palmitoyl CoA (K1=8.9 μM), oleoyl CoA (K1=5.5 μM), and stearoyl CoA (K1=0.94 μM). The inhibition by these CoA thioesters was non-competitive against nucleoside triphosphates. Short chain fatty acyl CoAs, such as acetyl CoA, propionyl CoA, acetoacetyl CoA, butyryl CoA, and decanoyl CoA, failed to inhibit RNA polymerase. CoA, Na-myristate, Na-palmitate, Na-oleate, Na-stearate, palmitoyl carnitine, and carnitine did not inhibit the enzyme. The inhibition of RNA polymerase by long chain fatty acyl CoAs was competitive against template DNA.View full abstractDownload PDF (357K) -
Yukiyasu TOYODA, Ichitomo MIWA, Jun OKUDA1983 Volume 94 Issue 2 Pages 421-431
Published: 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSComparative studies of mutarotase [aldose 1-epimerase, EC 5. 1. 3. 3] from the kidney, liver and small intestine of rats were performed placing the focus on the study of multiple forms. The findings obtained are as follows.
1. Mutarotases from the kidney and liver of adult rats were both separated into four forms (types I-IV) by DEAF-cellulose column chromatography, whereas only two forms (types I and II) were detected in the small intestine. Liver mutarotase type I was further separated into types I1 and I2, by column chromatography on hydroxylapatite.
2. Types I and II from the kidney and type II from the liver were purified to homogeneity as judged by isoelectric focusing on thin layer polyacrylamide gel. Of various physicochemical properties, only the Km for α-D-xylose and the isoelectric point were different among the multiple forms.
3. Liver mutarotase was immunohistochemically localized in the nuclei of parenchymal cells and small intestine enzyme in the nuclei of mucosal cells, indicating similarity with the localization of kidney enzyme (in the nuclei of epithelial cells of renal tubules and glomeruli) which was reported in our previous paper [E.xperientia (1979) 35, 1094-1097].
4. The kidney mutarotase level increased gradually after birth and reached a maximum near adult level within 20 days. This developmental pattern was essentially the same as that in the liver but clearly different from that in the small intestine, in which the mutarotase activity of suckling rats was several times higher than that of adult rats.
5. Distribution patterns of multiple forms (types I-IV) of the enzyme in the kidney and liver of 10-day-old rats were similar to those in respective tissues of adult rats. On the other hand, the small intestine of 10-day-old rats contained four forms (types I-IV), whereas there were only two forms (types I and II) in adult rats.View full abstractDownload PDF (1422K) -
Kuniharu OHNISHI, Kazuyoshi KIRITANI1983 Volume 94 Issue 2 Pages 433-441
Published: 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSTwo leucine-binding proteins isolated from osmotic shock fluid of Salmonella typhimurium LT2 were purified by DEAE-cellulose and DEAE-Sephadex A-50 chromatography, and subsequent isoelectric focusing. These purified binding proteins could be crystallized by adding 2-methyl-2, 4-pentanediol. One of the binding proteins, designated as LIVT-binding protein, binds L-leucine, L-isoleucine, L-valine, and L-threonine, while the other, L-binding protein, binds only L-leucine. The level of LIVT-binding protein in the shock fluid was about three-fold higher than that of L-binding protein. The molecular weight of the LIVT-binding protein was estimated to be 35, 000 by gel filtration, and 39, 000 by gel electrophoresis. The isoelectric point was pH 4.94. The dissociation constants of this protein for leucine, isoleucine, and valine were 0.43, 0.15, and 0.89 μM, respectively. For the L-binding protein, molecular weights of 34, 000 (gel filtration), and 38, 000 (gel electrophoresis) were obtained. The isoelectric point was pH 4.74. The dissociation constant of this protein for leucine was 0.54 μM. The LIVT-binding protein was more heatstable than the L-binding protein. These two binding proteins showed an antigenic similarity, they could cross-react with each other's antiserum. This similarity was also found between the binding proteins of Salmonella typhimurium and Escherichia coli K-12. Both LIVT- and L-binding proteins in a regulatory mutant, KA2313, were found to be about three-fold the levels in the wild-type strain.View full abstractDownload PDF (1769K) -
Toshinobu FUJIYOSHI, Makoto SASAKI, Kazuko ONO, Takanori NAKAMURA, Kaz ...1983 Volume 94 Issue 2 Pages 443-450
Published: 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA small packageable plasmid depending on the ColE1 replicon was constructed from ColE1-gal-cosλ:: Tn3, pKY2113 (T. Nakamura et al. (1981) J. Biochem. 90, 1013), and named pKY2662. This plasmid carries ampicillin resistance and colicin E1 immunity genes as selective markers, and has neither mobilization function nor movable transposon. The molecular size of pKY2662 is 8.7 kb, and it has a single cleavage site each for BamHI, BglII, ClaI, EcoRI, HpaI, PstI, and Tth111I.
By using pKY2662 as a vector, a 32 kb Escherichia coli DNA fragment covering thyA, recC, recB, and argA genes was cloned. This new small cosmid is among the most efficient vectors hitherto found for in vitro cloning of large DNA fragments.View full abstractDownload PDF (1309K) -
Akiyo MATSUMOTO, Toru KAMATA, Kazuo MATSUDA1983 Volume 94 Issue 2 Pages 451-458
Published: 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA branching enzyme was extracted from the mycelia of Neurospora crassa and was purified to electrophoretic homogeneity by procedures including DEAE-Sephacel column chromatography, 6-aminohexyl-Sepharose 4B column chromatography and gel filtration on Toyopearl HW-55S. The final yield of the branching enzyme activity was 15.1%, and the final purified enzyme preparation showed a specific activity of 702 units per mg of protein. The molecular weight of this enzyme was estimated to be 80, 000 by electrophoresis in sodium dodecyl sulfate-polyacrylamide gel. The amino acid composition and the carbohydrate content of this enzyme were analyzed. The isoelectric point of this enzyme determined by polyacrylamide gel isoelectrofocusing was 5.6. The branching activity of the enzyme was confirmed by its action on amylopectin as well as by the combined action of this enzyme and N. crassa glycogen synthase. The action of this enzyme on amylopectin decreased the wavelength of the absorption maximum of the glucan-iodine complex, and increased the amount of the short unit chains of the debranched product. The product obtained by the combined action yielded β-limit dextrin upon hydrolysis with β-amylase. No multiplicity was found for the branching activity either by chromatography or by electrophoresis.View full abstractDownload PDF (1664K) -
Youichi YACHIDA, Osamu MINARI1983 Volume 94 Issue 2 Pages 459-471
Published: 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe present investigation was on the particle species of human plasma high density lipoprotein (HDL) characterized by the stoichiometry of their apoprotein components. HDL2-1, HDL2-2, HDL3-1, and HDL3-2 isolated from normal human plasma by sequential ultracentrifugal flotation were further subfractionated by Bio Gel A-5m gel chromatography or hydroxyapatite column chromatography, and three distinct subtractions were obtained. Subfraction I was obtained from all the HDL fractions and it contained mostly apolipoprotein A-I (A-I). Subfraction 2 was obtained from HDL2-2 and HDL3-1 and it contained A-I and apolipoprotein A-II (A-II) in the molar ratio of one to one, and subfraction 3 from HDL2-2 and HDL3-1 contained A-I and apolipoprotein C (C). Each subfraction was treated with bifunctional cross-linking reagents, and the intraparticle cross-linked products of apolipoproteins were examined by SDS-polyacrylamide gel electrophoresis. The results of the cross-linking studies indicated that the HDL2 fraction consisted mainly of lipoprotein particles of the (A-I)4 type and a few of the (A-I)5, (A-I)2(A-II)2, and (A-I)4(C)2 types, and that the HDL3 fraction consisted mainly of (A-I)2(A-II)2 type particles and a few (A-I)4, (A-I)3, (A-I)2, (A-I), and (A-I)3(C)2 type particles. From the results of analyses of the lipid components in the HDL of each type, it was suggested that the function of the particle species of the (A-I)n type (n=1-5), which contained more cholesteryl ester than the (A-I)2(A-II)2 type, was concerned mainly with cholesterol metabolism.View full abstractDownload PDF (4763K) -
Tsunehiro KANNO, Morio SETAKA, Toshio HONGO, Takao KWAN1983 Volume 94 Issue 2 Pages 473-477
Published: 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSSurface pressure was found to be produced spontaneously at the interface between air and a suspension containing fragmented sarcoplasmic reticulum (FSR) from rabbit white muscle. Large and stable surface pressure was formed only in a limited concentration range of FSR in the suspension and the pressure formation was proved to be an irreversible phenomenon, suggesting the formation of a monolayer membrane resulting from the disruption of FSR vesicles. Monolayer formation was directly confirmed by analyzing the components included in the membrane and by calculating the surface area occupied by these components. The monolayer included phospholipids, cholesterol and proteins, and appeared to originate from FSR vesicles since the molecular ratios of these components as well as the results of the SDS polyacrylamide gel electrophoresis were similar in both membranes.
This phenomenon can be utilized as a method of monolayer preparation from biological membrane vesicles, and should be very useful for the reconstitution of planar biological membranes.View full abstractDownload PDF (922K) -
Masayuki OZAWA, Masahiro SATO, Takashi MURAMATSU1983 Volume 94 Issue 2 Pages 479-485
Published: 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSLaminin was purified to homogeneity from the extracellular matrix and soluble fraction of teratocarcinoma OTT6050 and also partially purified from the ascitic fluid of the mice carrying the teratocarcinoma. These laminin preparations were found to agglutinate trypsinized, glutaraldehyde-fixed rabbit erythrocytes. The hemagglutinating activity was inhibited by porcine gastric mucin, while invertase and mannan were not inhibitory. Heparin and heparan sulfate also inhibited the hemagglutination. Simple saccharides such as D-galactose, N-acetyl D-glucosamine, and N-acetyl D-galactosamine were not inhibitory, but D-glucosamine and D-galactosamine were. The hemagglutinating activity required Ca2+ and was dependent upon temperature. These results raised the possibility that laminin functions also in cell-cell interactions such as cell-cell adhesion. In addition, we report that laminin synthesized by the teratocarcinoma did not carry the large carbohydrate chain characteristic of early embryonic cells.View full abstractDownload PDF (2549K) -
Akihiko KAWAGUCHI, Kohei HOSAKA, Toshiro KIKUCHI, Kenji SODA, Shigenob ...1983 Volume 94 Issue 2 Pages 487-492
Published: 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA method for measuring free fatty acids by enzymic cycling is described. Free fatty acids are converted to acyl-CoAs by acyl-CoA synthetase, then the acyl-CoAs are hydrolyzed back to the free fatty acids by acyl-CoA hydrolase in a cyclic fashion. The amounts of AMP produced during this cyclic reaction are determined from the absorbance at 340 nm in the presence of AMP deaminase and glutamate dehydrogenase. This method is sensitive to as low as 0.1 nmol of free fatty acids, and the standard curve is linear up to 1.0 nmol. This method shows a broad specificity for long-chain fatty acids (C12-C20) and the recoveries of fatty acids added to bacterial cell-free extracts are more than 90%.View full abstractDownload PDF (434K) -
Fusao WATANABE, Wataru KAMIIKE, Tadashi NISHIMURA, Tadao HASHIMOTO, Ku ...1983 Volume 94 Issue 2 Pages 493-499
Published: 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe process of mitochondrial dysfunction in ischemic rat liver was studied. A close correlation was found between decrease in the mitochondrial adenine nucleotide content and deterioration of oxidative phosphorylation capacity. The level of total adenine nucleotides, which was 15-20 nmol/mg protein in mitochondria isolated from normal liver, fell to 1-2 nmol/mg protein with concomitant loss of oxidative phosphorylation capacity after anoxic incubation in vitro or in vivo for 120min. However, neither the permeability barrier to adenine nucleotides nor matrix enzymes were affected under these conditions. The loss of adenine nucleotides was ascribed to degradation of AMP to adenosine and then leakage of the latter. Conventional procedures for maintenance of oxidative phosphorylation capacity of isolated mitochondria, preservation in the cold and addition of ATP or a respiratory substrate under aerobic conditions, were very effective in maintaining the intramitochondrial levels of adenine nucleotides. Of the three species of adenine nucleotides, only AMP was ineffective in maintaining mitochondrial function; mitochondria containing more than 5 nmol of ATP plus ADP/mg protein exhibited normal activity of oxidative phosphorylation, but with less than 2 nmol they showed no activity.View full abstractDownload PDF (553K) -
Shingo HATA, Yoshihisa ODA, Tokuzo NISHINO, Hirohiko KATSUKI, Yuri AOY ...1983 Volume 94 Issue 2 Pages 501-510
Published: 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSAnalysis of sterols was made with a Saccharomyces cerevisiae mutant, N22, which was resistant to nystatin and defective in ergosterol synthesis, and with its parent strain, M10 (haploid type, methionineless, petite). The main sterol of M10 was ergosterol, whereas that of N22 was ergosta-5, 7-dien-3β-ol. A small amount of ergosterol was found also in N22. This indicates that the Δ22-desaturation reaction in N22 is blocked, though not completely. Ergosta-5, 8-dien-3β-ol, an unusual sterol, was detected in N22 but not in M10. Variations in the amounts and compositions of free and esterified sterols of both strains were examined during cultivation and the subsequent aerobic adaptation. The contents of free sterols, which were mostly composed of the respective main sterols of both strains, did not change markedly. In contrast, the contents and compositions of esterified sterols of both strains varied depending on the growth conditions. When the cells of both strains were aerobically adapted, the total contents of esterified sterols increased, as did, as the intermediate sterols. Upon aerobic adaptation in the presence of [methyl14C]methionine, [28-14C]ergosta-5, 7-dien-3β-ol accumulated markedly in N22 cells. Using this mutant, we devised a convenient method for preparation of the radioactive sterol in high yield.View full abstractDownload PDF (667K) -
Akira MURAYAMA, Fumio FUKAI1983 Volume 94 Issue 2 Pages 511-519
Published: 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA detailed study of the molecular mechanism of the translocation of estrogen receptor (ER) from the cytoplasm into the nucleus was undertaken in an in vitro system of porcine uterus. The capabilities of vero-ER•E (basic ER molecule bound with estradiol) (sedimentation coefficient 4.5S; Stokes radius 44 A) and the complexes [“5S” ER•E, (vero-ER•E)•(component A); “6S” ER•E, (vero-ER•E)•(component B)6; “8S” ER•E, (vero-ER•E)•(component B)6•(component A)] with ER-binding factors (ERBFs) to translocate into the isolated nuclei were estimated by subtracting the amounts of ER adsorbed by the nuclear envelopes from those of ER bound to the whole nuclei. The results strongly supported our previous assumption that vero-ER•E translocates into the nuclei, and the complexes with ERBFs do not. The results suggested also that the binding site of vero-ER to ERBFs is required to be unoccupied in the process of the translocation of ER from the cytoplasm into the nucleus. The presence of a cytoplasmic factor (component C) which binds specifically with “5S” ER•E under low salt conditions was indicated. The complex, (“5S” ER•E)•(component C), was shown to possess relatively high affinity towards nuclear envelopes, but not to translocate into the nuclei. It was assumed that this adsorption of (“5S” ER•E)•(component C) to the nuclear envelopes was taken as translocation into the nucleus in the previous studies. The ER-fragments [secto-ER•E (sedimentation coefficient, 4.5S; Stokes radius, 35 A) and “3.8S” ER•E (sedimentation coefficient, 3.8S; Stokes radius, 32 A)] obtained by the proteolysis of vero-ER•E by the endogenous proteases, in which the binding sites to ERBFs are destroyed, did not translocate into the nuclei. These results indicated that the binding site (or the adjacent region) of vero-ER to ERBFs plays an indispensable role in the translocation of vero-ER•E from the cytoplasm into the nucleus.View full abstractDownload PDF (719K) -
Takashi AGUI, Bunei SYUTO, Keiji OGUMA, Hiroo IIDA, Shuichiro KUBO1983 Volume 94 Issue 2 Pages 521-527
Published: 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe binding of Clostridium botulinurn type C neurotoxin to rat brain synaptosomes was determined by the use of 125I-neurotoxin. The binding was independent of the incubation temperature (0°C and 37°C) and was equilibrated in 10min. The dose dependence of 125I-toxin binding to synaptosomes at 0°C showed that there were two kinds of toxin receptors on the synaptosomal membrane; the association constants and maximum binding values were 1.05×1010 M-1, 5.25×10-13 mol/mg of synaptosomal protein and 5.00×108 M-1, 5.00×10-12 mol/mg of synaptosomal protein, respectively.
When the incubation of toxin with synaptosomes was continued at 37°C after 125I-toxin had been pre-incubated with synaptosomes at 0°C for 10 min, the displacement of labeled toxin by the addition of excess amounts of unlabeled toxin decreased slightly with increasing incubation time, and finally 0.4% of the bound 125I-toxin was not displaced from synaptosomes. The binding of 125I-toxin to synaptosomes was inhibited by anti-heavy chain IgG and a monoclonal antibody which neutralized toxin and recognized heavy chain. These results suggest that the binding sites of toxin to synaptosomes are localized on heavy chain and a small amount of the bound toxin is incorporated into the synaptosomal membrane or synaptosomes.View full abstractDownload PDF (566K) -
Shoko MIYAZAWA, Hisashi OZASA, Takashi OSUMI, Takashi HASHIMOTO1983 Volume 94 Issue 2 Pages 529-542
Published: 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe activities of carnitine octanoyltransferase (COT) and carnitine palmitoyltransferase (CPT) in rat liver were markedly increased by administration of di(2-ethylhexyl)phthalate. COT and CPT were purified from the enzyme-induced rat liver. COT was a 66, 000-dalton polypeptide. The molecular weight of native CPT was 280, 000-320, 000 daltons, and the enzyme consisted of 69, 200-dalton polypeptides. CAT, COT, and CPT were immunologically different. COT exhibited activity with all of the substrates tested (acyl-CoA's and acylcarnitines of saturated fatty acids having carbon chain lengths of C2-C20), though maximum activity was observed with hexanoyl derivatives. CPT exhibited catalytic activity with medium- and longchain acyl derivatives. 2-Bromo-palmitoyl-CoA inactivated COT but not CPT. Malonyl-CoA inhibited CPT but not COT. CPT was confined to mitochondria, whereas COT was found in peroxisomes and the soluble compartment but not in mitochondria.View full abstractDownload PDF (2398K) -
Hisashi OZASA, Shoko MIYAZAWA, Takashi OSUMI1983 Volume 94 Issue 2 Pages 543-549
Published: 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSMale Wistar rats were fed a diet with or without di (2-ethylhexyl) phthalate (DEHP) for 2 weeks. Carnitine octanoyltransferase (COT) in the liver was increased 23.5-fold in rats given DEHP. It was found by in vivo experiments using L-[4, 5-3H]leucine and the immunoprecipitation technique that the rate of synthesis of COT was 14.1-fold higher and that of its degradation was 1.5-fold lower in the DEHP group. COT was translated much more effectively in free polysomes than in membranebound polysomes. The molecular size of the in vitro product was the same as that of the mature enzyme. The translation activity of mRNA coding for COT measured with total hepatic RNA was 16.6-fold higher in the DEHP group. Carnitine palmitoyltransferase (CPT) was increased 5.9-fold after administration of DEHP. The rate of synthesis of CPT measured in the in vivo experiment was 5.0-fold higher in the DEHP group. The rate of its degradation was the same in the two groups. CPT was also translated much more effectively in free polysomes. The size of the preenzyme was larger than that of the subunit of the mature enzyme by about 2, 400 daltons. In contrast to COT, the increase in the translation activity of mRNA for CPT by administration of DEHP was markedly higher than the increase in the rate of its synthesis measured in the in vivo experiment.View full abstractDownload PDF (1051K) -
Sueko HAYASHI, Masaru SUZUKI, Satoshi NAKAMURA1983 Volume 94 Issue 2 Pages 551-558
Published: 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSSarcosine oxidase [sarcosine: oxygen oxidoreductase (demethylating) EC 1. 5. 3. 1] from Corynebacterium contained 8 sulfhydryl groups per mol of enzyme as determined with 5, 5'-dithiobis (2-nitrobenzoic acid) (DTNB) in the presence of 0.2 SDS and by titration with p-chloromercuribenzoate (PMB). Among them, 2 groups were easily modified by iodoacetamide (IAA) and the modification resulted in complete loss of enzymatic activity. The inactivation by IAA followed first-order kinetics with respect to IAA concentration. The presence of acetate, a competitive inhibitor (1), protected the enzyme from inactivation by IAA. However, the protection was only approximately 50%.
The enzyme was also inactivated by PMB, but in this case, there was practically no recovery of activity after treatment with thiol compounds.
The enzyme was also rapidly inactivated by incubation with diethylpyrocarbonate (DEP). The absorbance change accompanying the inactivation showed that a single histidyl residue was modified by DEP, resulting in a complete loss of enzymatic activity. In the presence of acetate, the enzyme was completely protected from DEP-inactivation. Furthermore, DEP-inactivated enzyme recovered its enzymatic activity on treatment with hydroxylamine. These observations seem to imply that the modified histidine is essential for enzyme activity. In addition, modification by DEP changed the absorption spectrum in the visible region. This strongly suggests that the modified histidyl residue is present in the vicinity of the flavin moiety of the enzyme molecule.View full abstractDownload PDF (640K) -
Satoko KITAJIMA, Akiko SETO-OHSHIMA, Mamoru SANO, Kanefusa KATO1983 Volume 94 Issue 2 Pages 559-564
Published: 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe production of anti-calmodulin antibodies in rabbit was examined with the use of performic acid-oxidized calmodulin from bovine brain or an emulsion of native calmodulin and methylated bovine serum albumin as the antigen. The antibodies in rabbit sera were determined serially by means of a sensitive enzyme immunoassay method that uses a mini-column of goat (anti-rabbit IgG) IgG-coupled Sepharose 4 B for separation. The antibodies could be produced in rabbits with either antigen. Although the native calmodulin with methylated bovine serum albumin seemed to be a better antigen, the titer of antiserum was not raised to levels detectable in the double immunodiffusion test. Monospecific antibodies were purified from the antiserum, and a competitive enzyme immunoassay method for the assay of calmodulin was developed with the use of the column-separation technique employed in the assay of anti-calmodulin. The method could determine from 10 ng to 10 μg (0.6 pmol to 0.6 nmol) of rat calmodulin and gave no cross-reaction with S-100 protein. Calmodulin contents in rat brain determined by the present method were consistent with those measured by the bioassay using calmodulin-deficient phosphodiesterase.View full abstractDownload PDF (491K) -
Shin-ichi NIHIRA, Jiro KOYAMA1983 Volume 94 Issue 2 Pages 565-573
Published: 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSInhibitory effects of various amino acid esters on the phagocytic activity of guinea pig peritoneal macrophages were studied with sensitized 51Cr-sheep erythrocytes (51Cr-EAb) as well as 125I-α-amylase complexed with homologous IgG2 antibody (Ag-Ab complex). The intracellular uptake of 51Cr-EAb was markedly inhibited by N-acetyl-L-phenylalanine ethyl ester (Ac-Phe-OEt), N-acetyl-L-tryptophan ethyl ester (Ac-Trp-OEt) and N-benzoyl-L-tyrosine ethyl ester (Bz-Tyr-OEt), but not by N-acetyl-L-tyrosine ethyl ester (Ac-Tyr-OEt), N-α-acetyl-L-arginine methyl ester (Ac-Arg-OMe), N-α-benzoyl-L-arginine ethyl ester (Bz-Arg-OEt) or N-α-acetyl-L-lysine methyl ester (Ac-Lys-OMe).
When phagocytosis of the Ag-Ab complex was assayed by measuring the amount of digested products released from macrophage cells, Ac-Tyr-OEt also inhibited it as markedly as Ac-Phe-OEt, Ac-Trp-OEt, and Bz-Tyr-OEt did, whereas Bz-Arg-OEt again did not show any effect. The results of analysis of the intracellular fate of the Ag-Ab complex taken up by macrophages through the use of analytical density gradient fractionation of the homogenized cells suggest that AcPhe-OEt inhibits the ingestive process since the distribution of Ag-Ab complex showed a single peak, closely accompanying the plasma membrane. Ac-Tyr-OEt, on the other hand, caused a marked accumulation of Ag-Ab complex in the lysosome fraction, reflecting the inhibition of intralysosomal digestion of the complex.
These results may classify the chymotrypsin substrates tested, into three groups: 1) Ac-Phe-OEt and Ac-Trp-OEt inhibiting the ingestive process in phagocytosis, probably more strongly than the digestive process; 2) Bz-Tyr-OEt inhibiting both the ingestive and digestive processes; and 3) Ac-Tyr-OEt inhibiting the digestive process alone. In addition, this classification of the chymotrypsin substrates may support the hypothesis that a certain chymotrypsin-like serine protease with a high substrate-specificity is involved in the ingestive process of immune complexes in macrophages.View full abstractDownload PDF (771K) -
Fuyuki IWASA, Hideo MOHRI1983 Volume 94 Issue 2 Pages 575-587
Published: 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSCalmodulin and calmodulin-binding proteins in the cytosol extract of eggs from the sea urchins Hemicentrotus pulcherrimus and Strongylocentrotus intermedius were studied in an attempt to elucidate the physiological role (s) of calmodulin in eggs. Calmodulin in the cytosol extract was found both in a free form and in complexes with other proteins, either Ca2+-dependently or Ca2+-independently. The extracts contained at least three calcium-dependent calmodulin-binding proteins. One was an NAD kinase of unknown molecular composition. The apparent molecular weights of the other two calmodulin-binding proteins were 50 K and 55K+17K daltons, as estimated by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. These three proteins formed complexes with calmodulin only in the presence of calcium as demonstrated by gel filtration. The 17K-dalton protein was found to be calmodulin itself; it did not dissociate from the 55K-dalton protein regardless of the presence or absence of calcium. The native molecular weights of these protein-calmodulin complexes obtained by gel filtration through a Sephacryl S-300 column were 190K for the NAD kinase, 130K for the 50K-dalton protein and 100 K daltons for the 55K+17K-dalton protein.View full abstractDownload PDF (2750K) -
Shigemi NORIOKA, Tokuji IKENAKA1983 Volume 94 Issue 2 Pages 589-599
Published: 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe amino acid sequences of four peanut protease inhibitors (A-I, A-II, B-I, and B-II) were determined by conventional methods and by comparison of peptide maps of their tryptic digests with that of B-III on HPLC. A-I, A-II, B-I, and B-III had the same amino acid sequence except for differences in their N-terminal regions. This suggests that the four inhibitors would be derived from an original inhibitor with a longer N-terminal amino acid sequence by proteolysis of its N-terminal region. But B-II possessed an extremely different amino acid sequence from those of the other peanut inhibitors and was thought to be biosynthesized from a gene different from that of the other inhibitors.
A phylogenetic tree of legume double-headed inhibitors was constructed on the basis of the matrix of amino acid differences among their sequences. The doubleheaded inhibitors whose sequences have been determined were classified into four groups.View full abstractDownload PDF (671K) -
Ken-ichi AMANO, Jim C. WILLIAMS1983 Volume 94 Issue 2 Pages 601-606
Published: 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe proteins associated with the peptidoglycan (PG) of Legionella pneumophila are resistant to proteolysis by trypsin, protease VI and proteinase K. These proteaseresistant proteins are associated with the PG noncovalently and covalently. Analysis of cell walls and PG-protein complex by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions revealed one major protein (38.5K) and several minor Coomassie and silver staining components. The 38.5K protein seemed to be a major component which was co-purified with the PG. The cleavage of the PG-protein complex by 1 N NaOH treatment yielded PG free of proteins which was subjected to alkali hydrolysis. This association of PG and proteaseresistant covalently-bound proteins may be an important structural and functional determiner of resistance to both environmental conditions and intracellular digestion of L. pneumophila by eukaryotic cells.View full abstractDownload PDF (1191K) -
Masaru TANOKURA, Kazuhiro YAMADA1983 Volume 94 Issue 2 Pages 607-609
Published: 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSEnthalpy titrations of calmodulin with Ca2+ (or Mg2+) in the presence and the absence of Mg2+ (or Ca2+) have been carried out by microcalorimetry. The binding reactions of both Ca2+ and Mg2+ to calmodulin are endothermic, indicating that the large entropy change gives rise to the strong binding of Ca2+ to calmodulin. The results also indicated that all the four binding sites are Ca2+-Mg2+ sites. These results are markedly different from the findings for troponin C.View full abstractDownload PDF (213K) -
Yutaka TANAKA, Yasuo TSUNOGAE, Yasuhiro HAYAKAWA, Isao TANAKA, Takashi ...1983 Volume 94 Issue 2 Pages 611-613
Published: 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSPreliminary crystallographic studies of a Bowman-Birk type protease inhibitor, AB-I, from adzuki beans (Phaseolus augularis) ‘Takara’, and its complex with trypsin were carried out. AB-I, MW 9100 with 82 amino acid residues, crystallizes in a trigonal space group, P3121 (or P3221), with the following unit cell dimensions: a=68.7, c=99.7 Å. The asymmetric unit contains two dimer molecules. Structure analysis at 5 Å resolution revealed the rough appearance of the dimer. The complex between AB-I and trypsin also could be crystallized in a tetragonal space group, P41212 (or P43212), with the unit cell dimensions, α=55.4 and c=181.5 Å, and Z=8. The crystallinity seems to be much better than that of the crystals of the inhibitor alone. The other type of inhibitor from adzuki bean, AB-IIa, was also crystallized.View full abstractDownload PDF (1006K) -
Makoto KINOSHITA, Mitsuyo OKAZAKI, Hirokazu KATO, Tamio TERAMOTO, Teru ...1983 Volume 94 Issue 2 Pages 615-617
Published: 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA simple and rapid method has been developed for the separation of apolipoproteins in high density lipoprotein (HDL) fractions by high performance liquid chromatography (HPLC) with gel permeation columns (G3000SW TSK GEL). The HPLC pattern monitored by A280 for a mixed solution of the HDL fraction (10 μl) and an eluent buffer (200 μl, 0.1M sodium phosphate buffer containing 0.1% SDS) incubated at 60°C for 5min showed two completely separated peaks which corresponded to the major components of human HDL, apolipoprotein A-I and A-II. Moreover, quantitation of apolipoprotein A-I by our method was found to correlate well with that by a single radial immunodiffusion (SRID) assay.View full abstractDownload PDF (222K) -
Tadashi YOSHIMOTO, Daisuke TSURU1983 Volume 94 Issue 2 Pages 619-622
Published: 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSCommercial preparations of aminopeptidases M split Gly-Pro-β-naphthylamide (Gly-Pro-2-NNap) into Gly-Pro and β-naphthylamine, and Ala-Pro into Ala and Pro. The activities on Gly-Pro-2-NNap and Ala-Pro were completely inhibited by diisopropyl phosphoro fluoridate (DFP) and p-chloromercuribenzoate (PCMB), respectively. When the substrate specificity was analyzed with tuftsin, Thr and Lys-Pro-Arg were released, and then Lys-Pro-Arg was split into Lys-Pro and Arg. Thereafter, slow liberation of Lys and Pro from Lys-Pro took place. The DFP-treated enzyme released only Thr from tuftsin and no hydrolysis of Lys-Pro-Arg was observed. With the enzyme treated with PCMB, tuftsin was converted into Thr and Lys-Pro-Arg, followed by the liberation of Arg, but no release of Lys and Pro was observed, contrary to the case of the untreated-enzyme. These results show that commercial aminopeptidase M contains dipeptidyl aminopeptidase IV and prolidase. Contamination by dipeptidyl aminopeptidase IV was confirmed by an immunological method.View full abstractDownload PDF (882K)
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