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Volume 80 (1976)
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- Issue 5 Pages 903-
- Issue 4 Pages 653-
- Issue 3 Pages 417-
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Volume 80, Issue 6
Displaying 1-35 of 35 articles from this issue
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Ikuo FUNAKOSHI, Ikuo YAMASHINA1976 Volume 80 Issue 6 Pages 1185-1193
Published: December 25, 1976
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSPlasma membranes were isolated from AH 66 cells, some of which had been labeled with [14C]glucosamine, by the following procedure: homogenization of cells which had been hardened by treatment with Zn ions, fractionation of the homogenate by sucrose density gradient centrifugation and purification of the membranes by partition in an aqueous twophase polymer system. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS) of the plasma membranes and subsequent staining of the gel for protein and carbohydrate, and determination of radioactivity on the gel eluates indicated the presence of at lease 10 bands of glycoprotein. The major band contained 27% of the total radioactivity incorporated into the plasma membranes and was most heavily stained with the periodate-Schiff reagent.
To isolate the major glycoprotein, the membranes were solubilized with 0.6 M lithium diiodosalicylate containing 0.5% Triton X-100, then the solution was treated with phenol. The major glycoprotein, obtained in the aqueous phase, was further purified mainly by repeated chromatographies on Sepharose 6B. The purified preparation was practically homogeneous on SDS-polyacrylamide gel electrophoresis, as judged by radioactivity determination and by carbohydrate staining, but contained small amounts of carbohydrate-free proteins. The major glycoprotein had an apparent molecular weight of 160, 000, as determined by SDS-polyacrylamide gel electrophoresis. The final preparation contained about 44%. carbohydrate on a weight basis, and the carbohydrate moiety was composed of glucosamine, galactosamine, galactose, mannose, fucose, and sialic acid. This composition indicates that the major glycoprotein contains both N- and O-glycosidically linked oligosaccharide moieties.View full abstractDownload PDF (1936K) -
Moriaki AKASAKI, Motoko SUZUKI, Ikuo FUNAKOSHI, Ikuo YAMASHINA1976 Volume 80 Issue 6 Pages 1195-1200
Published: December 25, 1976
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSβ-Galactosidase [EC 3.2.1.23] was isolated from a partially purified preparation obtained from cultured cells of a special strain of Aspergillus oryzae, RT 102 (FERM-P1680). The enzyme preparation gave a single protein band on polyacrylamide gel electrophoresis and was free from α-galactosidase, α- and β-mannosidase, α- and β-N-acetylhexosaminidase, and protease activities.
The β-galactosidase was capable of acting on aryl β-galactosides, lactose, and lactosides. It also hydrolyzed β-galactosyl linkages in urinary glycoasparagines and asialo α1-acid glyco-protein.
The enzyme was rather stable in aqueous solution, retaining full activity at 4° for at least several months. At pH 4.5, the optimum pH for the enzyme activity, and 37°, full activity was maintained for several days.View full abstractDownload PDF (1143K) -
I. The Mode of Action of Two Chondroitinase-AC Preparations of Different originKeiichiro HIYAMA, Shigetaka OKADA1976 Volume 80 Issue 6 Pages 1201-1207
Published: December 25, 1976
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe modes of action of two chondroitinases-AC [EC 4.2.2.5] from Arthrobacter aurescens and Flavobacterium heparinum were examined. By comparison of the increase of viscosity and by analyses of digests using paper chromatography and gel filtration, it was shown that the Arthrobacter enzyme (A-Chase) degrades the substrate by a stepwise attack, while the Flavobacterium enzyme (F-Chase) exhibits a more random attack, though the first attack of both enzymes is of endo-type.
Further study was carried out of the initial rate and final extent of the enzymic degradation of various mucopolysaccharides. The order of the initial rates at which the native mucopolysaccharides are degraded was similar for both enzymes. For A-Chase, the initial rate and final extent of degradation of modified chondroitin sulfate C or chondroitin methyl ester, however, were low compared with those of F-Chase. These results also suggested that A-Chase degrades the substrate by stepwise attack and F-Chase by random attack.View full abstractDownload PDF (904K) -
II. Numerical Calculation of the Degree of Muitiple AttackKeiichiro HIYAMA1976 Volume 80 Issue 6 Pages 1209-1214
Published: December 25, 1976
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSFurther investigation was carried out on the action patterns of two chondroitinase-AC [EC 4. 2. 2. 5.] preparations obtained from Arthrobacter aurescens and Flavobacterium heparinum. To infer the action patterns of the chondroitinases, we proposed a new method for the calcu-lation of the degree of multiple attack, based on the concept established by Robyt and French ((1967) Arch. Biochem. Biophys. 122, 8-16).
It was shown that the degree of multiple attack (DM) is represented by the ratio of the initial velocity of number-average degree of scission to that of viscosity-average degree of scission. By this method, DM for A-Chase was estimated to be 3.03 and for F-chase, 1.31.View full abstractDownload PDF (382K) -
Hiroshi HATANAKA, Fujio EGAMI1976 Volume 80 Issue 6 Pages 1215-1221
Published: December 25, 1976
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSRadioactivity was significantly incorporated from ascorbate 2-[35S] sulfate into chondroitin sulfate by embryonic chick cartilage epiphyses. The extent of incorporation was comparable with that from inorganic [35S]sulfate. The radioactive chondroitin sulfate formed from ascor-bate 2-[35S]sulfate gave two radioactive disaccharides on chondroitinase-ABC [EC 4.2.2.4] digestion.
The incorporation was markedly decreased by inorganic sulfate. The time course of incorporation from ascorbate 2-[35S]sulfate and inorganic [35S]sulfate into chondroitin sulfate and the constituent disaccharides suggest that the incorporation rates from the two radioactive substances are different.View full abstractDownload PDF (1052K) -
Core Structures of Oligosaccharides Obtained from Porcine Thyroglobulin and Pineapple Stem BromelainMinoru FUKUDA, Toshiaki KONDO, Toshiaki OSAWA1976 Volume 80 Issue 6 Pages 1223-1232
Published: December 25, 1976
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSHydrazinolysis of porcine thyroglobulin glycopeptides and of pineapple stem bromelain [EC 3.4.22.4] permitted the isolation of almost intact carbohydrate chains of these glycoproteins. On the basis of permethylation analyses of the released oligosaccharides after reduction with NaBH4, the core structures of Unit A-type and Unit B-type carbohydrate chains of porcine thyroglobulin were deduced to be Manαl→6 [Manαl→3] Manβl→4G1cNAcβ1→4 [Rαl→6] GlcNAc→Asn (Unit A-type, R=H; Unit B-type, R=Fuc), and that of bromelain was found to be Manαl→6 [R'1→2] Manβ1→4G1cNAcβ1→4 [R1→3] G1cNAc→Asn (R'=Xy1β and R=Fucα, or R'=Fucα and R=Xylβ). From these results, it appears that the hydrazinolysis method is applicable to a wide variety of glycoproteins which have an N-glycosylamine linkage between the carbohydrate and peptide moieties, regardless of the type of linkage to the most proximal N-acetylglucosamine residue which is bound to asparagine.View full abstractDownload PDF (721K) -
Shigenori SONOKI, Hiroh IKEZAWA1976 Volume 80 Issue 6 Pages 1233-1239
Published: December 25, 1976
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThree kinds of phospholipase C [EC 3.1.4.3] were used to selectively hydrolyze phospho-lipids in rat liver microsomes, and their effects on the acyl-CoA: glycerophosphate and acyl-CoA: lysophospholipids acyltransferase systems were examined.
The glycerophosphate acyltransferase [EC 2.3.1.15] system was inactivated rapidly by treatment with phospholipase C of Ps. aureofaciens or B. cereus and the loss of activity paralleled the degradation of phosphatidylcholine and phosphatidylethanolamine. The 1-acylglycerylphosphorylcholine acyltransferase [EC 2.3.1.23] system was only partially inactivated under the same conditions, whereas the 1-acylglycerophosphate acyltransferase [EC 2.3.1.51] system retained most of its activity even when more than 95% of phosphatidylcholine and phosphatidylethanolamine had been hydrolyzed. The results demonstrate the heterogeneity of acyltransferase systems with respect to their dependence on the intact membrane phos-pholipids. Hydrolysis of more than 80% of phosphatidylinositol by phosphoinositidase of B. cereus did not significantly affect these acyltransferase systems. The specificity for various acyl-CoA's of I-acylglycerophosphate acyltransferase in microsomes treated with phospholipase C of Ps. aureofaciens was apparently different from that in untreated microsomes, while the specificity of 1-acylglycerylphosphorylcholine acyltransferase was unchanged. Saturation profiles of the acceptors were significantly different between the acyltransferase systems in phospholipase C-treated and untreated microsomes.
These results suggest that 1-acylglycerophosphate and 1-acylglycerylphosphorylcholine acyltransferase systems do not require specific phospholipids such as phosphatidylcholine, phosphatidylethanolamine, and phosphatidylinositol for their catalytic activities, but the integrity of these phospholipids is necessary for the proper functioning and stability of the enzymes.View full abstractDownload PDF (548K) -
Norio SUZUKI, Kazuo DEGUCHI, Nobuo UETA, Hiroshi NAGANO, Ryoiti SHUKUY ...1976 Volume 80 Issue 6 Pages 1241-1246
Published: December 25, 1976
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe chemical properties of very low density and high density lipoproteins of adult bullfrog serum were determined. This serum contained extremely low levels of both very low density lipoprotein (10-30mg/100ml) and high density lipoprotein (5-10mg/100ml).
The constituents of very low density lipoprotein, on a weight percentage basis, were found to be 48.1% triglyceride, 17.3% cholesterol ester, 8.8%cholesterol, 11.6% phospholipid, and 12% protein. These constituents were also present in high density lipoprotein with weight percentage values of 3.7%, 19.3%, 11.9%, 25.2%, and 36.8%, respectively.
The fatty acid compositions of the triglycerides, cholesterol esters, and phosphatidyl-choline were quite similar in the very low density lipoprotein and high density lipoprotein. However, shingomyelin fatty acid composition was appreciably different in the two lipopro-teins.
Disc gel electrophoresis in sodium dodecyl sulfate-polyacrylamide gels produced pat-terns with one major (approximate molecular weight, 7, 000) and several minor bands for the apoprotein of very low density lipoprotein and one major (approximate molecular weight, 28, 000) and several minor bands for that of high density lipoprotein.View full abstractDownload PDF (1059K) -
Osamu DOI, Shoshichi NOJIMA1976 Volume 80 Issue 6 Pages 1247-1258
Published: December 25, 1976
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESS1. Escherichia coli K-12 mutants deficient in detergent-resistant (DR) and detergent-sensi-tive (DS) phospholipases A and deficient in DS phospholipase A were isolated.
2. The growth, compositions of phospholipids and fatty acids and turnover of phospholipids of three mutants (DR-, DS-, DR-DS-) were compared with those of their parent (DR- was isolated in a previous study).
3. Autodegradations of membrane phospholipids of 18, 000×g supernatants and precipitates of the homogenates of these three mutants were also compared with those of the parent, and the effects of various detergents and organic solvents on these activities were examined.
4. We could not identify any significant physiological role for DR or DS phospholipase A.View full abstractDownload PDF (865K) -
XX. Specific Inactivation of Ribonuclease T1 by Reaction with TosylglycolateHaruyuki OSHIMA, Kenji TAKAHASHI1976 Volume 80 Issue 6 Pages 1259-1265
Published: December 25, 1976
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESS1. Ribonuclease T1 [EC 3.1.4.8] was inactivated by reaction with tosylglycolate (carboxy-methyl p-toluenesulfonate). At pH 5.5 and 8.0, alkylation of the γ-carboxyl group of glutamic acid-58 appeared to be the predominant reaction and the major cause of inactivation by tosylglycolate, as in the case of the iodoacetate reaction, although the rate of inactivation was slower than that by iodoacetate. At pH 8.0, histidine residues were also alkylated to some extent.
2. The maximal rate of inactivation was observed at around pH 5.5 and the pH dependence of the rate of inactivation suggested the implication of two groups in the reaction, with apparent pKa values of about 3-4 (possibly glutamic acid-58) and about 7-8 (possibly histidine residue (s)).
3. In the presence of substrate analogs, ribonuclease T1 was markedly protected from inactivation by tosylglycolate at pH 5.5. The extent of protection corresponded to the binding strength of the substrate analog, except for guanosine. Ribonuclease T1 was much less protected from inactivation by guanosine than by 3'-AMP or 3'-CMP, which has a lower binding strength toward ribonuclease T1. This may indicate that glutamic acid-58 is situated in the catalytic site, at which the phosphate moiety of these nucleotides directly interacts.
4. Enzyme which had been extensively inactivated with tosylglycolate at pH 5.5 scarcely reacted with iodoacetate at pH 5.5, suggesting that these reagents react at the same site, i.e. glutamic acid-58. On the other hand, enzyme which had been inactivated almost completely with tosylglycolate at pH 8.0 still reacted with iodoacetate to some extent at pH 8.0, and the modes of reaction of tosylglycolate and iodoacetate toward ribonuclease T1 appeared to be somewhat different.View full abstractDownload PDF (492K) -
XXI. Modification of Histidine Residues in Ribonuclease T1 with IodoacetamideKenji TAKAHASHI1976 Volume 80 Issue 6 Pages 1267-1275
Published: December 25, 1976
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESS1. When ribonuclease T, [EC 3.1.4.8] (0.125% solution) was treated with a 760-fold molar excess of iodoacetamide at pH 8.0 and 37°, about 90% of the original activity was lost in 24hr. The half-life of the activity was about 8hr. The binding ability for 3'-GMP was lost simultaneously. Changes were detected only in histidine and the amino-terminal alanine residues upon amino acid analyses of the inactivated protein and its chymotryptic peptides. The inactivation occurred almost in parallel with the loss of two histidine residues in the enzyme.
The pH dependences of the rate of inactivation and that of loss of histidine residues were similar and indicated the implication of a histidine residue or residues with pKa 7.5 to 8 in this reaction. 3'-GMP and guanosine showed some protective effect against loss of activity and of histidine residues. The reactivity of histidine residues was also reduced by prior modifica-tion of glutamic acid-58 with iodoacetate, of lysine-41 with maleic or cis-aconitic anhydride or 2, 4, 6-trinitrobenzenesulfonate or of arginine-77 with ninhydrin.
2. Analyses of the chymotryptic peptides from oxidized samples of the iodoacetamide-inactivated enzyme showed that histidine-92 and histidine-40 reacted with iodoacetamide most rapidly and at similar rates, whereas histidine-27 was least reactive. Alkylation of histidine-92 was markedly slowed down when the GluSe-carboxymethylated enzyme was treated with iodoacetamide. On the other hand, alkylation of histidine-40 was slowed down most in the presence of 3'-GMP. These results suggest that histidine-92 and histidine-40 are involved in the catalytic action, probably forming part of the catalytic site and part of the binding site, respectively, and that histidine-27 is partially buried in the enzyme molecule or interacts strongly with some other residue, thus becoming relatively unreactive.View full abstractDownload PDF (759K) -
Chiaki MORIWAKI, Kyosuke MIYAZAKI, Yoshifumi MATSUDA, Hiroshi MORIYA, ...1976 Volume 80 Issue 6 Pages 1277-1285
Published: December 25, 1976
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe contents of kallikrein [EC 3.4.21.8] in the kidneys of various animals were estimated and the activity was found to be most potent in dogs. The dog renal kallikrein (DRK) was located mainly in the kidney cortex. Following the activation of a dog kidney cortex homogenate with acetone, kallikrein was purified about 2, 000-fold with an overall yield of 18% by diethylaminoethyl (DEAE)-cellulose adsorption, acetone fractionation, and chromatography on Sephadex G-75 and DEAE-Sephadex A-50.
The final purified preparation of dog renal kallikrein had a vasodilator activity of 65.5 KU per A280, and appeared to be homogeneous both in disc electrophoresis and ultracentrifugal analysis. Its molecular weight was estimated to be approximately 3.8×104 from the sedimentation coefficient obtained by ultracentrifugation, and by Sephadex gel filtration. However, isoelectric fractionation of the purified DRK preparation gave three isoelectric point, 3.9, 4.1, and 4.3.
The DRK had an optimum pH of about 8.6 and was stable at pH 8. This enzyme was hardly inhibited by Trasylol, soybean trypsin inhibitor, ovomucoid trypsin inhibitor or potato kallikrein inhibitors. These properties were compared with those of kallikrein from other sources; DRK appeared to be similar to urinary kallikrein.View full abstractDownload PDF (624K) -
Nobuyuki YAMASAKI, Takahiro TSUJITA, Fumio SAKIYAMA, Nobuhito MASUDA1976 Volume 80 Issue 6 Pages 1287-1292
Published: December 25, 1976
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSAcid-catalyzed hydrolysis of the formamide linkage of N-acetyl-N'-formyl-L-kynurenineamide in frozen dilute hydrochloric acid solution followed first-order kinetics, yielding N-acetyl-L-kynurenineamide as the sole reaction product. The maximum rate of reaction in the frozen solution was found at around -7.5° and approximated that of the reaction in liquid solution at 40°. By freezing the dilute acid solution at -8° the reaction was accelerated by 60 times compared with that in super-cooled liquid solution at the same temperature.View full abstractDownload PDF (441K) -
Masaaki YAMADA, Misao TASHIRO, Hiroko YAMAGUCHI, Hisao YAMADA, Fumio I ...1976 Volume 80 Issue 6 Pages 1293-1297
Published: December 25, 1976
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe reactive site peptide bend of the eggpla.zt inhibitor against trypsin [EC 3. 4. 21. 4] was identified by chemical modifications with 1, 2-cyclohexanedicne, 2, 4, 6-trinitrobenzencsulfcnic acid., acetic anhydride and glyoxal, and by sequential treatments with trypsin and carboxy-peptidase B [EC 3. 4. 12. 3]. The inhibitor was significantly inactivated by chemical modifica-tions of arginine residues, but was riot affected by lysine modifications. Free arginine was released from the trypsin-modified inhibitor by carboxypeptidase B digestion, accompanied by a marked less of inhibitory activity. A serine residue was newly exposed as the N-terminal amino acid of the inhibitor after modification with trypsin. The reactive site of the inhibitor against trypsin was concluded to be an arginylseryl bond. The inhibitor was completely inactivated by full rcducticn of its disulfide bonds.View full abstractDownload PDF (994K) -
Structural Differences between Heavy and Light Chains Derived from the Kinin-free ProteinsHisao KATO, Yong Nam HAN, Sadaaki IWANAGA, Tomoji SUZUKI, Masanobu KOM ...1976 Volume 80 Issue 6 Pages 1299-1311
Published: December 25, 1976
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSKinin-free proteins which were produced by the incubation of high-molecular-weight (HMW) kininogen with plasma kallikrein [EC 3.4.21.8] and of low-molecular-weight (LMW) kinino-gen with snake venom kininogenase were prepared. Each of the isolated proteins yielded two polypeptide chains, named heavy and light chains, after cleavage of their disulfide bridges. The heavy chains derived from the two kinin-free proteins were found to have almost the same molecular weight of 48, 000. However, the molecular weights of their light chains differed markedly; that of the HMW-light chain was 16, 000 and that of the LMW-light chain was 4, 800. These heavy and light chains were located in the amino and carboxyl terminal portions of the parent molecules and were held together by a single disulfide bridge, as judged from their terminal residues and amino acid compositions.
As regards the overall amino acid compositions and terminal residues, the heavy chains from the two kinin-free proteins were very similar, and they cross-reacted immunologically with an antibody against the heavy chain isolated from kinin-free HMW protein. Further, tryptic peptide mappings of the two heavy chains supported their structural similarity. How-ever, there was a slight difference in carbohydrate content; HMW-heavy chain contained 12.6%, whereas LMW-heavy chain contained 19.3%. On the other hand, the light chains derived from the two kinin-free proteins showed clear differences not only in molecular weight but also in their chemical properties, including amino acid and carbohydrate compositions, terminal residues and N-terminal sequences.
These results indicate that the major structural difference between HMW and LMW kininogens exists in the large C-terminal portions following the bradykinin moieties along the polypeptide chains. Based on the gross molecular structures of HMW and LMW kininogens, their possible functional roles are discussed.View full abstractDownload PDF (3133K) -
II. Kinetic Analysis of Protease DigestionTaiji IMOTO, Ken-ichi FUKUDA, Kazuyoshi YAGISHITA1976 Volume 80 Issue 6 Pages 1313-1318
Published: December 25, 1976
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSKinetic analyses of the protease digestion of several chemical derivatives of lysozyme [EC 3.2.1.17] showed that only the D (denatured) state of the protein is digested and that the reaction velocity is proportional to the equilibrium constant (K0) of the N_??_D transition of the protein. Alteration of the net charge of lysozyme by acetylation caused a shift of the N=D transition to the right (ten-fold increase in KD compared to that of native enzyme). Both the formation of a lysozyme-inhibitor complex and the introduction of a covalent bond in the lysozyme molecule restricted the transition.
The magnitude of the N_??_D transition is related to the susceptibility of lysozyme to protease digestion and it is estimated that the N_??_D transition in proteins is generally im-portant in the intracellular catabolism of proteins.View full abstractDownload PDF (417K) -
III. Effect of Alteration of Net Charge by AcetylationTaiji IMOTO, Sigeru MORIYAMA, Kazuyoshi YAGISHITA1976 Volume 80 Issue 6 Pages 1319-1325
Published: December 25, 1976
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSMeasurement of the enzymic activity and fluorescence properties showed that the gross con-formation of acetylated lysozyme [EC 3.2.1.17] is very similar to that of the native enzyme.
On the other hand, protease digestion, t-butyl hypochloride modification and thermal denaturation experiments performed on native, acetylated, and guanidinated lysozymes showed that acetylation caused a small but significant shift of the N_??_D transition to the right. Thus it can be concluded that charge balance in a protein plays an important role in maintaining its conformation.
The difference between equilibrium and kinetic methods of monitoring protein denatura-tion was also clarified.View full abstractDownload PDF (456K) -
Isolation and Characterization of the Exchange ProductsSusumu ITO, Taro NAKAMURA, Yoshitomo EGUCHI1976 Volume 80 Issue 6 Pages 1327-1334
Published: December 25, 1976
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSGel-electrophoretically homogeneous methioninase [L-methionine methanethiol-lyase (deami-nating), EC 4.4.1.11] of Pseudomonas putida, which catalyzes α, β- and α, γ-eliminations from S-substituted amino acids, could also catalyze a variety of β- and γ-exchange reactions, according to the following equations:
RSCH2CH(NH2)000H+R'SH_??_R'SCH2CH(NH2)COOH+RSH (β-exchange) and RSCH2CH2CH(NH2)COOH+R'SH_??_R'SCH2CH2CH(NH2)COOH+RSH (γ-exchange),
where R'SH represents an exogeneously added alkanethiol or a substituted thiol. Related amino acids not available for elimination reactions appeared to be inert as substrates for exchange.
The maximum activity for the exchange reactions was observed at pH 8.5 in potassium pyrophosphate buffer. The activity increased linearly with the increase in protein concentration from zero to 3.0 μg per ml, and with incubation time up to at least 15min at 30°.
Some of the exchange reaction products were purified by a combination of paper and ion exchange chromatographies, and charcoal treatment: their structures were confirmed by physicochemical methods including elemental analysis and proton magnetic resonance, in-frared, and mass spectrometries.View full abstractDownload PDF (611K) -
Mitsuaki SAKODA, Ketaro HIROMI1976 Volume 80 Issue 6 Pages 1335-1341
Published: December 25, 1976
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA novel method is proposed to determine deductively and uniquely the values of three param-eters, a, b, and c in a fractional function of the form,
y=a+bx/(c+x)
where x and y are experimentally obtainable variables. This type of equation is frequently encountered in chemistry and biochemistry involving relaxation kinetics.
The method of least squares with the Taylor expansion is employed for direct curve fitting of observed data to the fractional function. Approximate values of the parameters, which are always necessary prior to commencing the above procedure, can be obtained by the method of rearrangement after canceling the denominator of fractional functions. This procedure is very simple, but very effective for estimating provisional values of the parameters. Deductive and unique determination of the parameters involved in the fractional function shown above can be accomplished for the first time by the combination of these two procedures.
This method is extended to include the analysis of relaxation kinetic data such as those of temperature-jump method where the determination of equilibrium concentrations of react-ants in addition to the three parameters is also necessary.View full abstractDownload PDF (460K) -
Emi KUSUNOSE, Kosuke ICHIHARA, Yojiro NODA, Masamichi KUSUNOSE1976 Volume 80 Issue 6 Pages 1343-1352
Published: December 25, 1976
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESS1. A superoxide dismutase [EC 1. 15. 1. 1] was purified about 275-fold with a yield of 34% from Mycohacteritun tuberculosis, strain H37Ra (attenuated strain), grown on a Sauton medium for two months. The purified enzyme was homogeneous as judged by polyacrylamide gel electrophcresis, and by analytical ultracentrifugation and sedimentation equilibrium studies.
2. The molecular weight of the enzyme was estimated to be approximately 88, 000 by sedimentation equilibrium analysis. Since the molecular weight of the subunit was 21, 000 as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis, the enzyme appears to be composed of four subunits of equal size.
3. Electron spin resonance (ESR) spectra showed that the enzyme contained ferric iron, and metal analysis showed that approximately 3.7 atoms of iron were present per mole of the enzyme, indicating the occurrence of I atom of iron per subunit.
4. The amino acid composition was apparently similar to those of the iron-containing superoxide dismutases from Escherichia coli, luminous bacteria, Pseudornonas ovalis, and bluegreen alga.
5. Antibodies against the enzyme were raised in rabbits and immunological studies were performed. The enzyme from M. tuberculosis, strain H37Rv (virulent strain), was found to have antigenic structures identical with those of the H37Ra enzyme. On the other hand, the manganese-containing superoxide dismutases from other species of mycobacteria, i.e., Mycobacteriurn species, strain Takeo, M. phlei and M. lepraemurium, showed only partial immunological identity with the H37Ra enzyme.
6. During the growth of M. tuberculosis, strain H37Ra, the enzyme was found to be secreted into the culture medium.View full abstractDownload PDF (2765K) -
I. Binding of Adenosine Diphosphate to Myofibrils during the Adenosinetriphosphatase ReactionToshiaki ARATA, Yuji TONOMURA1976 Volume 80 Issue 6 Pages 1353-1358
Published: December 25, 1976
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESS1. The myosin content of myofibrils was found to be 51% by SDS-gel electrophoresis.
2. The initial burst of Pi liberation of the ATPase [EC 3.6.1.3] of a solution of myofibrils in 1M KCl was measured in 0.5M KCl, and found to be 0.93 mole/mole of myosin.
3. The amount of ADP bound to myofibrils during the ATPase reaction and the ATPase activity were measured by coupling the myofibrillar ATPase reaction with sufficient amounts of pyruvate kinase [EC 2.7.1.40] and PEP to regenerate ATP. The maximum amount of ADP bound to myofibrils in 0.05M KCl and in the relaxed state was about 1.5 mole/mole of myosin. On the other hand, the ATPase activity exhibited substrate inhibition, and the amount of ATP required for a constant level of ATPase activity was smaller than that required for the maximum binding of ADP to myofibrils.
4. The maximum amount of ADP bound to myofibrils in 0.5M KCl was about 1.9 mole/ mole of myosin. When about one mole of ADP was bound to 1 mole of myosin in myofibrils, the myofibrillar ATPase activity reached the saturated level, and with further increase in the concentration of ATP one more mole of ADP was bound per mole of myosin.View full abstractDownload PDF (400K) -
II. Separation of the Two Fractions of Subfragment-1 of Myosin by Affinity Column Chromatography on Immobilized F-Actin: Direct Evidence for Acceleration by F-Actin of the DecompositionAkio INOUE, Yuji TONOMURA1976 Volume 80 Issue 6 Pages 1359-1369
Published: December 25, 1976
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSF-Actin (FA) and pyruvate kinase (PK) [EC 2.7.1.40] were immobilized on PAB-cellulose. HMM-Subfragment-1 (S-1) was applied to a column of immobilized FA and PK, and eluted with 1-1.5 μM ATP and 1 mat PEP in 50mM KCl, 2mM MgCl2, and 10mM Tris-HCl at pH 7.8 and 4°. The size of the initial burst of Pi liberation of S-1 applied to the column was 0.5 mole/mole. The size of the initial burst of S-1 in early fractions from the column was 0.8-0.9 molelmole S-1. The burst size of S-1 decreased with increase in the fraction number, and S-1 in later fractions showed a burst size of 0.1-0.3 mole/mole. On the other hand, the rate of the ATPase [EC 3.6.1.3] reaction in the steady state was almost independent of the burst size, and increased slightly with increase in the fraction number.
The ATPase activity of S-1 with a burst size of less than 0.2 mole/mole was scarcely activated by FA. Usually, the dependence on the burst size of S-1 of its ATPase activity in the presence of FA was sigmoidal, and marked activation by FA was observed when the burst size was larger than 0.3-0.4 mole/mole. Similar results were obtained with S-1 fractions separated by the ultracentrifugation method described in our previous paper ((1976) J. Biochem. 79, 419-434).View full abstractDownload PDF (771K) -
III. Cooperativity of the Two Heads of the Myosin Molecule, Shown by the Effect of Modification of Head A with p-Chloromercuribenzoate on the Interaction of Head B with F-ActinKazuko SHIBATA-SEKIYA, Yuji TONOMURA1976 Volume 80 Issue 6 Pages 1371-1380
Published: December 25, 1976
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSSubfragment-1 of HMM was prepared by tryptic [EC 3.4.21.4] digestion of HMM, which had been modified with 1 mole of CMB per mole of HMM at a specific SH group, SHr. S-1(T) obtained from CMB-HMM retained almost all the CMB, and the amount of bound CMB was about 0.8-0.9 mole per 2 moles of S-1(T). S-2 of CMB-HMM contained no bound CMB.
The ATPase [EC 3.6.1.3] activity of HMM increased gradually with increase in the concentration of FA, and the acto-HMM ATPase was inhibited by excess substrate or removal of Ca2+ ions in the presence of RP. The ATPase activity of CMB-HMM increased to a maximum level on adding a small amount of FA, and the acto-CMB-HMM ATPase showed neither substrate inhibition nor Ca2+ sensitivity in the presence of RP. On the other hand, the dependence on the concentration of FA of the ATPase activity of acto-S-1(T) was unaffected by modification of S-1 with CMB. The Ca2+ sensitivity of the ATPase activity of acto-S-1(T) in the presence of RP was also unaffected by the modification.
Acto-S-1(T) dissociated almost completely, while acto-CMB-S-1(T) was only 50% dissociated on adding ATP. More than 80%. of the bound CMB was contained in S-1(T) undissociated from FA. Furthermore, superprecipitation of actomyosin induced by ATP was completely inhibited by adding about 2 moles of CMB-S-1(T) per mole of actin monomer. On the other hand, about 90% of the burst size of P_??_ liberation was retained in S-1(T) dis-sociated from FA.
It was concluded that the two heads of the myosin molecule are different: one shows the initial burst of P_??_ liberation, and does not contain the SHr group which binds CMB (head B), and the other does not show the initial burst and contains the SHr group (head A). It was also concluded that modification of head A of HMM or myosin with CMB increases its binding strength to FA, and consequently the substrate inhibition and Call sensitivity of acto-HMM or actomyosin ATPase at head B are lost on modification of head A with CMB.
CMB-S-I (CT) was prepared by chymotryptic [EC 3.4.21.1] digestion of CMB-myosin, and separated into two fractions by ultracentrifugation of acto-CMB-S-1(CT) in the presence of ATP. Three components of CMB-S-1(CT) with molecular weights of 9, 2.4, and 1.2×104 were separated by SDS-polyacrylamide gel electrophoresis. The ratios of the peak areas of the three components in electrophoretograms were the same in CMB-S-1(CT) and in the two fractions (1:0.18:0.09), indicating that heads A and B have the same subunit structure.View full abstractDownload PDF (768K) -
IV. Physiological Functions of Various Reaction Intermediates in Myosin Adenosinetriphosphatase, Studied by the Interaction between Actomyosin and 8-Bromoadenosine TriphosphatelHitoshi TAKENAKA, Morio IKEHARA, Yuji TONOMURA1976 Volume 80 Issue 6 Pages 1381-1392
Published: December 25, 1976
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe kinetic properties of the hydrolyses of 8-Br ATP and 8-SCH3 ATP by myosin [EC 3.6.1.3] and actomyosin were compared with those of ATP, and the following results were obtained.
The Ca-NTPase activities of myosin using these two ATP analogs as substrates were smaller than that of ATPase, and the NTPase activities toward these analogs were strongly suppressed by EDTA. The Mg-NTPase activities toward these analogs were higher in a medium of high ionic strength than in a medium of low ionic strength, in contrast to the activity of Mg-ATPase. These analogs did not produce any initial burst of P_??_ liberation, activation of myosin NTPase by F-actin, or superprecipitation of actomyosin.
The interactions between 8-Br ATP and HMM, acto-HMM, actomyosin, and myofibrils were studied in detail in the presence of Mg2+ in medium of low ionic strength. The Michaelis constant, Km, and the maximum rate, Vm, of 8-Br ATPase of HMM were 27 μM and 21 min-1, respectively. The fluorescence change of HMM induced by 8-Br ATP also followed the Michaelis-Menten equation, and the Michaelis constant, Kf1, was as low as 4 μM. Acto-HMM and acto-S-1 were fully dissociated by the addition of 8-Br ATP. The relation between the extent of dissociation of acto-HMM and the concentration of 8-Br ATP followed the Michaelis-Menten equation, and the apparent dissociation constant, Kd, was 22 μm. This Kd value is almost equal to the Km, value of 8-Br ATPase of HMM described above. Myofibrillar contraction was not supported by 8-Br ATP.
It was concluded that in the myosin NTPase reaction with 8-Br ATP as a substrate, M2NTP but not MNDPP is formed in route (1), while MNTP is formed in route (2). It was also concluded that the key intermediate for the actomyosin NTPase reaction is MNDPP, and that dissociation of acto-HMM is induced by the formation of M2NTP and MNTP in routes (1) and (2), respectively.View full abstractDownload PDF (878K) -
Keizo NOMURA, Yoshiaki NAKAMARU1976 Volume 80 Issue 6 Pages 1393-1399
Published: December 25, 1976
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe intravesicular pH (pHi) of fragmented sarcoplasmic reticulum (SR) of the skeletal muscle was determined from the distribution of 5, 5-dimethyl-2, 4-oxazolidinedione (DMO), a weak organic acid, between the intra- and extravesicular spaces. The pHi's thus obtained were found to be slightly lower (0.02-0.17 pH unit) than the pH's of the external medium (pHe, ) at pH 6.5-8.5 in the presence of 105 mM KCl and 40 mM Tris-maleate buffer. The higher the pHe, the greater the pH gradient. When pHe was changed, pHi attained equilibrium within about 20 min, the time necessary for the separation of the SR by centrifugation. When 0.25 M sucrose and 5 mM Tris-maleate buffer were used instead of 105 mM KCl and 40 mM buffer, the pH gradient increased to 0.56.
It was also demonstrated by direct measurements of pHe, with a glass-electrode pH meter that K+ ions added to the external medium exchanged the intravesicular H+ ions.
From these results it appears that the pH gradient across the SR membrane was at the Donnan equilibrium. In this state, the Donnan potentials corresponding to pH gradients of 0.17 and 0.56 were -9.3 and -30.6 mV, respectively.View full abstractDownload PDF (517K) -
Yoshinori HASEGAWA, Hisami YAMADA, Shoji MIZUSHIMA1976 Volume 80 Issue 6 Pages 1401-1409
Published: December 25, 1976
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe outer membrane proteins O-8 and O-9 were specifically bound to the peptidoglycan sacculus in sodium dodecyl sulfate (SDS) solution. Other cellular proteins failed to interact with the peptidoglycan sacculus under the same conditions. When the outer membrane was preheated in SDS solution, the binding did not take place. Optimum binding was observed at pH 8 in the presence of 5 mm Mg2+. A high concentration of sodium chloride strongly inhibited the binding. The effects of these factors on the bindings of O-8 and O-9 were very similar. The binding of O-8 and O-9 required neither the bound nor the free form of Braun's lipoprotein, nor was the binding of either protein necessary for the binding of the other.
Proteins O-8 and O-9 were also found in the peptidoglycan sacculus when it was prepared from cells in SDS solution at 60°. A dilution experiment showed that the complex was not an artifact. The mode of interaction between these proteins and peptidoglycan in the preparation was similar to that in the reassembled O-8•O-9-peptidoglycan complex, as judged from the sensitivity to sodium chloride and temperature.
The physiological importance of the complex is discussed in relation to the assembly of the outer membrane on the cell surface.View full abstractDownload PDF (697K) -
Kenzo NAKAMURA, Shoji MIZUSHIMA1976 Volume 80 Issue 6 Pages 1411-1422
Published: December 25, 1976
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSMajor outer membrane proteins of Escherichia coli K-12 with apparent molecular weights ranging from 30, 000 to 40, 000 were resolved into four distinct bands by electrophoresis on an improved urea-sodium dodecyl sulfate (SDS)-polyacrylamide gel containing a high concentration of N, N'-methylenebisacrylamide. The electrophoretic mobilities of three of these proteins, O-8, O-9, and O-10, changed when they were heated in SDS solution.
Proteins O-8, O-9, and O-10 were purified to near homogeneity without heating in SDS solution. Electrophoretic profiles of the purified proteins changed depending on the solubilization temperature in SDS solution.
Infrared and CD spectra of these proteins revealed that they were extremely rich in β-structured polypeptide, which is stable in SDS solution at room temperature. On the other hand, CD spectra typical of α-helix structure were obtained when the proteins were heated in SDS solution, indicating that a gross conformational change occurred in the protein molecules upon heating in SDS solution. The conformational change was confirmed by the abnormal profiles of Ferguson plots in gel electrophoretic analysis.
It was concluded that conformational changes in the protein molecules are responsible for the heat modifiability of these proteins in SDS gel electrophoresis.View full abstractDownload PDF (2611K) -
Emiko ISHIZAKI, Yuko YOSHIOKA, Yoki MORI, Jiro KOYAMA1976 Volume 80 Issue 6 Pages 1423-1427
Published: December 25, 1976
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSTwo forms of activated Cls, a subcomponent of the first component of complement, were present in preparations of C_??_specifically purified from rabbit serum by affinity chromatography on IgG-Sepharose 6B and were separated by DEAE-cellulose chromatography in the presence of EDTA. These two activated Cls, designated Cl_??_(I) and Cl_??_(II), were indistinguishable with regard to hemolytic activity as well as Cl_??_esterase activity, though they had different molecular weights. Cl_??_(I) had a molecular weight of 106, 000, consisting of H and L chains connected by disulfide bonds; the molecular weights of the chains were 70, 000 and 36, 000, respectively. On the other hand, Cl_??_(II), with a molecular weight of 72, 000, consisted of two chains each with a molecular weight of about 37, 000, which were also connected by disulfide bonds. These results suggest that, in the case of rabbit Cls, the primary product of activation with Cl_??_, Cl_??_(I), may be susceptible to further cleavage of its H chain without any loss of Cl_??_activity, resulting in the formation of Cl_??_(II), though the active principle responsible for this conversion remains to be elucidated.View full abstractDownload PDF (1124K) -
Yoshimi OHNO, Ikuya YANO, Masamiki MASUI1976 Volume 80 Issue 6 Pages 1429-1433
Published: December 25, 1976
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA glucosyl group from uridine diphosphate [U-14C]glucose is incorporated into a phosphoglycolipid, probably a glucosylphosphatidylglycerol, by a disrupted membrane enzyme prepa-ration from a gram-negative, moderately halophilic bacterium, Pseudomonas halosaccharolytica ATCC 29423. The conversion of [14C]phosphatidylglycerol into phosphoglycolipid by the particulate preparation was also enhanced in the presence of non-labelled UDP-glucose. A chemical degradation study of labelled phosphoglycolipid showed the bulk of the radioactivity from UDP [U-14C]glucose to be associated with the glucose moiety, which also appeared to be attached to the hydroxyl group of a second glycerol.View full abstractDownload PDF (1182K) -
Toru NAKANO, Masamichi TSUBOI1976 Volume 80 Issue 6 Pages 1435-1438
Published: December 25, 1976
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWhen nuclei isolated from rat liver in a low salt buffer were washed with 0.1 M NaCl solution, the supernatant showed a deoxyribonuclease (DNase) activity. The activity required Mg2+ and in addition spermine or spermidine, and its optimal pH was 7.2-7.4. The activity was higher on denatured (single stranded) DNA than on double-helical DNA. With both sub-strates the activity was highest at a polyamine concentration at which the DNA-polyamine complex began to precipitate. No Mg2++Ca2+ dependent DNase activity was detected in the preparation.View full abstractDownload PDF (228K) -
Kazuko NISHIKURA, Yoshiki SUGITA1976 Volume 80 Issue 6 Pages 1439-1441
Published: December 25, 1976
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe spectral changes of nitrosyl hemoglobin on addition of inositol hexaphosphate were stud-ied in hybrid-heme hemoglobins. The results showed that the decrease in absorption in the Soret region was mainly due to a spectral change in α chains, and that the tension on heme in the quaternary T structure was much stronger in α than in β chains.View full abstractDownload PDF (244K) -
Kazuhiro SOGAWA, Kenji TAKAHASHI1976 Volume 80 Issue 6 Pages 1443-1446
Published: December 25, 1976
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA novel neutral protease(s), which is presumably membrane-bound, was found in monkey liver using heat-denatured casein as a substrate and was separated from other major catheptic proteases by successive procedures of gel filtration on Ultrogel AcA 22, solubilization by deoxycholate and gel filtration on Sepharose 6B. The enzyme(s) showed maximal activity at pH 8.0, and was strongly inhibited by DFP and PMSF. Many other reagents tested, includ-ing TPCK, TLCK, pCMB, iodoacetic acid, and EDTA, were without marked effect on the activity. Activation of the enzyme(s) by NaCI was not observed.View full abstractDownload PDF (308K) -
Yukihiro OZAKI, Teizo KITAGAWA, Yoshimasa KYOGOKU, Hideo SHIMADA, Tets ...1976 Volume 80 Issue 6 Pages 1447-1451
Published: December 25, 1976
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSResonance Raman spectra of cytochrome P-450cam (P-450cam) and its enzymatically inactive form (P-420) in various oxidation and spin states were measured for the first time. The Raman spectrum of reduced P-450cam was unusual in the sense that the “oxidation-state marker” appeared at an unexpectedly lower frequency (1346 cm-1) in comparison with those of other reduced hemoproteins (_??_1355-1365 cm-1), whereas that of oxidized P-450cam was located at a normal frequency. This anomaly in the Raman spectrum of reduced P-450cam can be explained by assuming electron delocalization from the fifth ligand, presumably a thiolate anion, to the antibonding π orbital of the porphyrin ring. The corresponding Raman line of reduced P-420 appeared at a normal frequency (1360 cm-1), suggesting a status change or replacement of the fifth ligand upon conversion from P-450cam to P-420. The Raman spec-trum of reduced P-450cam•metyrapone complex was very similar to that of ferrous cyto-chrome b5.View full abstractDownload PDF (317K) -
Kanji FUJIKI, Akio FUKUDA1976 Volume 80 Issue 6 Pages 1453-1455
Published: December 25, 1976
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSPeptidoglycan of a gram-negative stalked bacterium, Caulobacter crescentus CB13, contained alanine, diaminopimelic acid, and glutamic acid, in molar ratios of 2:1:1. The amino acid compositions of peptidoglycans isolated from cultures enriched in swarmer and stalked cells, and from a stalk-less mutant were similar. This finding conflicts with a previous observation that swarmer peptidoglycan does not contain diaminopimelic acid (Goodwin and Shed-larski (1975) Arch. Biochem. Biophys. 170, 23-36). It appears that, despite the morphological differences, the Caulobacter cells all contain a similar peptidoglycan in the cell wall.View full abstractDownload PDF (187K) -
Shukuko IKAWA, Takehiko SHIBATA, Tadahiko ANDO1976 Volume 80 Issue 6 Pages 1457-1460
Published: December 25, 1976
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA new site-specific endonuclease (DNase) was isolated from the cells of Bacillus pumilus AHU 1387 strain. This enzyme (endonuclease R.Bpu 1387) introduced double-stranded scissions at unique sites on DNA's of coli phage λ, λdvl, coil phage T7, Bacillus phage φ105C, Bacillus phage SP10, and Simian Virus 40, in the presence of magnesium ion. The activity was stimulated by the presence of NaCl.View full abstractDownload PDF (1400K)
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