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41.
The substrate specificity of rice embryo benzoyl-L-argininep-nitroanilide hydrolase (BAPAase) was examined. No endopeptidaseactivity toward protein substrates was detectable. Small peptides(less than 8 residues) and amide, ester substrates, however,were hydrolyzed very well at the carboxyl side of the lysineor arginine residue. No other peptide bond was hydrolyzed. TheN-terminal arginine of the substrates was released very slowly.Peptides with lysine or arginine penultimate to the C-terminalposition were hydrolyzed well and released an amino acid. Theoxidized insulin B chain (30 residues) was cleaved very slowlyat the C-terminal Lys-Ala bond, whereas an Arg-Gly bond at aninner position was not cleaved. The hydrolytic rate increasedafter the chain length was shortened by chymotryptic digestion.These results show that the rice embryo BAPAase is a novel enzymewhich has mixed endopeptidase-carboxypeptidase activity towardthe Arg-X and Lys-X bonds of small peptides, a characteristicintermediate between trypsin and serine carboxypeptidase. Thisenzyme may act in the breakdown of small peptides that havephysiological functions. (Received May 26, 1984; Accepted August 29, 1984) 相似文献
42.
Benzoyl-L-arginine p-nitroanilide hydrolase (BAPAase), whichhas both endopeptidase and carboxypeptidase activity towardthe Arg-X or Lys-X bond of small peptides [Shibata and Doi (1984)Plant & Cell Physiol. 25: 1421], was purified from riceembryos by ammonium sulfate and polymin fractionations and byion exchange, gel exclusion and hydrophobic chromatographies.The purified enzyme was homogeneous when analyzed by polyacrylamidegel electrophoresis. It was unstable in the absence of surface-activereagents such as Triton X-100. Maximum activity for benzoyl-Largininep-nitroanilide (L-BAPA) or carboxypeptidase activity towardbutoxycarbonyl-Gly-Lys-Leu was obtained at pH 9.0. L-BAPA athigh concentrations inhibited the enzyme's activity. Di-isopropylphosphofluoridate, N-tosyl-L-lysine chloromethyl ketone, leupeptinand antipain, which are specific inhibitors of trypsin, inhibitedBAPAase activity, but soybean and rice bran trypsin inhibitorhad no effect on it. Sulfhydryl reagents strongly inhibitedthe BAPAase activity. (Received May 26, 1984; Accepted August 29, 1984) 相似文献
43.
The effect of polyamines on ribonucleases in the presence of various inhibitors (poly(G), heparin, and rat liver RNase inhibitor) has been studied. Bovine pancreatic RNas A and a ribonuclease from horse submaxillary gland (RNase HS) were inhibited by the inhibitors, but RNase T1 and RNase M were not inhibited. Polyamines were found to restore the activites of RNase A and RNase HS inhibited by poly(G) or heparin but not those activities inhibited by rat liver RNase inhibitor. When poly(U) and poly(C) were used as substrates, the inhibitory effects of poly(G) and heparin were greater with poly(U) than poly(C) as a substrate. However, when poly(C) was used as a substrate in the presence of either of the above inhibitors, the restoration of RNase activity by sperimine was more efficient. In fact, a stimulatory effect was observed. From the double-reciprocal plots, it was concluded that polyamines restored the activiities of RNases by increasing the availability of the substrate and enzyme to each other. The restoration of enzyme activity by polyamines occurred through the binding of the polyamines to the inhibitor and the subsequent release of enzyme from the inhibitor. 相似文献
44.
Chlorella vulgaris 11h cells grown in air enriched with 4% CO2(high-CO2 cells) had carbonic anhydrase (CA) activity whichwas 20 to 90 times lower than that of algal cells grown in ordinaryair (containing 0.04% CO2, low-CO2 cells). The CO2 concentrationduring growth did not affect either ribulose 1,5-bisphosphate(RuBP) carboxylase activity or its Km for CO2. When high-CO2 cells were transferred to low CO2 conditions,CA activity increased without a lag period, and this increasewas accompanied by an increase in the rate of photosynthetic14CO2 fixation under 14CO2-limiting conditions. On the otherhand, CA activity as well as the rate of photosynthetic 14CO2fixation at low 14CO2 concentrations decreased when low-CO2cells were transferred to high CO2 conditions. Diamox, an inhibitor of CA, at 0.1 mM did not affect photosynthesisof low-CO2 cells at high CO2 concentration (0.5%). Diamox inhibitedphotosynthesis only under low CO2 concentrations, and the lowerthe CO2 concentration, the greater was the inhibition. Consequently,the CO2 concentration at which the rate of photosynthesis attainedone-half its maximum rate (Km) greatly increased in the presenceof this inhibitor. When CO2 concentration was higher than 1%, the photosyntheticrate in low-CO2 cells decreased, while that in high-CO2 cellsincreased. Fractionation of the low-CO2 cells in non-aqueous medium bydensity showed that CA was fractionated in a manner similarto the distribution of chlorophyll and RuBP carboxylase. These observations indicate that CA enhances photosynthesisunder CO2-limiting conditions, but inhibits it at CO2 concentrationshigher than a certain level. The mechanism underlying the aboveregulatory functions of CA is discussed.
1This work was reported at the International Symposium on PhotosyntheticCO2-Assimilation and Photorespiration, Sofia, August, 1977 (18).Requests for reprints should be addressed to S. Miyachi, RadioisotopeCentre, University of Tokyo, Bunkyo-ku, Tokyo 113, Japan. (Received December 11, 1978; ) 相似文献
45.
Ayaaki Ishizaki Tomoko Ueda Kenji Tanaka Peter F. Stanbury 《Biotechnology letters》1992,14(7):599-604
Summary The growth, substrate utilisation and L-lactate production ofLactococcus lactis IO-1 were examined on xylose, and glucose and xylose media. The yield of lactate on xylose was 0.47 g lactate/g xylose at an initial xylose concentration of 51.2 g/l and the max was 0.72 h–1. Xylose cultures were more susceptible to lactate inhibition than were glucose cultures but showed similar kinetic behaviour. The organism was capable of complete sugar utilisation when grown on a mixture of 20 g/l xylose and 20 g/l glucose and synthesised 0.66 g lactate/g sugar. 相似文献
46.
Toshio Sugimoto Tsutomu Kawasaki Tomohiko Kato Robert F. Whittier Daisuke Shibata Yukio Kawamura 《Plant molecular biology》1992,20(4):743-747
A full-length cDNA encoding a subunit of phosphoenolpyruvate carboxylase (PEPC) was isolated from a developing seed expression library of the C3 plant Glycine max. The corresponding mRNA is present at similar levels in leaf, stem, root and developing seed. Two potential start codons exist, and the activity of protein initiated from the first such codon could be subject to regulation by protein kinase. Sequence comparison shows a similar upstream start codon in the case of the Ppc2 gene from Mesembryanthemum crystallinum, previously assumed to lack the sequences necessary for phosphorylation. The soybean encoded protein tends to resemble other C3-type PEPC proteins more closely than those implicated in C4 or crassulacean acid metabolism. 相似文献
47.
Jun Ishizaki Mikio Tamaki Masaru Shin Hiroshige Tsuzuki Kazumasa Yoshikawa Hiroshi Teraoka Nobuo Yoshida 《Applied microbiology and biotechnology》1992,36(4):483-486
Summary Recombinant human glucagon was succesfully produced with a high level of expression in Escherichia coli as a fusion protein with human interferon . The synthetic gene was designed to release glucagon, which does not contain glutamic acid residues, from fusion protein with the Staphylococcus aureus strain V8 protease that specifically cleaves the peptide bond on the carboxyl side of the glutamic acid residue. The resulting glucagon was purified to homogeneity by a combination of C18 reverse-phase HPLC and ion-exchange HPLC. The yield of intact glucagon obtained from 11 of culture was approximately 12 mg. The structure of recombinant human glucagon was confirmed by HPLC and amino acid composition/sequence analyses.
Offprint requests to: J. Ishizaki 相似文献
48.
49.
Kazunori Maruyama Kouji Nagata Minoru Tanaka Hiromichi Nagasawa Akira Isogai Hironori Ishizaki Akinori Suzuki 《The protein journal》1992,11(1):1-12
We report the synthesis of bombyxin-IV, a disulfide-linked, heterodimeric, insulin superfamily peptide from the silkworm,Bombyx mori. The two chains (A- and B-chains) were synthesized separately by the solid-phase method using fluoren-9-ylmethoxycarbonyl (Fmoc) group as a protecting group for α-amino group. Three disulfide bonds were bridged step by step (A6–A11, A20–B22, and A7–B10) in a good yield. Synthetic bombyxin-IV was identical with natural one with regard to the retention time on a reversed-phase column and the molecular weight measured by mass spectrometry. Circular dichroism (CD) spectrum of the synthetic bombyxin-IV was very similar to that of the natural one. The specific activity of synthetic bombyxin-IV is equal to that of natural one (0.1 ng/Samia unit). These results suggest that the synthetic bombyxin-IV has the tertiary structure identical with the natural peptide. Our method developed for synthesis of bombyxin-IV would be generally applicable to the synthesis of insulin-like heterodimeric peptides. 相似文献
50.
Tetsuo Kunieda Eiji Kobayashi Hiroshi Ikadai Tomonori Imamichi Nobuo Nomura Ryotaro Ishizaki 《Biochemical genetics》1990,28(11-12):631-642
Novel restriction fragment length polymorphisms (RFLPs) in inbred rats were revealed with the human N-ras gene as probe. Three fragments hybridizing to the probe were detected by Southern blot hybridization under highly stringent conditions, and one of the fragments showed variation in inbred rat strains. Furthermore, on hybridization under low-stringency conditions, an additional fragment hybridizing to the probe was observed, and this fragment also showed interstrain variation. These two variant fragments showed different distributions in 27 inbred rat strains and segregated in backcross progeny as codominant alleles of independent single autosomal loci. Therefore, the loci for these RFLPs were named Nras-1 and Nras-2, respectively. Analyses of linkages between the RFLPs and 11 other loci revealed that the Nras-2 locus was closely linked to the c locus (3.7 +/- 2.6%), which belongs to rat linkage group I. 相似文献