Abstract: Cyclic GMP (cGMP) is an important signaling molecule that controls a range of cellular functions. So far, however, only a few genes have been found to be regulated by cGMP in higher plants. We investigated the cGMP-responsiveness of several genes encoding flavonoid-biosynthetic enzymes in soybean (Glycine max L.) involved in legume-specific isoflavone, phytoalexin and anthocyanin biosynthesis, such as phenylalanine ammonia-lyase, cinnamate 4-hydroxylase, 4-coumarate:CoA ligase, chalcone synthase, chalcone reductase, chalcone isomerase, 2-hydroxyisoflavanone synthase, 2-hydroxyisoflavanone dehydratase, anthocyanidin synthase, UDP-glucose:isoflavone 7-O-glucosyltransferase, and isoflavone reductase, and found that the majority of these genes were induced by cGMP but not by cAMP. All cGMP-induced genes were also stimulated by sodium nitroprusside (SNP), a nitric oxide (NO) donor, and illumination of cultured cells with white light. The NO-dependent induction of these genes was blocked by 6-anilino-5,8-quinolinedione, an inhibitor of guanylyl cyclase. Moreover, cGMP levels in cultured cells were transiently increased by SNP. Consistent with the increases of these transcripts, the accumulation of anthocyanin in response to cGMP, NO, and white light was observed. The treatment of soybean cotyledons with SNP resulted in a high accumulation of isoflavones such as daidzein and genistein. Loss- and gain-of-function experiments with the promoter of chalcone reductase gene indicated the Unit I-independent activation of gene expression by cGMP. Together, these results suggest that cGMP acts as a second messenger to activate the expression of genes for enzymes involved in the flavonoid biosynthetic pathway in soybean.
Abstract: P450 (cytochrome P450) enzymes catalyse the mono-oxygenation of a wide range of compounds such as steroids, fatty acids, vitamins and drugs. In the present paper we demonstrate a system for bioconverting diverse compounds [flavanone, DHEA (dehydroepiandrosterone) and 7-ethoxycoumarin] using P450 species expressed in Escherichia coli. First, we expressed four P450 species: rabbit CYP2B (P450 family 2, subfamily B), fruitfly (Drosophila) CYP317A, rat CYP3A23 and mouse CYP2J5. Next, we added substrates directly to the incubation medium. The resulting metabolites were extracted and analysed by HPLC and spectrofluorimetry. The first substrate, 7-ethoxycoumarin, was de-ethylated by CYP2B; CYP2J5 and CYP3A23 showed weak activity, and CYP317A had no activity for 7-ethoxycoumarin. We next used flavanone, a flavonoid, as a substrate for these four P450 species and other P450 species expressed previously. As a result, CYP2B, CYP2C43 and CYP2C29 catalysed flavanone 2-hydroxylation. CYP2A5 catalysed 2- and 4-hydroxylations. Finally, to produce diverse modified compounds, variants of CYP2A5 with point mutations were incubated with a steroid (DHEA) and an antioxidant (flavanone) in vivo. HPLC analysis indicated that two P450 species produced a 7-beta-hydroxy-DHEA and two P450 species produced a 2-alpha-hydroxy-DHEA. Four P450 species catalysed flavanone 2- and 4-hydroxylations. These results indicate that bioconversion by P450 is a useful technique to modify small molecules (steroids, coumarin and flavanone) and produce new, diverse hydroxylated compounds, which could be used for high-throughput screening for drug discovery.
Abstract: We indicated that two P450s (1A9 and 1C1) from Japanese eel (Anguilla japonica) metabolized 7-ethoxycoumarin, 7-ethoxyresorufin, and flavanone. At first, we constructed expression vectors for two types of P450 (1A9 and 1C1). The reduced CO-difference spectra of Escherichia coli cells transformed with these plasmids showed Soret peaks (450 nm) that were typical of P450s. We performed bioconversion experiments in which substrates were added directly to incubation medium. The resulting metabolite(s) were extracted and analyzed by high-performance liquid chromatography and spectrofluorometer. Incubation of 50 nmol 7-ethoxyresorufin with P450 1C1 yielded 0.773 nmol of deethylated product, whereas 50 nmol 7-ethoxycoumarin resulted in 4.76 nmol. P450 1A9 metabolized 50 nmol of 7-ethoxyresorufin and 7-ethoxycoumarin to yield 6.54 and 20.9 nmol of deethylated product, respectively. Incubation of 50 nmol flavanone with P450 1C1 yielded 1.46 nmol and 0.69 nmol of products, whereas 50 nmol flavanone with P450 1A9 resulted in 1.10 nmol. In this system, 4'-hydroxy flavanones were formed by P450 1A9 and P450 1C1. P450 1A9 also metabolized 50 nmol of 17 beta-estradiol to yield 4.25 nmol of product. In this system, 2-hydroxy estradiol was formed by P450 1A9 using 17 beta-estradiol as a substrate. This study is the first to identify the substrates that P450 1C1 and 1A9 metabolize.
Abstract: The small GTPases known as Rab proteins are key regulators of membrane trafficking. We used RT-PCR to isolate cDNA clones of insect-specific Rab proteins (BRabN1 and BRabN2) showing low homology with known Rab proteins from other animals, from mRNA of Bombyx mori. These 2 Rabs were produced in Escherichia coli and purified. BRabN1 bound [(3)H]-GDP and [(35)S]-GTPgammaS with dissociation constants of 0.087 x 10(-6) M and 1.02 x 10(-6) M, respectively, whereas those of BRabN2 were 0.546 x 10(-6) M and 1.02 x 10(-6) M, respectively. Binding of [(35)S]-GTPgammaS to BRabN1 and N2 was inhibited by GDP and GTP. The GTP-hydrolysis activities of BRabN1 and N2 were 154 and 35.5 mmol/min/mole, respectively, and bound [(35)S]-GTPgammaS was exchanged efficiently with GTP. BRabN1 also showed ATPase activity and exchange of [(35)S]-GTPgammaS with ATP. Monoclonal antibodies against BRabN1 and N2 did not recognize any other Rab proteins, and Western blotting using the anti-BRabN1 antibody revealed a single band in the testis of B. mori. These results suggest that BRabN1 and N2 of B. mori bind GTP, convert from the GTP-bound state to the GDP-bound state by intrinsic GTP hydrolysis activity, and return to the GTP-bound state with the exchange, and that BRabN1 is specifically expressed in testis. Arch. Insect Biochem. Physiol. 2008. (c) 2008 Wiley-Liss, Inc.
Abstract: The cold-active protein tyrosine phosphatase found in psychrophilic Shewanella species exhibits high catalytic efficiency at low temperatures as well as low thermostability, both of which are characteristics shared by many cold-active enzymes. The structure of cold-active protein tyrosine phosphatase is notable for the presence of three hydrophobic sites (termed the CA, Zn-1 and Zn-2 sites) behind the loop structures comprising the catalytic region. To identify the structural components responsible for specific enzyme characteristics, we determined the structure of wild-type cold-active protein tyrosine phosphatase at high resolution (1.1 A) and measured the catalytic efficiencies of enzymes containing mutations in the three hydrophobic sites. The bulkiness of the amino acid side chains in the core region of the Zn-1 site strongly affects the thermostability and the catalytic efficiency at low temperatures. The mutant enzyme I115M possessed a higher kcat at low temperatures. Elucidation of the crystal structure of I115M at a resolution of 1.5 A revealed that the loop structures involved in retaining the nucleophilic group and the acid catalyst are more flexible than in the wild-type enzyme.
Abstract: The Rab family of small GTPases are key regulators of membrane trafficking. Partially purified Rab8 from Bombyx mori (BRab8) was phosphorylated by protein kinase C in mammalian cells in vitro. To determine which of the seven serines and four threonines are phosphorylated, we generated deletion and site-directed mutants of BRab8, inserted them in Escherichia coli, partially purified the encoded fusion proteins by affinity chromatography, and examined their phosphorylation by protein kinase C in vitro. We found that Ser-132 of BRab8 was specifically phosphorylated by protein kinase C. In addition, Western blotting using an antiserum against BRab8 and in-gel staining for phosphorylated proteins revealed that BRab8 is phosphorylated in vivo.
Abstract: The complete nucleotide sequences of the cDNA and its gene that encode a bifunctional alpha-amylase/subtilisin inhibitor of rice (Oryza sativa L.) (RASI) were analyzed. RASI cDNA (939 bp) encoded a 200-residue polypeptide with a molecular mass of 21,417 Da, including a signal peptide of 22 amino acids. Sequence comparison and phylogenetic analysis showed that RASI is closely related to alpha-amylase/subtilisin inhibitors from barley and wheat. RASI was found to be expressed only in seeds, suggesting that it has a seed-specific function. A coding region of RASI cDNA without the signal peptide was introduced into Escherichia coli and was expressed as a His-tagged protein. Recombinant RASI was purified to homogeneity in a single step by Ni-chelating affinity column chromatography and characterized to elucidate the target enzyme. The recombinant inhibitor had strong inhibitory activity toward subtilisin, with an equimolar relationship, comparable with that of native RASI, and weak inhibitory activity toward some microbial alpha-amylases, but not toward animal or insect alpha-amylases. These results suggest that RASI might function in the defense of the seed against microorganisms.
Abstract: We developed a system for bioconverting diverse compounds using P450s produced in Escherichia coli. Vectors for the expressing various P450 cDNAs quickly and easily in E. coli were developed by using several restriction enzyme sites. Three types of P450 (2C2, 2C29, and 2D22) were produced using these plasmids. Substrates were directly added to the incubation medium and metabolized. To obtain pure product from the medium, we first tried production of P450 in synthetic medium. The amount of another P450 2C43 produced in the synthetic medium was similar to the amount produced in Luria broth (LB) medium. Next, estradiol, a steroid, was added as a substrate, incubated, and the metabolite was extracted and analyzed by high-performance liquid chromatography. The metabolite extracted from synthetic medium was purer than that obtained from LB medium. Three P450s (2C29, 2C2, and 2A4) metabolized testosterone at different positions. P450 2C29 metabolized 7-ethoxycoumarin, androstendione, and dehydroepiandrosterone in this medium. P450s produced in the synthetic medium may be useful for producing various modified compounds for high-throughput screening.
Abstract: A cDNA clone encoding methyl DNA binding domain-containing protein (bMBD2/3) was obtained by homology searches using a Bombyx mori fat body cDNA library. The cDNA encoded a polypeptide with 249 amino acids sharing 54% similarity with the methyl DNA binding protein from Drosophila melanogaster. To characterize the biochemical properties of bMBD2/3, the clone was expressed in Escherichia coli as His-tagged protein. The recombinant protein was purified to homogeneity using Ni-NTA superflow resin and heparin agarose. The protein showed specific methyl DNA binding activity and was phosphorylated by protein kinase in vitro. Immunoblotting using the purified antibody indicated that bMBD2/3 was expressed in almost all tissues. Using west-western blotting analysis, some proteins that interact with bMBD2/3 were identified in the brain. This is the first report that insect MBD is phosphorylated and is present in adult tissues. These results suggest that bMBD2/3 plays important roles in the DNA methylation-specific transcription of Bombyx mori.
Abstract: Protein-tyrosine phosphatase [EC 3.1.3.48] from a psychrophile, Shewanella sp. shows high activity at low temperatures and has the conserved amino acid sequence of protein-Ser/Thr-phosphatases. Site-directed mutagenesis with the conserved amino acid residues indicated that His148 could be important as a general acid catalyst and Asp115 assists the protonation with His148 of the leaving group of a substrate, and that Asp76 and Asp112 were involved in binding to magnesium ions.
Abstract: Cucumisin, a subtilisin-like serine protease, is expressed at high levels in the fruit of melon (Cucumis melo L.) and accumulates in the juice. We investigated roles of the promoter regions and DNA-protein interactions in fruit-specific expression of the cucumisin gene. In transient expression analysis, a chimeric gene construct containing a 1.2-kb cucumisin promoter fused to a beta-glucuronidase (GUS) reporter gene was expressed in fruit tissues at high levels, but the promoter activities in leaves and stems were very low. Deletion analysis indicated that a positive regulatory region is located between nucleotides -234 and -214 relative to the transcriptional initiation site. Gain-of-function experiments revealed that this 20-bp sequence conferred fruit specificity and contained a regulatory enhancer. Gel mobility shift experiments demonstrated the presence of fruit nuclear factors that interact with the cucumisin promoter. A typical G-box (GACACGTGTC) present in the 20-bp sequence did not bind fruit protein, but two possible cis-elements, an I-box-like sequence (AGATATGATAAAA) and an odd base palindromic TGTCACA motif, were identified in the promoter region between positions -254 and -215. The I-box-like sequence bound more tightly to fruit nuclear protein than the TGTCACA motif. The I-box-like sequence functions as a negative regulatory element, and the TGTCACA motif is a novel enhancer element necessary for fruit-specific expression of the cucumisin gene. Specific nucleotides responsible for the binding of fruit nuclear protein in these two elements were also determined.
Abstract: A psychrophilic alkaline phosphatase (EC 3.1.3.1) from Shewanella sp. is a cold-active enzyme that has high catalytic activity at low temperature [Ishida et al. (1998) Biosci. Biotechnol. Biochem., 62, 2246-2250]. Here, we identified the nucleotide sequence of a gene encoding the enzyme after cloning with the polymerase chain reaction (PCR) and inverted PCR techniques. The deduced amino acid sequence of the enzyme contained conserved amino acids found among mesophilic alkaline phosphatases and showed some structural characteristics including a high content of hydrophobic amino acid residues and the lack of single alpha-helix compared with the alkaline phosphatase of Escherichia coli, which were possibly efficient for catalytic reaction at low temperatures. The recombinant enzyme expressed in E. coli was purified to homogeneity with the molecular mass of 41 kDa. The recombinant enzyme had a specific activity of 1,500 units/mg and had high catalytic activity at low temperatures.
Abstract: The signal transduction processes involved in the regulation of SAMDC gene expression by blue and red light were examined using pharmacological inhibitors of signalling pathways. Calcium and calmodulin positively regulated SAMDC gene expression in red light, whereas in blue light they regulated negatively. These results indicate that calcium homeostasis is involved in both red and blue light induction of SAMDC expression. Both signal transduction pathways also require new protein synthesis.
Abstract: The expression of asparagine synthetase (AS; EC 6.3.5.4) in response to externally supplied nitrogen was investigated with respect to enzyme activity and protein levels as detected immunologically in rice (Oryza sativa) seedlings. The asparagine content was very low in leaves and roots of nitrogen-starved rice plants but increased significantly after the supply of 1 mM NH4+ to the nutrient solution. While neither AS activity nor AS protein could be detected in leaves and roots prior to the supply of nitrogen, levels became detectable in roots but not in leaves within 12 h of the supply of 1 mM NH4+ or 10 mM glutamine. Other nitrogen compounds, such as nitrate, glutamate, aspartate and asparagine had no effect. Methionine sulfoximine completely inhibited the NH4+-induced accumulation of AS protein but did not affect the glutamine-induced accumulation of the enzyme. The results suggested that glutamine or glutamine-derived metabolites regulate AS expression in rice roots.
Abstract: Light induced rapid and transient activation of a 46-kDa protein kinase in soybean photomixotrophic cell culture. This kinase was designated as LAP kinase (light signal-activated protein kinase). Activation of LAP kinase in response to light was associated with tyrosine phosphorylation of the kinase, and treatment of the kinase with protein tyrosine phosphatase abolished its activity. The LAP kinase efficiently phosphorylated myelin basic protein and histone, but did not phosphorylate casein. Phospho-amino acid analysis indicated that the LAP kinase was a serine/threonine protein kinase. These results indicated that the LAP kinase is related to the MAP kinase family of protein kinases.
Abstract: The complete nucleotide sequences of a cDNA (RSP1) that encodes a subtilisin-like serine protease (subtilase) of rice (Oryza sativa L.) and a gene (ASP48) for Arabidopsis subtilase were analyzed. The RSPI cDNA and ASP48 DNA encoded 736- and 757-residue pre-pro-polypeptides including a signal peptide with molecular masses of 78,668 Da and 79,414 Da, respectively. RSP1 is the first known serine protease in rice, and ASP48 is a gene for ara12 cDNA. Sequence comparison and phylogenetic analysis showed that RSP1 is distantly related to all other plant subtilases and ASP48 is closely related to a tomato subtilase, SBT1. The ASP48 gene was found to lack introns. The Arabidopsis subtilase gene appears to consist of a small gene family. The RSP1 was found to be expressed in seed and shoots of seedlings while ASP48 transcripts was found to be accumulated in immature silique and flowers, indicating that both RSP1 and ASP48 are organ-specific and may be involved in the specific proteolytic events that occur during organ development.
Abstract: From the soybean cDNA library, we isolated and analyzed the chlH gene encoding a subunit of Mg-chelatase. The subunit was a polypeptide of 1,383 amino acids with a molecular mass of 153,491 Da, which shared 90% identity with the olive gene from Antirrhinum majus. The regulation of the expression of chlH was investigated in photomix-otrophic soybean suspension cells (SB-P). The expression was light-inducible, and the induction was more rapid than those of chlI and cab2. Furthermore, the levels of the transcripts and products of chlH appeared to be regulated by a circadian oscillation. The subchloroplastic localization of ChlH was investigated by immunoblot analyses with antiserum against recombinant ChlH. Depending on the concentration of Mg2+ in the lysis buffer, the localization of ChlH protein migrated between the stroma and the envelope membrane; ChlH was localized on the envelope membrane, a major site of chlorophyll biosynthesis, when the Mg2+ concentration of the lysis buffer was high (above 5 mM). These results indicated that the activity of Mg-chelatase was regulated by modulation of the expression and subchloroplastic localization of ChlH protein.
Abstract: A cDNA encoding glutamine-dependent asparagine synthetase was isolated from dark-adapted Glycine max cell culture. The deduced amino acid sequence showed 76-89% identity with other plant sequences. The gene for asparagine synthetase is expressed predominantly in shoots as compared to roots of etiolated plants and the level of expression decreases following light treatment, suggesting that the gene expression is down-regulated by light.
Abstract: A bifunctional alpha-amylase/subtilisin inhibitor (RASI) was purified to electrophoretic homogeneity from rice (Oryza sativa L.) bran. Its molecular mass was 21 kDa by SDS-PAGE and its isoelectric point was 9.05. Purified RASI inhibited subtilisin Carlsberg strongly and inhibited alpha-amylase from germinating rice seeds weakly. It inhibited rice alpha-amylase more than barley alpha-amylase, and the inhibition of rice alpha-amylase was greater at higher pHs. RASI did not inhibit trypsin, chymotrypsin, cucumisin, or mammalian alpha-amylase. The RASI was in the outermost part of the rice grain and its subcellular site seemed to be aleurone particles in aleurone cells. SDS-PAGE and western blotting showed that RASI was synthesized in the late milky stage in developing seeds, and it remained fairly constant during the first 7 days of germination.
Abstract: The plant photoreceptor phytochrome A utilizes three signal transduction pathways, dependent upon calcium and/or cGMP, to activate genes in the light. In this report, we have studied the phytochrome A regulation of a gene that is down-regulated by light, asparagine synthetase (AS1). We show that AS1 is expressed in the dark and repressed in the light. Repression of AS1 in the light is likely controlled by the same calcium/cGMP-dependent pathway that is used to activate other light responses. The use of the same signal transduction pathway for both activating and repressing different responses provides an interesting mechanism for phytochrome action. Using complementary loss- and gain-of-function experiments we have identified a 17 bp cis-element within the AS1 promoter that is both necessary and sufficient for this regulation. This sequence is likely to be the target for a highly conserved phytochrome-generated repressor whose activity is regulated by both calcium and cGMP.
Abstract: A cDNA for early light-inducible protein (ELIP) was isolated from Glycine max L. and the nucleotides were sequenced. The cDNA encodes a 192-residue polypeptide of 20.3 kDa. The deduced amino acid sequence included a transit peptide in the amino-terminus and three hydrophobic regions long enough to span the thylakoid membranes, and had a high similarity to those of ELIPs and related polypeptides from other plants.
Abstract: Mg-insertion is the first committed step in chlorophyll synthesis and is catalyzed by Mg-chelatase. In photosynthetic bacteria, bchI gene product was suggested to be a subunit of Mg-chelatase. We isolated a bchI homolog from a soybean cDNA library and designated it as chlI. CHLI consisted of 421 amino acid residues and the sequence exhibited a high similarity to other BchI homologs. CHLI contained an ATP-binding motif found in other BchI homologs. CHLI was localized in the soluble fraction in soybean chloroplasts, suggesting that it was a stromal subunit of Mg-chelatase. chlI mRNA in cell culture (SB-P) of soybean was reversibly induced by light.
Abstract: Three signal transduction pathways, dependent on cGMP and/or calcium, are utilized by phytochrome to control the expression of genes required for chloroplast development and anthocyanin biosynthesis in plant cells. For example, chs is controlled by a cGMP-dependent pathway, cab is controlled by a calcium-dependent pathway, and fnr is regulated by a pathway that requires both cGMP and calcium. Using a soybean photomixotrophic cell culture and microinjection into the cells of a phytochrome-deficient tomato mutant, we have studied the regulatory mechanisms acting within and between these three signaling pathways. We provide evidence that changes in cGMP levels mediate the observed induction and desensitization of chs gene expression in response to light and demonstrate that high cGMP concentrations cause negative regulation of both the calcium- and the calcium/cGMP-dependent pathways. Conversely, high activity of the calcium-dependent pathway can negatively regulate the cGMP-dependent pathway. We have termed these opposing regulatory mechanisms reciprocal control. In all cases, the molecules that are involved appear to be downstream components of the signal transduction pathways, rather than calcium and cGMP themselves. Furthermore, we have found that the calcium/cGMP-dependent pathway has a lower requirement for cGMP than does the cGMP-dependent pathway. The role of these phenomena in the regulation of plant photoresponses is discussed.
Abstract: We have previously used single-cell assays in a phytochrome-deficient tomato mutant to demonstrate that phytochrome signaling involves heterotrimeric G proteins, calcium, and calmodulin. While G protein activation could stimulate full chloroplast development and anthocyanin pigment biosynthesis, calcium and calmodulin could not induce anthocyanins and were only able to stimulate the development of immature chloroplasts lacking cytochrome b6f and photosystem I core components. We now report that cyclic GMP is able to trigger the production of anthocyanins, and that a combination of cyclic GMP with calcium can induce the development of fully mature chloroplasts containing all the photosynthetic machinery. Furthermore, using reporter genes for these different pathways (cab-gus, chs-gus, and fnr-gus) we demonstrate that cGMP and calcium act primarily by modulating gene expression.
Abstract: Cucumisin is a thermostable alkaline serine protease that is found in the juice of melon fruits (Cucumis melo L.). We have determined the complete nucleotide sequence of a cucumisin cDNA (2,552 nucleotides) and deduced the corresponding amino acid sequence. The open reading frame of the cDNA consists of 731 codons and encodes a large precursor (molecular weight, 78,815) relative to the observed size of mature native cucumisin (67 kDa). Sequence comparisons reveal that cucumisin has several features in common with the microbial proteases of the subtilisin family. The highly conserved sequences to the proximal regions of the catalytic triad amino acids Asp, His, and Ser, together with the substrate binding site in subtilisin, can be found within the deduced amino acid sequence of the protease domain of the cucumisin precursor. Cucumisin is the first known plant protease with such characteristics. Examination of the primary structure of cucumisin revealed that it is synthesized as a precursor, consisting of four functional domains: a possible signal peptide (22 amino acid residues), an NH2-terminal pro-sequence (88 residues), a 54-kDa protease domain (505 residues), which is the active enzyme domain of the 67-kDa native cucumisin, and a 14-kDa COOH-terminal polypeptide (116 residues), which arises by limited autolysis of the 67-kDa native cucumisin. This structure of cucumisin suggests that it is probably synthesized as an inactive precursor.
Abstract: The complex of an adzuki bean subtilisin inhibitor (ASI-II) with its target enzyme, prepared at pH 7.6, was subjected to reversed-phase HPLC in a trifluoroacetic acid-acetonitrile system. Two peptide fragments derived from the reactive site-modified ASI-II were obtained. The analyses of the amino acid composition and sequence of these two fragments revealed that one corresponded to the region from the amino-terminal Lys to the reactive site P1 Ala and the other, to the region from the reactive site P1' Asp to the carboxyl-terminal Gly of the inhibitor. Although neither fragment alone showed inhibitory activity against subtilisin, an equimolar mixture of both fragments was found to inhibit strongly the target enzyme, as did the intact inhibitor. Thus, it was suggested that the two fragments have strong specific affinity with each other, regenerating the reactive site-modified ASI-II, to inhibit the target enzyme.
Abstract: The complete amino acid sequence of a major molecular form of subtilisin inhibitor from adzuki beans (Vigna angularis) was established by manual analysis using 4-N,N-dimethylaminoazobenzene-4'-isothiocyanate (DABITC). Sequencing was performed on the peptides which were derived by digesting the inhibitor with lysyl-endopeptidase and Staphylococcus aureus V8-protease. The inhibitor consisted of 92 amino acid residues and the molecular weight was calculated to be 10,800. A minor form of subtilisin inhibitor was found, which lacked the amino-terminal 19 residues of the major one. Comparison of amino acid sequences revealed that the adzuki bean subtilisin inhibitors were 29-68% homologous in sequence to the inhibitors of so-called "potato inhibitor I family."
Abstract: Preworkshift and postworkshift urinary fluoride concentrations were measured in 142 hydrofluoric acid (HF) workers and 82 unexposed workers aged 18 to 59. Postshift urinary fluoride concentration in HF workers was significantly higher than that in preshift or control workers. A linear relationship was observed between the mean values in urinary fluoride concentration and the HF concentration in the air. The mean urinary fluoride concentration of 4 ppm and its lower fiducial limit (95%, P = 0.05) of 2 ppm (specific gravity 1.024) were estimated corresponding to the atmospheric HF concentration of 3 ppm, which is the maximal allowable concentration recommended by the Japanese Association of Industrial Health and also the threshold limit value suggested by the American Conference of Governmental Industrial Hygienists. The results suggest that exposure to HF can be monitored by determining the urinary fluoride concentration.
Abstract: Sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis of the starchy endosperm protein of rice (Oryza sativa L. Japonica cv Koshihikari) during seed development confirmed that storage protein begins to accumulate about 5 days after flowering. Two polypeptide groups, 22 to 23 and 37 to 39 kilodaltons, the components of glutelin, the major storage protein in rice seed, appeared 5 days after flowering. A 26-kilodalton polypeptide, the globulin component, also appeared 5 days after flowering. Smaller polypeptides (10- to 16-kilodaltons) including prolamin components, appeared about 10 days after flowering. In contrast, the levels of the 76- and 57-kilodalton polypeptides were fairly constant throughout seed development. Transmission electron microscopy and fractionation by sucrose density gradient centrifugation of the starchy endosperms at various stages of development showed that protein body type II, the accumulation site of glutelin and globulin, was formed faster than protein body type I, the accumulation site of prolamin.The 57-kilodalton polypeptide but not the glutelin subunits was labeled in a 2-hour treatment with [(14)C]leucine given between 4 and 12 days after flowering to developing ears. In vivo pulse-chase labeling studies showed the 57-kilodalton polypeptide to be a precursor of the 22 to 23 and 37 to 39 kilodalton subunits. The 57-kilodalton polypeptide was salt-soluble, but the mature glutelin subunits were almost salt insoluble.In vitro protein synthesis also showed that the mRNAs directly coding the 22 to 23 and 37 to 39 kilodalton components were absent in developing seeds and that the 57-kilodalton polypeptide was the major product. Thus, it was concluded that the two subunits of rice glutelin are formed through post-translational cleavage of the 57-kilodalton polypeptide.
