ABSTRACT
Cardiovascular disease is a major cause of morbidity and mortality in patients with diabetes who are receiving dialysis. It is known that cardiac mortality is high in the first year of hemodialysis (HD). The purpose of this study was to clarify the prevalence of coronary artery disease (CAD) in patients with diabetes at the initiation of HD, and to investigate the relationship between short-term outcomes after starting hemodialysis and CAD. We performed coronary angiography (CAG), whether or not patient had angina, within one month of initiation of maintenance HD in 17 of 34 consecutive unselected diabetic patients. We studied the two-year survival rate of those with CAD. Eleven patients (65%) were classified CAD-positive. Nine (82%) of these 11 had multivessel disease. Clinical and hematologic factors did not differ significantly between the groups with and without CAD. During the two-year follow-up period, 28 (82%) of 34 patients survived. In patients with CAD the two-year survival rate was 60%. None of the patients without CAD died within 2 years after starting HD. These results suggest that the presence of CAD at the initiation of HD may play a critical role in short-term outcomes after starting HD. We believe that an evaluation of CAD should be performed at the initiation of HD.
Subject(s)
Coronary Disease/etiology , Diabetes Complications , Kidney Failure, Chronic/complications , Renal Dialysis/adverse effects , Chi-Square Distribution , Coronary Angiography , Coronary Disease/epidemiology , Female , Humans , Kidney Failure, Chronic/therapy , Male , Middle Aged , Prevalence , Risk Factors , Statistics, Nonparametric , Survival RateABSTRACT
The glycinin gene family encoding the glycinin subunits in soybean plants is composed of at least 5 gene members, i.e.: Gy1-Gy5. A genomic clone containing the Gy5 gene from a genomic library of cv. Williams was isolated by using Gy5 cDNA probe. The complete nucleotide sequence of this gene has been determined. It is 2819 bp long consisting of four exons and three introns. These exons and introns are as follows: exonl(292 bp), intronl(358 bp), exon2(263 bp), intron2(425 bp), exon3(645 bp), intron3(485 bp), exon4(351 bp). The gene encodes 517 amino asids. This is the first time to report the complete Gy5 gene sequence from a genomic library.
Subject(s)
DNA/chemistry , Globulins/genetics , Base Sequence , Cloning, Molecular , Exons , Introns , Molecular Sequence Data , Soybean ProteinsABSTRACT
A soybean protein was purified from mature dry seeds. Amino-acid sequencing of the nine internal peptides derived from this N-terminally blocked protein showed that it has a significant similarity to the soluble epoxide hydrolases known to date. A degenerate series of 23-mer oligonucleotides with sequences corresponding to an internal region of eight amino-acid residues was synthesized as a probe mixture for detection of a putative epoxide hydrolase cDNA in a developing cotyledon cDNA library. The 1332-bp cDNA obtained was found to have an open-reading frame encoding the seed epoxide hydrolase-like precursor consisting of 341 amino-acid residues, suggesting that 25 amino-acid residues upstream from the second methionine correspond to a transit peptide. Employing an Escherichia coli expression system, the putative mature epoxide hydrolase-like protein was overexpressed and purified to homogeneity. This recombinant protein was confirmed to exhibit its epoxide-diol converting activity using styrene oxide as substrate. The Vmax and Km values for styrene oxide are 1.36 micromol x min-1 x mg-1 and 1500 microM, respectively. Sedimentation equilibrium experiments showed that the active form of this epoxide hydrolase is monomeric in solution. Using the above cDNA as a probe, a 12-kb genomic clone was selected and the sequence of a 1933-bp fragment from this clone was found to cover the entire coding region together with 5'- and 3'-flanking regions of the soybean epoxide hydrolase gene. The coding region of the gene, interrupted by two short introns, was identical to the corresponding regions of the cDNA. Northern blot analyses showed that this epoxide hydrolase gene was expressed strongly at a very early stage (13 days after flowering) and then the level of expression gradually decreased and almost ceased at a very late stage (58 days after flowering) of seed development, whereas its expression was markedly up-regulated by ethylene treatment. In stems (hypocotyl portion), the epoxide hydrolase transcript was detected at significant levels and was also up-regulated in response to ethylene. On the other hand, it is hardly expressed in leaves, even though they were treated with the phytohormone. Overall, the results obtained may indicate that soluble-type epoxide hydrolase mRNA is expressed at the maximum level in an early stage of seed development. Later, oil bodies are formed and subsequently epoxy fatty acids, naturally occurring metabolites, accumulate within those bodies. The temporal induction of this epoxide hydrolase transcript in some tissues in response to ethylene also indicates that this epoxide hydrolase may play a crucial role in self-defense systems of plant.
Subject(s)
Epoxide Hydrolases/isolation & purification , Ethylenes/pharmacology , Gene Expression Regulation, Enzymologic/drug effects , Gene Expression Regulation, Plant/drug effects , Glycine max/enzymology , Amino Acid Sequence , Base Sequence , Blotting, Southern , Cloning, Molecular , DNA Primers , DNA, Complementary , Enzyme Induction , Epoxide Hydrolases/chemistry , Epoxide Hydrolases/genetics , Escherichia coli/genetics , Molecular Sequence Data , Seeds/enzymology , Sequence Homology, Amino Acid , SolubilityABSTRACT
A soybean seed-specific PR-8 chitinase, named Chib2, has a markedly extended C-terminal segment compared to other plant Chib1 homologues of the PR-8 chitinase family known to date. To further characterize the molecular structure and the expression pattern of this chitinase family, we cloned two typical Chib1-similar cDNAs (Chib1-1 and Chib1-2) from soybeans by PCR-cloning techniques. The deduced primary sequence of Chib1-1 chitinase is composed of a signal peptide segment (26 amino acid residues) and a mature 273 amino acid sequence (calculated molecular mass 28,794, calculated pI 3.7). This Chib1-1 enzyme is more than 90% identical to Chib1-2 chitinase but is below 50% identical to Chib2 enzyme. Thus, we confirmed the occurrence of two distinct classes, Chib1 and Chib2 in the plant PR-8 chitinase family. The Chib1 genes, interrupted by one intron, were found to be up-regulated in response to ethylene in stems and leaves, but scarcely expressed in developing soybean seeds. Chib1 chitinases may be responsible for protecting the plant body from various pathogenic attacks.
Subject(s)
Chitinases/genetics , Gene Expression Regulation, Plant , Glycine max/genetics , Plant Proteins/genetics , Amino Acid Sequence , Base Sequence , Blotting, Northern , Blotting, Southern , Chitinases/biosynthesis , Chitinases/chemistry , Cloning, Molecular , DNA Primers/chemistry , DNA, Plant/chemistry , Ethylenes/metabolism , Molecular Sequence Data , Molecular Weight , Phylogeny , Plant Growth Regulators/metabolism , Plant Proteins/biosynthesis , Plant Proteins/chemistry , Polymerase Chain Reaction , RNA, Plant/chemistry , Sequence Alignment , Sequence Analysis, DNA , Sequence Homology, Amino Acid , Glycine max/enzymologyABSTRACT
Authentic soybean ß-amylase preparation, purified to homogeneity as judged by SDS-PAGE by using an affinity purification step, was composed of four pI-differing isoforms. By chromatofocusing, these isoforms were separated into three fractions, designated as fractions 1-3 in the order of elution. Fraction 1 contained two isoforms having the same molecular mass (55,989 Da), as measured by mass spectrometric analysis, with different pIs, 5.32 (Isoform I) and 5.22 (Isoform II). Fraction 2 showed a single isoform having a molecular mass of 55,994 Da and having a pI of 5.09. This component, named Isoform III, existed rather in excess in a mixture of the authentic enzyme isoforms. The remainder (fraction 3) also contained a single component (Isoform IV) which has a molecular mass of 56,310 Da with a pI of 4.97. Chemical analyses indicated that the N-termini and the C-terminal tripeptides of four pI-separated isoforms mentioned are similar to one another, and are blocked and are NH2-Val-Asp-Gly-COOH, respectively. Moreover, enzymic properties involving specific activity and the value of kcat/Km for the above three fractions are almost the same, and also agreed completely with those of an unfractionated authentic ß-amylase preparation.
ABSTRACT
Assuming that the amount of superoxide radicals generated in vivo correlates with the production of ergastic substances such as storage proteins, the coordinated response of detoxication enzymes such as superoxide dismutases is largely exploited to understand the self-defense systems of plant. Here we examined expression of the genes for superoxide dismutases during seed development of soybean. The cDNAs encoding a cytosolic copper/zinc form and an iron form of the above enzyme have been cloned and then employed as probes, separately. Northern blotting results suggested that both superoxide dismutase mRNAs are expressed at the maximum level, preceding a developmental stage when mRNA encoding glycinin, soybean 11S-storage protein, at the maximum.
Subject(s)
Cloning, Molecular , Gene Expression Regulation, Plant , Glycine max/enzymology , Seeds/enzymology , Superoxide Dismutase/genetics , Amino Acid Sequence , Base Sequence , DNA Primers , Molecular Sequence Data , Seeds/genetics , Seeds/growth & development , Glycine max/geneticsABSTRACT
A soybean chitinase which has an apparent molecular mass of 28 kDa by SDS-PAGE, and has chitinase specific activity of 133 units per mg protein at pH 5.2 and an apparent pI of 5.7, was purified from mature dry seeds. Based upon the selected part (the residue positions 10-17) of the determined N-terminal 38 amino acid sequence, a 23-mer degenerate oligonucleotide was synthesized and used for the PCR cloning of the chitinase cDNA. The resulting 1340 bp cDNA was comprised of a 5'-untranslated region of 39 bases, a coding region corresponding to a 25 amino acid signal sequence, followed by a mature 308 amino acid sequence (calculated molecular mass 34,269, calculated pI 4.7), and a 235 nucleotide 3'-terminal untranslated region including 24 bases of the poly(A) tail. By comparing the deduced primary sequence with those of plant chitinases known to date, this enzyme was more than 50% identical to every class III acidic chitinase, but has no significant similarity to other families of chitinases. The comparison also showed that the C-terminal region of this chitinase is markedly extended, by at least 31 residues. Northern blot analysis demonstrated that this mRNA species is remarkably transcribed from the early stage until the late middle stage of seed development, whilst it is hardly expressed in the leaves and the stems of soybean. Spatial and temporal expression of this single gene imply that this class III chitinase is mainly devoted to the seed defense, not only in development but also in dormancy of soybean seed. This is the first reported isolation and cDNA cloning of a class III acidic endochitinase from seeds. According to the chitinase nomenclature we propose that this enzyme would be classified into a new class of chitinase PR-8 family, together with a Sesbania homologue.
Subject(s)
Chitinases/biosynthesis , Gene Expression Regulation, Plant , Glycine max/enzymology , Amino Acid Sequence , Base Sequence , Chitinases/chemistry , Chitinases/metabolism , Cloning, Molecular , Consensus Sequence , Gene Expression Regulation, Developmental , Gene Expression Regulation, Enzymologic , Kinetics , Molecular Sequence Data , Plant Proteins , Polymerase Chain Reaction , Recombinant Proteins/biosynthesis , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Seeds/enzymology , Sequence Alignment , Sequence Homology, Amino Acid , Glycine max/growth & developmentABSTRACT
Soybean beta-amylase, comprising a (beta/alpha)8-barrel core with a mobile loop, similar to that of triose phosphate isomerase, was mutated by site-directed mutagenesis at residues Glu380 and Leu383. X-ray crystallographic findings suggest that Glu380 is the counterpart of the catalytic site (Glu186) and that Leu383, located near the active-site cavity, forms an inclusion complex with cyclomaltohexaose. Separate substitutions of Glu380 by Gln and Asp completely eliminated the activity without inducing any significant changes in the circular dichroic spectra nor in the binding affinity for cyclomaltohexaose. Glu380, in cooperation with Glu186, therefore, is clearly indispensable for the liberation of beta-maltose from starch. Substitutions of Leu383 by Ile and Gln, in contrast, led to remarkable increases in the Km values of both mutants when compared to that of the non-mutant enzyme. The mutants also showed marked reductions in their binding affinities to cyclomaltohexaose. Overall, it would appear that the kcat/Km of soybean beta-amylase increases in proportion to the length of the substrate molecule, and depends also on the characteristics of the side chain of the residue at position 383. Leu383, therefore, may be important for both substrate penetration and subsequent retention at the active site. Based on the foregoing, we propose an action mechanism of soybean beta-amylase involving the interactions of three essential amino acid residues (Asp101, Glu186 and Glu380) in concert with Leu383, and assumed an indispensable role for Asp101.
Subject(s)
Glycine max/enzymology , alpha-Cyclodextrins , beta-Amylase/chemistry , beta-Amylase/metabolism , Amino Acid Sequence , Binding Sites/genetics , Circular Dichroism , Crystallography, X-Ray , Cyclodextrins/metabolism , Glutamic Acid/chemistry , Kinetics , Leucine/chemistry , Models, Molecular , Molecular Sequence Data , Mutagenesis, Site-Directed , Protein Conformation , Sequence Homology, Amino Acid , Glycine max/genetics , beta-Amylase/geneticsABSTRACT
A protein fraction was isolated from defatted soybean flour by extraction at acid pH, 40% ammonium sulfate precipitation, hydrophobic interaction chromatography, and gel filtration chromatography. SDS-PAGE, under reducing conditions, confirmed it as a homogeneous preparation. This conclusion was consistent with N-terminal amino acid sequence data (20 cycles) which showed a major sequence identical to those reported for soybean Bowman-Birk-type protease inhibitor (BBI), and also indicated a minimum 95% purity based on recoveries of PTH-amino acid residues. The purified fraction inhibited both trypsin and chymotrypsin with average specific activities of 350 and 672 units mg(-1), respectively. Compared with classical BBI purification, this procedure is very rapid requiring only 72-96 h to achieve a yield of 37 mg purified BBI per 200 g starting material.
Subject(s)
Trypsin Inhibitor, Bowman-Birk Soybean/isolation & purification , Amino Acid Sequence , Caseins/chemistry , Chromatography, Gel , Chymotrypsin/analysis , Electrophoresis, Polyacrylamide Gel , Molecular Sequence Data , Trypsin Inhibitor, Bowman-Birk Soybean/metabolismABSTRACT
In beta-escin-permeabilized cultured pig aortic smooth muscle cells GTP gamma S dose-dependently enhances Ca(2+)-induced, wortmannin-sensitive phosphorylation of 20 kDa myosin light chain (MLC20). GTP gamma S does not potentiate thiophosphorylation of MLC20, but does inhibit its dephosphorylation. Pretreatment with C. botulinum exotoxin C3, which specifically ADP-ribosylates and inactivates the rho family of the small molecular weight G proteins, completely abolishes the effects of GTP gamma S. These results indicate that rho is involved in the GTP gamma S-induced enhancement of Ca(2+)-dependent MLC20 phosphorylation in aortic smooth muscle cells, and strongly suggest that this effect of rho is due to inhibition of protein phosphatase activity toward MLC20.
Subject(s)
Guanosine 5'-O-(3-Thiotriphosphate)/pharmacology , Muscle, Smooth, Vascular/metabolism , Myosins/metabolism , Animals , Botulinum Toxins/pharmacology , Calcium/pharmacology , Cells, Cultured , Escin/pharmacology , GTP-Binding Proteins/metabolism , In Vitro Techniques , Phosphoprotein Phosphatases/antagonists & inhibitors , Phosphorylation , SwineABSTRACT
By reducing the amount of ginkgo water-soluble polysaccharides, which occupy about 35% of the wet seed mass and interfere with the extraction of RNA, cDNA-quality mRNA was obtained from developing seeds of Ginkgo biloba. Based on the NH2-terminal 17-amino acid sequence and an internal 12-amino acid sequence derived from the basic subunit of ginnacin, 11S-seed storage protein family of ginkgo, two degenerate oligonucleotide primers were synthesized and used for polymerase chain reaction (PCR). The resulting PCR product was used for screening the above endosperm cDNA library, and a plaque carrying the 1614 bp cDNA insert, which contained the entire coding region for a precursor of ginnacin was isolated. This is the first reported cloning of cDNA from ginkgo seeds. The deduced primary sequence is composed of a signal peptide segment (25 amino acid residues) and an acidic subunit (248 residues) followed by a basic subunit (187 residues). It was also found that the post-translational cleavage site in the ginnacin precursor is the Asn-Asn rather than the Asn-Gly bond found in a variety of the major subunit precursors in 11S seed protein family known to date. We showed that a purified soybean extract and an extract of ginkgo seeds can specifically hydrolyze -Asn248-Asn249- but not -Asn249-Val250-, in the heptapeptide Gly-Asn248-Asn-Val-Glu-Glu-Leu that corresponds to the ginnacin cleavage region.
Subject(s)
Asparagine , Plant Proteins/biosynthesis , Plants/metabolism , Protein Processing, Post-Translational , RNA, Messenger/metabolism , Seeds/metabolism , Amino Acid Sequence , Base Sequence , DNA Primers , Gene Library , Molecular Sequence Data , Oligopeptides/metabolism , Plants/enzymology , Polymerase Chain Reaction , Seeds/enzymology , Glycine max/enzymologyABSTRACT
The soybean embryo factor binding sequence in the glycinin A2B1a gene promoter was delimited to an A/T-rich 9 bp sequence, 5'-TAATAATTT-3', designated as the glycinin box, by DNA footprinting and gel mobility shift assay using synthetic oligonucleotides. It was shown that the interaction with the factor takes place at a defined DNA sequence rather than at random A/T-rich sequence blocks in the glycinin 5' flanking region. There are four glycinin boxes in the quantitative regulatory region between positions -545 and -378 of the glycinin A2B1a promoter. Multiple nonamer motifs similar to the glycinin box were also found in the equivalent regions of other glycinin and legumin promoters, suggesting that they must be conserved as a binding site for the embryo factor that activates the differential and stage-specific expression of seed 11S globulin genes in leguminous plants.
Subject(s)
Gene Expression Regulation , Genes, Plant , Globulins/genetics , Glycine max/genetics , Regulatory Sequences, Nucleic Acid , Base Sequence , Deoxyribonucleases , Molecular Sequence Data , Promoter Regions, Genetic , Seeds , Soybean ProteinsABSTRACT
To determine which amino acid residues are essential for the catalytic activity of soybean beta-amylase, deoxyoligonucleotide site-directed mutagenesis was employed against aspartyl, glutamyl, and cysteinyl residues located in highly conserved regions found in beta-amylase family to date. Both substitution of aspartic acid at position 101 and that of glutamic acid at position 186 of the enzyme by neutral and acidic amino acids, respectively, led to the complete elimination of activity, but did not induce any significant changes in circular dichroic spectra or the binding affinity for cyclomaltohexaose, a substrate analogue. Taking account of the results obtained here, the above two amino acid residues are involved in the catalytic site of soybean beta-amylase. The replacement of glutamic acid at position 345 decreased activity to below 6% of the non-mutant level, implying that this residue may also play a crucial role in beta-amylase activity, although it may not be involved at the catalytic site itself. In contrast, substitution of cysteinyl residue at position 95 by a serinyl residue led to a drastic reducing of the optimal temperature (from 50 degrees C to 30 degrees C), suggesting that this cysteinyl residue is responsible for the thermal stability of the enzyme.
Subject(s)
Glycine max/enzymology , beta-Amylase/chemistry , Amino Acid Sequence , Aspartic Acid/chemistry , Base Sequence , Binding Sites , Circular Dichroism , Cysteine/chemistry , Electrophoresis, Polyacrylamide Gel , Glutamine/chemistry , Hydrogen-Ion Concentration , Molecular Sequence Data , Mutagenesis, Site-Directed , Plasmids , Temperature , beta-Amylase/genetics , beta-Amylase/metabolismABSTRACT
Peptidylarginine deiminase is a protein-modulating enzyme which converts the arginine residues in proteins to citrulline residues. This study describes the complete primary structure of mouse peptidylarginine deiminase, which was deduced from nucleotide sequence analysis of cDNA clones plus proteochemical analysis of the purified enzyme. The composite cDNA sequence contained a 5' untranslated region of 7 bases, an open reading frame of 2019 bases that encoded 673 amino acids, a 3' untranslated region of 2662 bases, and part of a poly(A) tail. The N-terminal and C-terminal sequences of the enzyme matched the sequences deduced from nucleotide analysis. Furthermore, we determined that the N-terminal sequence was N alpha-acetyl-Met-Gln-, a sequence which has never previously been reported among N alpha-acetyl-Met proteins. The Arg 352 of the enzyme was converted to a citrulline residue and the potential Asn-linked glycosylation site (Asn542-Glu543-Ser544) had no carbohydrate moiety. Thus, mouse peptidylarginine deiminase consists of 673 amino acids with a molecular mass of 76,260. Mouse peptidylarginine deiminase mRNA has two AU-rich structures in the 3' untranslated region which exhibit a high degree of similarity to those in lymphokine, cytokine and proto-oncogene mRNA species. Since the rat enzyme (previously reported) does not possess these characteristic structures, we compared the levels of enzyme activity and mRNA in the mouse and rat uterus at four defined phases of the estrous cycle. The degradation of peptidylarginine deiminase and its mRNA proceeded significantly faster in the mouse than in the rat. We speculate that the unusual structure of the mouse enzyme and its mRNA be involved in this species-specific rapid degradation.
Subject(s)
Estrus/metabolism , Hydrolases/genetics , Introns , RNA, Messenger/genetics , Uterus/enzymology , Amino Acid Sequence , Animals , Base Sequence , DNA , Female , Mice , Molecular Sequence Data , Protein-Arginine Deiminase Type 4 , Protein-Arginine Deiminases , RNA, Messenger/metabolism , Rats , Rats, Wistar , Sequence Homology, Amino Acid , Sequence Homology, Nucleic AcidABSTRACT
This paper reports a novel method of affinity purification of soybean and barley beta-amylase on cyclomaltohexaose-immobilized Sepharose. Until now, it has been shown that sweet potato beta-amylase can be purified using the above absorbent but beta-amylases from soybean and barley seeds cannot. We found that soybean and barley beta-amylase becomes adsorbed specifically on the above absorbent if it is in solution with 1 to 2 M ammonium sulfate, and the adsorbed enzyme can be easily eluted with a buffer containing no ammonium sulfate. Employing this procedure, soybean beta-amylase was demonstrated to be purified about 10-fold to homogeneity as judged from analysis of both a sodium dodecyl sulfate-gel electrophoresis and the specific activity, using a crude enzyme preparation (sp act 95 U/mg) as a starting material. The specific activity of this highly purified enzyme (950 U/mg) was almost the same as that of crystallized soybean beta-amylase at 37 degrees C.
Subject(s)
Chromatography, Affinity/methods , Plants/enzymology , alpha-Cyclodextrins , beta-Amylase/isolation & purification , Ammonium Sulfate , Cyclodextrins , Evaluation Studies as Topic , Hordeum/enzymology , Sepharose , Glycine max/enzymologyABSTRACT
During seed embryogenesis, glycinin, the 11-S seed storage protein found in soybeans, undergoes post-translational proteolytic processing, in which a proprotein molecule is cleaved into an acidic and a basic subunit by a one-point cleavage that occurs at the carboxyl side of the asparaginyl residue located at the junction of the subunits. To elucidate the mechanism of this very limited proteolysis, we purified the cysteine endoprotease and used purified proglycinin produced by Escherichia coli as a substrate. This enzyme was separated by isoelectric focusing into three isomeric forms: two had a molecular mass of 33 kDa and the third, 33.8 kDa. The cysteine protease was found both in the proteinaceous vacuoles of cotyledonary tissue of immature seeds and in mature seeds, and is the first proteolytic enzyme to be classified as an asparagine-specific endoprotease. The results also indicate that the above proteolysis is largely attributable to the conformational accessibility of the enzyme to the asparaginyl residue in the cleavage site of proglycinin. The conformation of this single enzyme-accessible region on the proglycinin molecule is relatively flexible and becomes unstable under low salt conditions, or when heat is applied, causing the enzyme to lose its specificity.
Subject(s)
Cysteine Endopeptidases/isolation & purification , Globulins/metabolism , Plant Proteins, Dietary/metabolism , Protein Precursors/metabolism , Amino Acid Sequence , Asparagine/metabolism , Base Sequence , Cysteine Endopeptidases/chemistry , Molecular Sequence Data , Mutation , Protein Conformation , Soybean ProteinsABSTRACT
The cDNA clones corresponding to soybean beta-amylase mRNA were isolated and sequenced. The cDNA contained an open-reading frame composed of 496 amino acids. The comparison of the amino acid sequence deduced from the cDNA with the N-terminal peptide sequence from mature enzyme proved that beta-amylase had no leader sequence. Employing the cDNA, the beta-amylase was directly synthesized in Escherichia coli by the expression vector pKK233-2 controlled by the tac promoter. The enzyme activity detected in E. coli lysate drastically increased with a lower cultivation temperature, and the total activity and specific activity of the enzyme in E. coli lysate cultured at 13 degrees C was 130-fold and 280-fold, respectively, the value at 37 degrees C. The enzyme produced in E. coli was purified by the affinity column chromatography of cyclomaltohexaose-immobilized Sepharose 6B. Employing the established expression and purification system of the enzyme, the functional ionizable groups in the active site were searched. His93, involving an imidazole, and Asp348, involving a carboxylate, in the highly conserved regions within the beta-amylases were replaced by Arg (H93R) and Ash (D348N) by site-directed mutagenesis, respectively. All beta-amylases, including the non-mutant and mutant beta-amylases, produced in E. coli exhibited lower Vmax values than that of beta-amylase isolated conventionally from soybean seeds. Especially the Vmax value of [H93R]beta-amylase was reduced drastically compared to that of the non-mutant; however, none of them lost their enzyme activities completely. Therefore, neither His93 nor Asp348 may participate in the catalytic reaction directly.
Subject(s)
Genes, Plant/genetics , Glycine max/genetics , Mutation , alpha-Cyclodextrins , beta-Amylase/genetics , Amino Acid Sequence , Base Sequence , Binding Sites/genetics , Chromatography, Affinity , Cloning, Molecular , Cyclodextrins , Escherichia coli/genetics , Gene Expression , Molecular Sequence Data , Mutagenesis, Site-Directed , RNA, Messenger/genetics , Sepharose , Sequence Analysis, DNA , Sequence Homology, Amino Acid , beta-Amylase/biosynthesis , beta-Amylase/isolation & purificationABSTRACT
A 2.2 kb fragment containing the 5'-flanking region of the soybean glycinin A2B1a gene and its successive deletions with a shorter 5'-flanking sequence were fused, in frame, to the beta-glucuronidase (GUS) reporter gene. The resultant fusions were introduced into tobacco plants via Agrobacterium tumefaciens. Assays of the GUS activity in seeds of transgenic tobacco showed that the upstream region, -657 to -327 (relative to the transcription initiation site [+1]), of the glycinin gene is required for optimal expression of the transformed gene. Interactions between embryo nuclear factors and DNA fragments covering the downstream region of -326, in which are included the TATA box and legumin boxes, were not apparent. The embryo factors capable of binding specifically to three subregions, -653 to -527, -526 to -422, and -427 to -321, of the upstream regulatory region were detected. Such factors appeared to be organ-specific and could be found solely in developing seeds at the early middle stage of embryogenesis (around 24 days after flowering). Evidence obtained by characterizing the nature of the binding proteins and by gel mobility shift assays established that the same factor does interact with a consensus motif 5'-ATA/TATTTCN-/CTA-3' which occurs four times in the cis-acting regulatory region between -657 and -327. Moreover, this conserved motif could also be found in the 5' regulatory region of another glycinin A1aB1b gene. Thus it is likely that the observed interaction between the nuclear factor and the conserved motifs would lead to activation of transcription from the glycinin genes in maturing soybean seeds.
Subject(s)
DNA-Binding Proteins/metabolism , Globulins/genetics , Glycine max/genetics , Plant Proteins, Dietary/genetics , Plant Proteins/metabolism , Regulatory Sequences, Nucleic Acid , Base Sequence , Binding, Competitive , Cloning, Molecular , DNA , Gene Expression Regulation , Molecular Sequence Data , Oligodeoxyribonucleotides/chemical synthesis , Oligodeoxyribonucleotides/metabolism , Promoter Regions, Genetic , Restriction Mapping , Seeds/metabolism , Soybean Proteins , Glycine max/embryologyABSTRACT
Apolipoprotein E (apoE) has a high affinity to cell-surface low density lipoprotein (LDL) receptor. To determine the role of apoE in plasma lipoprotein metabolism, transgenic mouse lines with integrated rat apoE gene under the control of the metallothionein promoter were established. We found that a high expressor line produced rat apoE mainly in the liver, and the gene product was almost entirely associated with plasma lipoproteins. The plasma level of rat apoE in homozygotes for the transgene was 17.4 mg/dl after zinc induction (vs. 4.56 mg/dl of mouse apoE in controls). In this group, plasma cholesterol and triglyceride levels were 43% and 68% reduced as compared with controls, respectively. Heterozygotes showed decreases in both lipids to a lesser extent. Gel filtration chromatography showed that lipid reduction was mainly due to decreased very low density lipoproteins (VLDL) and LDL. Especially in zinc-treated homozygotes, VLDL had almost disappeared, and a remarkable decrease in LDL and a slight decrease in high density lipoprotein were also observed. Consistently, the plasma level of apoB, a structural protein of VLDL and LDL, was 78% lower than that of controls, indicating a marked reduction in lipoproteins containing apoB. Furthermore, the transgenic mice, in contrast to controls, did not develop hypercholesterolemia when fed a high cholesterol diet. These results demonstrated that overexpression of apoE reduces plasma cholesterol and triglyceride levels and prevents diet-induced hypercholesterolemia. From dramatic and dose-related decreases in plasma lipoproteins in transgenic mice, we conclude that apoE plays a key role in plasma lipoprotein metabolism.
Subject(s)
Apolipoproteins E/physiology , Hypercholesterolemia/prevention & control , Lipoproteins/blood , Animals , Cholesterol/blood , Diet , Gene Expression , Genes , Lipoproteins, HDL/blood , Mice , Mice, Transgenic , RNA, Messenger/genetics , Rats , Triglycerides/bloodABSTRACT
To investigate the role of apolipoprotein E (apo E) secreted by peripheral tissues in local lipoprotein metabolism, we developed a cell strain that constitutively produced and secreted apo E. A fusion plasmid containing rat apo E genomic DNA under control of mouse metallothionein promotor was constructed and transfected into Chinese hamster ovary cells. A stable transformant designated CHO-MAEII constitutively secreted rat apo E mainly in the form of sialylated free protein. The secretion was further enhanced by metal induction up to 1 micrograms apo E/ml per 12 h. When incubated with 125I-labeled very low density lipoprotein (125I-VLDL) at 37 degrees C, CHO-MAEII took up and degraded 125I-VLDL with higher affinity than control cells. Furthermore, considerable amount of methylated 125I-VLDL was degraded by CHO-MAEII, while no methylated 125I-VLDL was degraded by control cells. No significant differences were found in the uptake of 125I-LDL. The data indicated that apo E molecules secreted by CHO-MAEII were transferred to 125-VLDL particles, which caused a higher affinity of these particles for LDL receptors on the cells. It is suggested that apo E secreted from peripheral tissues enhances the uptake of lipoproteins by themselves or by surrounding cells in the local environment which demand cholesterol and express LDL receptors. CHO-MAEII was a good model for these 'auto- or paracrine-like functions' of apo E.