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K Izui

Publications and source records attributed to K Izui.

At least 19 recordsLinked to original sources
PubMed1992

A Ca(2+)-dependent protein kinase phosphorylates phosphoenolpyruvate carboxylase in maize.

In C4 plants the activity of phosphoenolpyruvate carboxylase (PEPC; EC 4.1.1.31) is regulated by phosphorylation/dephosphorylation which is mediated by light/dark signals. The study using protein kinase inhibitors showed that the inhibition pattern of maize PEPC-protein kinase (PEPC-PK) is similar to that of myosin light chain kinase, a Ca(2+)-calmodulin-dependent PK. The kinase activity was also inhibited by EGTA and the inhibition was relieved by Ca2+. These results suggest that PEPC-PK is Ca(2+)-dependent in contrast with previous observations by other research groups.

Calcium↗
PubMed1992

MNF1, a leaf tissue-specific DNA-binding protein of maize, interacts with the cauliflower mosaic virus 35S promoter as well as the C4 photosynthetic phosphoenolpyruvate carboxylase gene promoter.

When gel shift assays were performed with maize nuclear extract and a DNA fragment containing the cauliflower mosaic virus (CaMV) 35S promoter, three DNA-protein complexes were observed. Analyses with nuclear extracts prepared from green leaves, etiolated leaves, stems and roots showed that the complexes resulted from the existence of at least two nuclear factors. One of them is presumably a constitutive nuclear factor found in all tissues tested, and another is a leaf-specific factor present both in green and etiolated leaves. This leaf-specific nuclear factor seemed to be identical to MNF1, previously identified as a factor interacting with the promoter of the maize gene for phosphoenolpyruvate carboxylase involved in the C4 photosynthesis. Deletion analysis revealed that MNF1 binds to the sequence from -281 to -235 relative to the transcription start site of the CaMV 35S promoter. MNF1-like nuclear protein was also found in tobacco nuclear extracts. The possibility that MNF1 participates as a positive trans-acting factor in the expression of genes in maize leaves is discussed.

Base Sequence↗
PubMed1992

Molecular evolution of phosphoenolpyruvate carboxylase for C4 photosynthesis in maize: comparison of its cDNA sequence with a newly isolated cDNA encoding an isozyme involved in the anaplerotic function.

Molecular events associated with the evolution of an enzyme for C4 photosynthesis were investigated. In maize, at least three isozymes of phosphoenolpyruvate carboxylase [EC 4.1.1.31] are known: the C4-form, the C2-form and the root-form, being named according to their physiological roles and pattern of tissue distribution [Ting, I.P. & Osmond, C.B. (1973) Plant Physiol. 51, 448-453]. A cDNA clone which presumably encodes the C3-form isozyme was newly isolated and analyzed. Comparison of the sequences of the C3-form and C4-form isozymes revealed that (i) the homologies in the nucleotide and deduced amino acid sequences were 71 and 77%, respectively, and (ii) the gene for the C4-form isozyme evolved under strong G/C pressure. The genes for these isozymes were found to be located apart on different chromosomes. A phylogenetic tree was constructed using 8 amino acid sequences of phosphoenolpyruvate carboxylases from various sources. The topology of the tree indicated that, at least in monocots such as maize and sorghum, the genes for the C4-form and C3-form isozymes diverged from their common ancestral gene earlier than the monocot-dicot divergence (about 2 x 10(8) yr ago), though the divergence of maize (C4 plant) from wheat (C3 plant) is supposed to have occurred much later (6 x 10(7) yr ago).

Amino Acid Sequence↗
PubMed1991

Site-directed mutagenesis of the conserved histidine residue of phosphoenolpyruvate carboxylase. His138 is essential for the second partial reaction.

Histidine residues have previously been suggested to be essential for the activity of phosphoenolpyruvate carboxylase as demonstrated by chemical modification of these residues. Although the location of these residues on the primary structure is not known, a comparison of nine phosphoenolpyruvate (P-pyruvate) carboxylases sequenced recently revealed that there are only two conserved histidine residues (His138 and His579, coordinates from the E. coli enzyme). Site-directed mutagenesis of these residues were undertaken with the E. coli P-pyruvate carboxylase and the properties of purified mutant enzymes were investigated. Mutation of His138 to asparagine (H138N) produced a protein which did not show carboxylase activity. However, this mutant enzyme catalyzed the bicarbonate-dependent dephosphorylation (Vmax = 1.4 mumol.min-1.mg-1) of the P-pyruvate. Since this reaction is due to one of the two partial reactions proposed for this enzyme, the results indicate that His138 is obligatory for the second-step reaction, i.e. the carboxylation of the enolate form of pyruvate by carboxyphosphate. Mutation of His579 to asparagine (H579N) produced an enzyme which had 69% of the wild-type carboxylase activity, but its affinity for P-pyruvate was decreased by 24-fold.

Amino Acid Sequence↗
PubMed1991

Site-directed mutagenesis of phosphoenolpyruvate carboxylase from E. coli: the role of His579 in the catalytic and regulatory functions.

Phosphoenolpyruvate carboxylases (PEPC) [EC 4.1.1.31] from a wide variety of organisms contain a unique and highly conserved sequence, 578FHGRGGSIGRGGAP591 (coordinates for the Escherichia coli enzyme), which has been presumed to participate in the binding of phosphoenolpyruvate (PEP). Since previous chemical modification studies had suggested the importance of His for the catalytic activity, the role of His579 was investigated by constructing variants of E. coli PEPC, in which this residue was substituted to Asn (H579N) or Pro (H579P). Kinetic studies with partially purified enzymes revealed the following: (1) The apparent maximal velocities in the presence of acetyl-CoA (CoASAc, one of the allosteric activators) were 29% and 5.4% of the wild-type enzyme, for H579N and H579P, respectively. (2) The half-saturation concentration for PEP was increased about 40-fold by the substitutions, while those for another substrate (HCO3-) and the metal cofactor (Mg2+) were increased only 2- to 4-fold. (3) The half-saturation concentrations of four kinds of allosteric activators and of dioxane, an artificial activator, were also changed to various extents. Among them the most remarkable increase was observed for CoASAc (28-fold). (4) The concentration of an allosteric inhibitor, aspartate, required for 50% inhibition remained substantially unchanged. It was concluded that the imidazole group of His579 is not obligatory for the enzyme catalysis, but plays important roles in catalytic and regulatory functions.

Amino Acid Sequence↗
PubMed1990

Multiple interactions between tissue-specific nuclear proteins and the promoter of the phosphoenolpyruvate carboxylase gene for C4 photosynthesis in Zea mays.

The expression of the phosphoenolpyruvate carboxylase (PEPC) gene involved in C4 photosynthesis is regulated in a highly organized manner. Nuclear factors interacting with DNA fragments from the 5' flanking region (from positions -1012 to +88 relative to the transcription start site) of the maize gene were identified by gel shift assays. Among the three kinds of such nuclear proteins (MNF1, MNF2a and MNF2b) found in the extract from maize leaves, MNF2a and MNF2b, which were distinguishable by their chromatographic behavior, interacted with the same motif of the repeated sequence (RS2) in the region from -432 to -201. MNF1 interacted with the region from -905 to -818 in which two copies of another kind of repeated sequence (RS1) reside. All of these nuclear factors were found only in the extracts from green and etiolated leaves but not in those from stems and roots. The relative content of MNF1 and MNF2b was almost equal in green and etiolated leaves, while that of MNF2a was significantly higher in etiolated leaves than green leaves. It is suggested that expression of the PEPC gene is controlled by the combined effects of these nuclear factors.

Base Sequence↗
PubMed1990

Phosphoenolpyruvate carboxylase prevalent in maize roots: isolation of a cDNA clone and its use for analyses of the gene and gene expression.

In search of the gene family for phosphoenolpyruvate carboxylase (PEPC) [EC 4.1.1.31] in C4 plants, we isolated from a maize root cDNA library a new cDNA clone that cross-hybridized with a cDNA for PEPC involved in the C4 photosynthesis (Yanagisawa et al. (1988) FEBS Lett. 229, 107-110). Alignment of the nucleotide sequence with that of the probe cDNA revealed the absence of sequence homology in the 3' non-coding region between the two cDNAs. Southern blot hybridization probed with this specific sequence indicated that the corresponding gene is unique in the maize genome. Northern blot hybridization using the same probe showed that this gene-family member was expressed most strongly in roots and also in green leaves to a lesser extent, but not significantly in etiolated leaves.

Base Sequence↗
PubMed1989

First crystallization of a phosphoenolpyruvate carboxylase from Escherichia coli.

Two different forms of crystal for a phosphoenolpyruvate carboxylase from Escherichia coli were obtained by the hanging-drop vapor diffusion technique, using polyethylene glycol 4000 as precipitant. The hexagonal crystal in space group P6(2)22 (or P6(4)22) has cell dimensions of a = 131 A and c = 325 A, whereas the orthorhombic crystal in space group I222 has a = 119 A, b = 252 A and c = 83 A. A tetrameric molecule (396,244 Mr), a subunit of which contains 883 amino residues, has a crystallographic 2 symmetry in the hexagonal crystal or 222 symmetry in the orthorhombic crystal, respectively.

Carboxy-Lyases↗
PubMed1989

Maize phosphoenolpyruvate carboxylase involved in C4 photosynthesis: nucleotide sequence analysis of the 5' flanking region of the gene.

To clone the genomic DNA fragment containing the putative promoter region of the gene for phosphoenolpyruvate carboxylase [EC 4.1.1.31] involved in C4 photosynthesis (C4-type PEPC), maize genomic libraries were screened. On probing with a 384-bp fragment from the N-terminal coding region of the maize cDNA for C4-type PEPC, four EcoRI-fragments differing in the restriction map were cloned, reflecting the presence of a small gene family. Southern blot analyses were carried out on the genomic DNA and the cloned DNA fragments using several segments of the cDNA for C4-type PEPC as probes. The results indicated that the C4-type PEPC is encoded by a single gene and the cloned 7.0-kb fragment was derived from this gene. The transcription start site, as determined in a primer extension experiment, was located at about 4 kb downstream of the 5' end of the cloned fragment. The nucleotide sequence was determined for the region which extended about 1 kb upstream from the transcription start site and possible signal sequences related to gene expression were found, including four classes of direct repeats. The sequences of the corresponding regions of the other two cloned fragments (8.9 and 12.9 kb) which strongly hybridized with the 384-bp probe were very similar to each other, but they were quite different in the 5' upstream region from the sequence of the gene for C4-type PEPC.

Base Sequence↗
PubMed1988

Further analysis of cDNA clones for maize phosphoenolpyruvate carboxylase involved in C4 photosynthesis. Nucleotide sequence of entire open reading frame and evidence for polyadenylation of mRNA at multiple sites in vivo.

Four clones of cDNA for phosphoenolpyruvate carboxylase [EC 4.1.1.31] were obtained from a maize green leaf cDNA library by colony hybridization. The largest cDNA was of full-length (3335 nucleotides), being 243 nucleotides longer than the cDNA cloned previously [(1986) Nucleic Acids Res. 14, 1615-1628]. Alignment of the sequence for the N-terminal coding region found in two of the four clones with the sequence reported previously, established the sequence of the entire coding region for the enzyme. The sequencing of 3'-untranslated region of the clones revealed that the poly(A) tract is attached at multiple sites in vivo.

Base Sequence↗
PubMed1987

Preparation and characterization of monoclonal antibodies against phosphoenolpyruvate carboxylase of Escherichia coli.

Twelve hybridoma clones which secrete monoclonal antibodies (mAb) against purified phosphoenolpyruvate carboxylase [EC 4.1.1.31] from Escherichia coli K-12 were obtained. These 12 mAb were prepared from the ascites fluids of mice. Six among the 12 mAb formed precipitin lines with the enzyme on immunodiffusion. Four mAb inhibited the activity of the enzyme and 2 mAb enhanced it. Four mAb altered the sensitivity of the enzyme to allosteric effectors. Competitive enzyme-binding experiments among the 12 different mAb were also performed. The results showed that the 12 mAb can be classified into at least 8 groups.

Allosteric Regulation↗
PubMed1986

Cloning and sequence analysis of cDNA encoding active phosphoenolpyruvate carboxylase of the C4-pathway from maize.

A recombinant clone, pM52, containing cDNA for maize phosphoenolpyruvate carboxylase (PEPCase, EC 4.1.1.31) was isolated from a maize leaf cDNA library constructed using an expression vector in Escherichia coli. The screening of the clone was conveniently performed through its ability to complement the phenotype (glutamate requirement) of PEPCase-negative mutant of E. coli. The enzyme encoded by this clone was identical with the major PEPCase in maize, a key enzyme in the C4-pathway, as judged from its allosteric properties and immunological reactivity. The cloned cDNA (3093 nucleotides in length) contained an open reading frame of 2805 nucleotides, the 3'-untranslated region of 222 nucleotides and the poly(dA) tract of 64 nucleotides. The deduced amino acid sequence (935 residues) of the enzyme showed higher homology with that of an enterobacterium, E. coli (43%) than that of a cyanobacterium (blue-green alga), Anacystis nidulans (33%).

Amino Acid Sequence↗
PubMed1986

Phosphoenolpyruvate carboxylase of Escherichia coli K-12. N- and C-terminal sequences and tentative assignment of the catalytically essential cysteine residue.

The N- and C-terminal amino acid sequences of phosphoenolpyruvate carboxylase [EC 4.1.1.31] from Escherichia coli K-12 were determined to establish the primary structure deduced from the nucleotide sequence of the cloned gene for the enzyme (Fujita, N., Miwa, T., Ishijima, S., Izui, K., & Katsuki, H. (1984) J. Biochem. 95, 909-916). As predicted from the nucleotide sequence, two polypeptides were produced upon treatment with hydroxylamine, which specifically cleaves the Asn-Gly bond, and their amino acid compositions were also in accordance with those predicted. The tryptic peptides which contained cysteine residues labeled with a fluorescent reagent, N-[7-(dimethylamino)-4-methylcoumarinyl]maleimide, were isolated by high-performance liquid chromatography and partially sequenced. All of them could be assigned on the deduced primary structure. The modified cysteine residues were Cys-157, Cys-385, Cys-458, Cys-568, Cys-665, and Cys-754. Furthermore, the essential cysteine residue which is presumably located at or near the active site was tentatively identified as Cys-568, since it was consistently protected against the modification by 2-phospholactate, a substrate analog.

Amino Acid Sequence↗
PubMed1985

Comparison of amino acid sequences between phosphoenolpyruvate carboxylases from Escherichia coli (allosteric) and Anacystis nidulans (non-allosteric): identification of conserved and variable regions.

Amino acid sequences of phosphoenolpyruvate carboxylases of Escherichia coli (allosteric) and a cyanobacterium Anacystis nidulans (non-allosteric) were aligned. The pattern of homology suggests that the enzyme molecule is comprised of two distinct regions, namely, a conserved region (C-terminal half) and a variable region (N-terminal half). Among the amino acid residues which have previously been presumed essential for the catalytic activity, three histidine residues were found to be conserved, but cysteine residues were not. Furthermore, the conserved sequence unique to the enzyme was identified by comparison of the enzyme sequence with amino acid sequences in our data bank.

Allosteric Regulation↗
PubMed1985

Promoter analysis of the phosphoenolpyruvate carboxylase gene of Escherichia coli.

In order to find the promoter region of phosphoenolpyruvate carboxylase [EC 4.1.1.31] gene (ppc), in vitro transcription was performed using truncated DNA fragments as templates. Transcription mapping showed three promoters as candidates, but only one of them could be assigned to the promoter of ppc gene, considering the nucleotide sequence of its coding region (Fujita, N., Miwa, T., Ishijima, S., Izui, K. and Katsuki, H. (1984) J. Biochem. 95, 909-916). Nuclease S1 mapping showed that the in vivo and in vitro transcription initiation sites are identical and that the site lies 91 or 92 nucleotides upstream the translation initiation site. No alteration of the transcription initiation site was observed whether the cells were starved for an amino acid or grown on various carbon sources. The sequences of the -10 and -35 regions were fairly in accordance with the consensus sequences hitherto reported. Some features of the sequence around the promoter region were discussed.

Base Sequence↗