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Query: UNIPROT:P06889 (Mol)
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Bovine liver aminolaevulate dehydratase (ALA-D) has been chemically attached to Sepharose 4B and its properties have been studied. The optimal conditions for coupling have been determined. It was found that the immobilized enzyme retained a significant percentage of the activity of the free enzyme. The coupling yield was rather high. The insolubilized enzyme requires both anaerobiosis and a thiol activator for maximal activity. It can be stored at 4 degrees C for long periods with little loss of activity and it can be repeatedly used without alteration of its enzymic capacity. Attachment of ALA-D to the gel has led to an enhanced thermal stability. pH optima of free and bound enzyme was the same while a small decrease in the Km of the matrix bonded ALA-D as compared to that of the soluble enzyme was observed. The use of the fixed-ALA-D for the preparation of PBG is described.
Mol Cell Biochem 1977 Jul 05
PMID:Porphyrin biosynthesis. Immobilized enzymes. IV. Studies on aminolaevulate dehydratase attached to Sepharose. 1 69

It is shown that the kinetics of DNA despiralization in the presence of beta-alanine--formaldehyde reaction product (beta-ALA-FORM) can be described in terms of theory of DNA despiralisation by "slowly reacting agents". Conditions are determined in which beta-ALA-FORM product can be used to establish the concentration of defects in DNA secondary structure. Possible advantages are discussed of using the new agent in the kinetic method of determining DNA defects as compared to formaldehyde, in particular in analysis of DNA complexes with proteins. The data obtained throw some light on the nature of the interaction between beta-alanine and formaldehyde in slightly acidic solutions and with the excess of aminoacid.
Mol Biol (Mosk)
PMID:[Use of the reaction product of beta-alanine and formaldehyde in the kinetic method of determining defects in secondary structure]. 57 55

The Rhodobacter capsulatus hemA gene, coding for the enzyme delta-aminolevulinic acid synthase (ALAS), was isolated from a genome bank by hybridization with a hemT probe from Rhodobacter sphaeroides. Subcloning of the initial 3.9 kb HindIII fragment allowed the isolation of a 2.5 kb HindIII-BglII fragment which was able to complement the delta-aminolevulinic acid-requiring (ALA-requiring) Escherichia coli mutant SHSP19. DNA sequencing revealed an open reading frame coding for a protein with 401 amino acids which displayed similarity to the amino acid sequences of other known ALASs. However, no resemblance was seen to the HemA protein of E. coli K12. Based on the sequence data, an ALA-requiring mutant strain of R. capsulatus was constructed by site-directed insertion mutagenesis. Introduction of a plasmid, containing the hemA gene of R. capsulatus on the 3.9 kb HindIII fragment, restored ALA-independent growth of the mutant indicating that there is only one gene for ALA biosynthesis in R. capsulatus. Transfer of the R' factor pRPS404 and hybridization analysis revealed that the ALAS gene is not located within the major photosynthetic gene cluster.
Mol Gen Genet 1990 May
PMID:Cloning and sequencing of the hemA gene of Rhodobacter capsulatus and isolation of a delta-aminolevulinic acid-dependent mutant strain. 238 18

The hemB gene of Escherichia coli K12, coding for porphobilinogen synthase (PBG-S; syn., 5-aminolevulinic acid dehydratase, ALA-D), was cloned following insertion of an EcoRI fragment of plasmid F'13 into the mobilizable vector pCR1. The hybrid plasmid carrying the hemB gene was able to complement a hemB mutant of E. coli K12: not only was the PBG-S activity of the mutant restored after the acquisition of the hemB gene, but it was about ten times higher than that of the wild type. Subcloning of the original EcoRI fragment (14.6 kb) enabled us to locate the hemB gene on an NruI-HpaI fragment of about 1.1 kb. The hemB promoter was located toward the NruI end of the fragment, as shown by the use of the pKO promoter-probe series of vectors. Sequencing of the hemB gene indicated the presence of an open reading frame (ORF) of 1051 nucleotides, which should correspond to the HemB protein. Primer extension experiments enabled us to identify the 5' end of the hemB mRNA, and to deduce the -10 and -35 regions of the hemB promoter. Protein synthesis performed by an in vitro coupled transcription-translation system, showed the presence of a protein of about 35 kDa. This is in agreement with the molecular weight of the HemB protein (35.6 kDa), as deduced from the nucleotide sequence of the gene. Comparison of the amino acid sequences of E. coli and human PBG-S allowed the detection of several regions of strong homology between the two proteins. Two of these regions correspond, as expected, to the putative zinc-binding and catalytic sites of the human PBG-S.
Mol Gen Genet 1988 Nov
PMID:Nucleotide sequence of the hemB gene of Escherichia coli K12. 246 27

cDNA clones for the alad gene encoding the chlorophyll biosynthetic enzyme ALA dehydratase (ALAD) from Chlamydomonas reinhardtii were isolated by complementation of an Escherichia coli ALAD mutant (hemB). The C. reinhardtii alad gene encodes a protein that has 50 to 60% sequence identity with higher plant ALADs, and includes a putative Mg(2+)-binding domain characteristic of plant ALADs. Multiple classes of ALAD cDNAs were identified which varied in the length of their 3'-untranslated region. Genomic Southern analysis, using an ALAD cDNA as a probe, indicates that it is a single-copy gene. This suggests that the differently sized ALAD cDNAS are not the products of separate genes, but that a primary ALAD transcript is polyadenylated at multiple sites. A time course determination of ALAD mRNA levels in 12-h light:12-h dark synchronized cultures shows a 7-fold increase in ALAD mRNA at 2 h into the light phase. The ALAD mRNA level gradually declines but continues to be detectable up to the beginning of the dark phase. ALAD enzyme activity increases 3-fold by 6 h into the light phase and remains high through 10 h. Thus, there is an increase in both ALAD mRNA level and ALAD enzyme activity during the light phase, corresponding to the previously observed increase in the rate of chlorophyll accumulation.
Plant Mol Biol 1995 Feb
PMID:Structure and expression of the Chlamydomonas reinhardtii alad gene encoding the chlorophyll biosynthetic enzyme, delta-aminolevulinic acid dehydratase (porphobilinogen synthase). 789 23

The gsa gene, which encodes glutamate 1-semialdehyde (GSA) aminotransferase (GSAT), an enzyme in the chlorophyll and heme biosynthetic pathway, has been cloned from Chlamydomonas reinhardtii by complementation of an Escherichia coli hemL mutant. The deduced C. reinhardtii GSAT amino acid sequence has a high degree of similarity to GSAT sequences from barley, tobacco, soybean and various prokaryotic sources. In vitro enzyme activity assays from E. coli transformed with the C. reinhardtii GSAT cDNA showed that higher levels of GSAT activity are associated with the expression of the cDNA insert. Analysis of changes in mRNA levels in light:dark synchronized C. reinhardtii cultures was done by northern blotting. The level of GSAT mRNA nearly doubled during the first 0.5 h in the light and increased over 26-fold after 2 h in the light. This increase is comparable to previously reported increases in GSAT activity in dark-grown cultures transferred to the light, and is the first report of induction by light of a gene encoding an ALA biosynthetic enzyme in plant or algal cells. The accumulation of GSAT mRNA follows the pattern of chlorophyll accumulation and the pattern of chlorophyll a/b-binding protein (cabII-1) mRNA accumulation in these cells, suggesting that the two genes may be regulated by light through a common mechanism. Additional evidence that the GSAT mRNA may be transcriptionally regulated by light is found in the genomic sequence of the gsa gene.(ABSTRACT TRUNCATED AT 250 WORDS)
Plant Mol Biol 1994 Feb
PMID:Structure and light-regulated expression of the gsa gene encoding the chlorophyll biosynthetic enzyme, glutamate 1-semialdehyde aminotransferase, in Chlamydomonas reinhardtii. 815 81

Alpha tubulin isotypes are encoded by at least four genes designated alpha-1 to alpha-4 in the nematode Caenorhabditis elegans. We describe here, molecular cloning of the alpha-2 tubulin gene, located on chromosome I, that encodes a protein of 449 amino acids that has high homology to human, mouse and Drosophila alpha tubulins, but relatively lower homology to the yeast alpha tubulins. The alpha-2 tubulin gene is trans-spliced to the SL1 leader sequence. Northern analysis shows that the gene is increasingly transcribed during the early (L1-L3) larval stages but has a lower level of transcription in L4 L4 larvae, adults, and embryos. Using an alpha-2-lacZ fusion gene expression in transgenic animals, we show that the gene is expressed in a tissue-specific manner in the intestine, pharyngeal muscle cells, and a subset of neurons which include a class of DB and VB motor neurons in the ventral nerve cord, posterior touch receptor neurons, PLML, PLMR, in the lumbar ganglia; PVT in the pre-anal ganglion, and ALA in the dorsal ganglion in the head. Our results support the notion that tubulin structure may contribute to the functional specialization of microtubules.
J Mol Biol 1993 Dec 20
PMID:Molecular cloning and developmental expression of the alpha-2 tubulin gene of Caenorhabditis elegans. 826 34

5-Aminolevulinic acid (ALA) is the universal precursor of tetrapyrroles (e.g., chlorophylls and hemes). In the chloroplasts of plants and in several eubacterial species ALA is formed in a two-step process known as the C5 pathway. In the first step, glutamyl-tRNA reductase (GluTR), converts glutamate of glutamyl-tRNA to glutamate 1-semialdehyde (GSA) which is rearranged to ALA by glutamate 1-semialdehyde-2,1-aminomutase (GSA-A) in the second step. Since ALA formation is a limiting step in chlorophyll biosynthesis, GluTR, which is encoded by the HEMA gene in Arabidopsis thaliana plays a vital role in that biosynthesis. Here we report the occurrence of a second functional HEMA gene (HEMA2) in A. thaliana. This gene was isolated by screening a genomic library with a probe from HEMA1. The nucleotide sequence of the cDNA and the corresponding genomic DNA indicates that the Arabidopsis HEMA2 gene contains two short introns (285 bp and 159 bp). The deduced amino acid sequence predicts a HEMA2 protein of 530 amino acids with 79% identity to the HEMA1-encoded GluTR. The 5'-flanking sequence of the HEMA2 gene includes several motifs (e.g., GT-1 boxes, GATA motifs) similar to light-responsive regulatory elements found in light-inducible genes. Unlike the HEMA1 transcript, which is present in all parts of the plant, HEMA2 is expressed in low levels in roots and flowers. The presence of a second functional HEMA gene in Arabidopsis raises the possibility that two C5 pathways exist in chloroplasts.
Plant Mol Biol 1996 Feb
PMID:A second and differentially expressed glutamyl-tRNA reductase gene from Arabidopsis thaliana. 860 95

Heme biosynthesis was studied in the segregants of Saccharomyces cerevisiae (DW10 tetrade 2) from D27 and D27/C6 mating, as a function of the carbon source in the growth medium and the physiological state of the cells. The effects of the HEM R+ gene on the 5-aminolevulinate synthase (ALA-S) and 5-aminolevulinate dehydratase (ALA-D) activities of heme biosynthesis in cells grown on nonfermentable and fermentable carbon sources were compared. Profiles obtained for both strains grown on a fermentable carbon source (glucose) were identical. However, in the presence of a nonfermentable carbon source (ethanol), they behave quite different, as if the mutation could only be expressed under these growth conditions. Moreover, their behavior is similar to that found for the parental strains, indicating that for the mutant its particular behavior might be inheritedly linked to the HEM R+ gene, which in turn affects some regulatory aspects of ALA synthesis explaining its characteristic phenotype.
Comp Biochem Physiol B Biochem Mol Biol 1996 Oct
PMID:Porphyrin biosynthesis in normal and haem mutants of Saccharomyces cerevisiae. Studies on the inheritance of the HEM R+ phenotype. 893 97

The significantly increased concentrations of granulocyte manganese in subjects with AIP may be an indication of overexpression of manganese-associated enzymes. In this study we present further observations related to this phenomenon and speculate that this may provide a rational basis for hypotheses attempting to explain the pathogenesis of the acute attack of porphyria. Such hypotheses are advanced with regard to pyruvate carboxylase, mitochondrial superoxide dismutase and glutamine synthetase, three manganese-dependent enzymes associated with either ALA-generating or ALA-dependent processes. The metabolic impacts in acute porphyria of these enzymes would be functions of the current energy charge of the organism, and would thus explain the protecting and ameliorating effects of glucose in these conditions. Although granulocytes from AIP subjects have elevated manganese concentrations, this did not appear to be associated with increased activities of two enzymes assayed, pyruvate carboxylase or mitochondrial superoxide dismutase. However, enzyme activities in white blood cells do not necessarily represent the levels of catalytic activity in cell types involved in the phenotypic expression of porphyria. Thus it proposed that hypotheses along these new lines of thinking are not flawed by the apparently missing correlations, and should not be therefore discarded. The possible roles of manganese-associated enzymes in the mechanisms behind the acute porphyric attack are discussed in some detail in the paper.
Cell Mol Biol (Noisy-le-grand) 1997 Feb
PMID:Pathogenic mechanisms of the acute porphyric attack: speculative roles of manganese associated enzymes. 907 84


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