Alas1 is essential for neutrophil maturation in zebrafish.

Neutrophils play essential roles in innate immunity and are the first responders to kill foreign micro-organisms, a function that partially depends on their granule content. The complicated regulatory network of neutrophil development and maturation remains largely unknown. Here we utilized neutrophil-deficient zebrafish to identify a novel role of Alas1, a heme biosynthesis pathway enzyme, in neutrophil development. We showed that Alas1-deficient zebrafish exhibited proper neutrophil initiation, but further neutrophil maturation was blocked due to heme deficiency, with lipid storage and granule formation deficiencies, and loss of heme-dependent granule protein activities. Consequently, Alas1-deficient zebrafish showed impaired bactericidal ability and augmented inflammatory responses when challenged with Escherichia coli These findings demonstrate the important role of Alas1 in regulating neutrophil maturation and physiological function through the heme. Our study provides an in vivo model of Alas1 deficiency and may be useful to evaluate the progression of heme-related disorders in order to facilitate the development of drugs and treatment strategies for these diseases.

Involvement of delta-aminolaevulinate synthase encoded by the parasite gene in de novo haem synthesis by Plasmodium falciparum.

The malaria parasite can synthesize haem de novo. In the present study, the expression of the parasite gene for delta-aminolaevulinate synthase (Pf ALAS ) has been studied by reverse transcriptase PCR analysis of the mRNA, protein expression using antibodies to the recombinant protein expressed in Escherichia coli and assay of ALAS enzyme activity in Plasmodium falciparum in culture. The gene is expressed through all stages of intra-erythrocytic parasite growth, with a small increase during the trophozoite stage. Antibodies to the erythrocyte ALAS do not cross-react with the parasite enzyme and vice versa. The recombinant enzyme activity is inhibited by ethanolamine and the latter inhibits haem synthesis in P. falciparum and growth in culture. The parasite ALAS is localized in the mitochondrion and its import into mitochondria in a cell-free import assay has been demonstrated. The import is blocked by haemin. On the basis of these results, the following conclusions are arrived at: PfALAS has distinct immunological identity and inhibitor specificity and is therefore a drug target. The malaria parasite synthesizes haem through the mitochondrion/cytosol partnership, and this assumes significance in light of the presence of apicoplasts in the parasite that may be capable of independent haem synthesis. The Pf ALAS gene is functional and vital for parasite haem synthesis and parasite survival.

Characterization of the rhodobacter sphaeroides 5-aminolaevulinic acid synthase isoenzymes, HemA and HemT, isolated from recombinant Escherichia coli.

The hemA and hemT genes encoding 5-aminolaevulinic acid synthase (ALAS) from the photosynthetic bacterium Rhodobacter sphaeroides, were cloned to allow high expression in Escherichia coli. Both HemA and HemT appeared to be active in vivo as plasmids carrying the respective genes complemented an E. coli hemA strain (glutamyl-tRNA reductase deficient). The over-expressed isoenzymes were isolated and purified to homogeneity. Isolated HemA was soluble and catalytically active whereas HemT was largely insoluble and failed to show any activity ex vivo. Pure HemA was recovered in yields of 5-7 mg x L-1 of starting bacterial culture and pure HemT at 10 mg x L-1 x HemA has a final specific activity of 13 U x mg-1 with 1 unit defined as 1 micromol of 5-aminolaevulinic acid formed per hour at 37 degrees C. The Km values for HemA are 1.9 mM for glycine and 17 microM for succinyl-CoA, with the enzyme showing a turnover number of 430 h-1. In common with other ALASs the recombinant R. sphaeroides HemA requires pyridoxal 5'-phosphate (PLP) as a cofactor for catalysis. Removal of this cofactor resulted in inactive apo-ALAS. Similarly, reduction of the HemA-PLP complex using sodium borohydride led to > 90% inactivation of the enzyme. Ultraviolet-visible spectroscopy with HemA suggested the presence of an aldimine linkage between the enzyme and pyridoxal 5'-phosphate that was not observed when HemT was incubated with the cofactor. HemA was found to be sensitive to reagents that modify histidine, arginine and cysteine amino acid residues and the enzyme was also highly sensitive to tryptic cleavage between Arg151 and Ser152 in the presence or absence of PLP and substrates. Antibodies were raised to both HemA and HemT but the respective antisera were not only found to bind both enzymes but also to cross-react with mouse ALAS, indicating that all of the proteins have conserved epitopes.

delta-Aminolevulinate synthase isozymes in the liver and erythroid cells of chicken.

Antibodies raised against the purified chicken liver delta-aminolevulinate synthase showed a partial cross-reactivity with the chicken erythroid delta-aminolevulinate synthase. delta-Aminolevulinate synthase synthesized in vitro using polysomes from erythroid cells showed a subunit molecular weight of 55,000, whereas the enzyme synthesized in vitro using liver polysomes had a subunit molecular weight of 73,000. delta-Aminolevulinate synthase isolated from mitochondria of erythroid cells showed a molecular weight of 53,000, while the enzyme in liver mitochondria had a value of 65,000. These observations imply that the erythroid delta-aminolevulinate synthase differs from the hepatic enzyme.

Delta-Aminolevulinic acid synthase from chick embryo liver mitochondria. II. Immunochemical correlation between synthesis and activity in induction and repression.

A specific rabbit antibody was prepared against chick embryo liver mitochondrial delta-aminolevulinic acid synthase (EC 2.3.1.37) and used to quantitate the amount of enzyme present in liver mitochondria from normal and drug-treated chick embryos. When increases in enzyme activity were produced by the drugs 2-allylisopropylacetamide, or 1,4-dihydro-3,5-dicarbethoxycollidine, quantitative immunotitrations and the Laurell electroimmunoassay showed proportional increases occurred in the amount of enzyme antigen. Conversely, decreases of induced enzyme activity produced by hemin were accompanied by corresponding decreases in enzyme antigen. The relative rate of synthesis of delta-aminolevulinic acid synthase was measured by pulse-labeling of liver proteins with L-[4,5-3H]leucine. delta-Aminolevulinic acid synthase was isolated by quantitative immunoprecipitation followed by electrophoresis of the dissolved immunoprecipitate on a sodium dodecyl sulfate-polyacrylamide gel. In normal and drug-treated chick embryo liver mitochondria, enzyme activity was closely correlated with the relative rate of enzyme synthesis. When a 360-fold increase in enzyme activity was produced in vivo by a combination of 2-allylisopropylacetamide and 1,4-dihydro-3,5-dicarbethoxycollidine in 12 hours, there was a 500-fold increase in relative synthesis, such that delta-aminolevulinic acid synthase constituted over 1% of the total intracellular protein synthesis and over 3% of the total labeled protein in liver mitochondria. Hemin administered after inducing chemicals was able to completely block the induced synthesis of delta-aminolevulinc acid synthase with no significant change in general protein synthesis. Mitochondria from untreated chick embryo livers contain delta-aminolevulinic acid synthase that migrates on sodium dodecyl sulfate gels identically with the induced enzyme.