Purification and characterization of {delta}-aminolevulinic acid dehydratase from Chlorella regularis

-Aminolevulinic acid dehydratase (-aminolevulinic acid hydrolyaseEC 4.2.1.24 [EC] ) which catalyzes the formation ofporphobilinogenfrom two molecules of -aminolevulinic acid (ALA) was purifiedfrom Chlorella regularis 737-fold by acetone and ammonium sulfatefractionations, DEAE-cellulose column chromatography, and SephadexG-200 gel filtration. The enzyme had an optimum pH of 8.5 inTris-HCl buffer and required either Mg²⁺ or Mn²⁺ for its maximumactivity. The Km values for Mg²⁺, Mn²⁺ and ALA were 15 µM,10µM, and 0.5 mM, respectively. The enzyme was not activatedby thiol compounds, but was inhibited by p-chloromercuribenzoate.The molecular weight estimated by gel filtration was 316,000and the isoelectric point was 5.25. (Received October 18, 1978; )     

Purification and characterization of 5-aminolevulinic acid dehydratase from Methanosarcina barken

Abstract 5-Aminolevulinic acid dehydratase from the archaebacterium Methanosarcina barken resembles the mammalian and yeast enzymes in its activation by Zn²⁺, whereas its activation by K⁺ resembles the characteristic of bacterial enzymes. This enzyme is activated with Ni²⁺ which is a component of F₄₃₀, a cofactor present mainly in methanogens. The M _r of 280000 for the native enzyme and 30 000 ± 2000 for the individual subunit suggest that the enzyme is composed of eight apparently indentical subunits similar to mammalian and yeast enzymes. The enzyme has two pH optima, at 8.5 and 9.4. Higher levels of 5-aminolevulinic acid dehydratase in acetate-grown cells suggest the possibility that regulation and control of this enzyme could be different on various growth substrates.     

Studies on {partial}-amino levulinic acid deydratase in radish cotyledons during chloroplast development

The subcellular localization and biosynthetic site of 8-aminolevulinic acid dehydratase [EC 4.2.1.24 [EC] , ALAD] were investigatedin relation to chloroplast development in radish cotyledons. ALAD was mainly located in the chloroplasts and cytoplasm. Mostof the ALAD in the chloroplasts was readily released by hypotonicshock. The enzyme was also found in the proplastids of etiolatedcotyledons. The normal increase in the activity of ALAD in the chloroplastsas well as the cytoplasm was inhibited by cycloheximide butunaffected by D-threo chloramphenicol and kanamycin during thegreening of radish cotyledons. We concluded that the ALAD inboth the cytoplasm and chloroplasts was synthesized on the cytoplasmic80S-ribosomes. This suggests that the ALAD formed on the 80S-ribosomesmight be incorporated into chloroplasts during their development. When etiolated radish seedlings were illuminated, ALAD in boththe cytoplasm and chloroplasts increased up to the point ofthe full development of the chloroplasts, and thereafter itdecreased. (Received August 20, 1975; )     

Syntheses of {delta}-aminolevulinic acid and chlorophyll during chloroplast formation in Chlorella protothecoides

Greening cells of Chlorella prolothecoides were assayed foractivity of the in vivo synthesis of ALA, which was markedlydeveloped during light-induced greening. Effects of CH on thesyntheses of ALA and chlorophyll were also examined. The resultsstrongly suggested that a labile enzyme is involved in ALA synthesis,and that continuous formation of the enzyme is required forthe greening of cells. However, the prompt suppression of chlorophyllsynthesis when CH was added to rapidly greening cells was foundto be attributable not to the blockage of ALA synthesis butto the suppression of some later process(es) in the course ofchlorophyll synthesis, under the conditions used. The valueof the Hill coefficient for the CH inhibition of chlorophyllsynthesis as well as the CH concentration which caused 50% inhibitionremained unaltered whether it was measured when the ALA synthesisactivity was greatly inhibited by CH or when the activity wasonly slightly suppressed. (Received November 11, 1974; )     

The development of structure and function in chloroplasts of greening mutants of Scenedesmus III. Biosynthesis of {delta}-aminolevulinic acid

Cells of the mutant C-2A' of Scenedesmus obliquus which requirelight for chlorophyll formation were assayed for in vivo activityof ALA synthesis. In general, ALA and chlorophyll syntheseswere coupled during the greening process. The action spectrafor ALA and chlorophyll syntheses both show the highest activitiesin the blue region, but were different in details. Under certainconditions, ALA synthesis occurred without a corresponding synthesisof chlorophyll. Reasons for these variances were discussed. The controlling action of light on ALA synthesis may occur throughthree different, but related, mechanisms. The principle mechanismappeared to be linked to lightenhanced respiration since itsinhibition by cycloheximide blocks ALA synthesis. The Hill coefficientof this inhibition is 2. After the light-induced enhancementof respiration had ceased, the Hill-coefficient of inhibitionof ALA synthesis became 1. Thus, in addition to enhanced respiration,ALA formation depends on its sensitizing enzyme having a half-lifetime of less than 1 hr. Finally, the dependence of the synthesisof ALA precursors on light was evident. ¹ On leave from the Institute of Applied Microbiology, Universityof Tokyo, Tokyo, Japan. (Received November 11, 1974; )     

Purification and properties of agmatine iminohydrolase from groundnut cotyledons

Agmatine iminohydrolase was purified ca 375-fold from groundnut cotyledons. The enzyme exhibited an optimum pH between 5.5 and 8.5 and the energy of activation was 22 kcal/mol. The K_m for agmatine was (7.57 ± 0.77) × 10^?4 M. The enzyme was inhibited by tryptamine, putrescine, cadaverine, spermidine and spermine. Inhibition by cadaverine and spermidine was competitive. The K_i values for cadaverine and spermidine were 4.1 × 10^?3 and 7.5 × 10^?4 M, respectively.     

Molecular cloning of the 5-aminolevulinic acid dehydratase gene from Rhodobacter sphaeroides.

A hemB mutant of Escherichia coli was used to clone the gene encoding 5-aminolevulinic acid dehydratase from Rhodobacter sphaeroides after physiological complementation of the mutation. A 2.9-kb DNA fragment was obtained and cloned in both orientations into the unique PstI restriction site of pUC19. This recombinant plasmid encodes a protein (Mr 39,000) that is immunoreactive with antibodies raised against the enzyme from higher plants.     

Purification and properties of D-gluconate dehydratase fromAchromobacter

d-Gluconate dehydratase fromAchromobacter, grown ond-gluconate, was purified 100-fold by a procedure involving ammonium sulfate fractionation and preparative acrylamide gel electrophoresis. The purified enzyme appeared to be homogeneous by disc gel electrophoresis. It is an inducible enzyme with an optimal activity in the pH region 8.4–8.8, a Km value of 2.08 × 10^–2 m ford-gluconate and a molecular weight of 270,000 ± 25,000. Only C₅ and C₆ aldonic acids possessing al-threo configuration at C₂ and C₃ are dehydrated. The dehydration products ofd-gluconate,d-xylonate,d-galactonate,d-fuconate andl-arabonate were identified as 2-keto-3-deoxy compounds by specific colour reactions and thin layer chromatography. Onemm Mg^{+ +} is a powerful activator, 0.1 mm Mn^{+ +} activates poorly and EDTA inhibits. Glutatione, dithiothreitol and mercaptoethanol had no effect, althoughp-chloromercuribenzoate (0.01 mm) decreased enzyme activity.We wish to thank Mr D. Dewettinck for skilful technical assistance. The senior author (J.D.L.) is indebted to the Fonds voor Kollektief en Fundamenteel Onderzoek (Belgium) for research and personnel grants. J.K.-M. is indebted to the Belgian government for a travel and study grant.     

Structural modifications and δ-aminolevulinate dehydratase synthesis/transport in cotyledons of far-red irradiated radish

δ-Aminolevulinate dehydratase (ALAD) is synthesized in the cytoplasm and subsequently transported into the plastids in cotyledons of tar-red irradiated radish ( Raphanus sativus L. ev. Longue Rave Saumonée). The intra-cellular localization of ALAD activity was determined under far-red irradiation and darkness. After 60 h far-red, ALAD was essentially eytoplasmic; 96 h far-red corresponded to a peak in total ALAD activity, as well as the onset of massive ALAD transfer into the plastids, and after 120 h a maximum enzyme activity was located in the plastids. Under dark conditions, essentially no transport of ALAD was noted.
Structural modifications of plastid-eytoplasmic relationships were analysed at the three developmental periods under dark and far-red conditions. 60 h far-red induced a change in the "elasticity" of plastid envelopes leading to envelope proliferations and amoeboid shaped plastids. After 96 h tar-red, membrane proliferations formed a highly evaginated plastid periphery. It is suggested that these membranes form a transport network between the two compartments. Also at 96 h, numerous vesicles, which appear to originate from the endoplasmic reticulum (ER), surround the plastid profile. On the basis of the structural observations, it is proposed that ALAD would be synthesized within the ER and transported toward the plastids via ER extensions, which take the form of smooth vesicles. Our hypothesis is that the transition vesicles and proliferations of the outer envelope membrane undergo a fusion process emptying the content of the vesicle into the intermembrane space of the envelope.     

14 C incorporation from exogenous compounds into -aminolevulinic acid by greening cucumber cotyledons

Greening cucumber cotyledons accumulate δ-aminolevulinic acid when treated with levulinic acid. A variety of specifically labelled compounds were applied to the tissue and label was measured in the δ-aminolevulinic acid. Glutamate, glutamine and α-ketoglutarate were found to be incorporated into δ-aminolevulinic acid to a much greater extent than were glycine and succinate. A new route of δ-aminolevulinic acid biosynthesis is proposed wherein the carbon skeleton of α-ketoglutarate is incorporated intact.     

Rapid purification and direct spectrophotometric assay for 5-aminolevulinic acid dehydratase

A two-step purification procedure for 5-aminolevulinic acid dehydratase (EC 4.2.1.24) from human red blood cells has been developed. It involves one ion exchange and one gel filtration step. The purification is about 1000-fold, and the yield is more than 85%. With the purified enzyme a direct spectrophotometric assay of product formation without subsequent reaction with Ehrlich's reagent is described.