Reconstitution of the two terminal enzymes of the heme biosynthetic pathway into phospholipid vesicles

Purified mouse protoporphyrinogen oxidase (EC 1.3.3.4) and ferrochelatase (EC 4.99.1.1), the two terminal enzymes of the heme biosynthetic pathway, have been reconstituted into phospholipid vesicles, and the kinetics of the enzymes in the reconstituted systems were compared with the values obtained with the free enzymes. The apparent Km for free protoporphyrinogen oxidase in detergent solution is 5.61 +/- 0.62 microM for free protoporphyrinogen. The Km was lower when the enzyme was inserted into phospholipid vesicles (0.78 +/- 0.28 microM) and when both enzyme and substrate were incorporated into phospholipid vesicles (0.61 +/- 0.14 microM). In the presence of cardiolipin, a phospholipid present mainly in the inner mitochondrial membrane, the value of the Km for the substrate decreased 3-fold (0.20 +/- 0.02 microM). For reconstituted ferrochelatase similar kinetic analyses were carried out and it was found that the apparent Km values were only weakly affected by the lipid environment. Studies on the orientation of ferrochelatase demonstrated that approximately 50% of the enzyme in the reconstituted system had the active site located in the inner face of the phospholipid vesicle. This is in contrast to intact mitochondria where the active site is located on the matrix side of the inner mitochondrial membrane. The activation energies for both enzymes were determined for free and reconstituted enzymes. It was found that for both enzymes the activation energies were lower for the reconstituted systems than for the free enzymes.     

Mouse protoporphyrinogen oxidase. Kinetic parameters and demonstration of inhibition by bilirubin.

The penultimate step of haem biosynthesis, the oxidation of protoporphyrinogen to protoporphyrin, was examined with purified murine hepatic protoporphyrinogen oxidase (EC 1.3.3.4) in detergent solution. The kinetic parameters for the two-substrate (protoporphyrinogen and oxygen) reaction were determined. The limiting Km for protoporphyrinogen when oxygen is saturating is 6.6 microM, whereas the Km for oxygen with saturating concentrations of protoporphyrinogen is 125 microM. The kcat. for the overall reaction is 447 h-1. The ratio of kcat. to the Km for protoporphyrinogen is approx. 20-fold greater than the kcat./Km,O2 ratio. The ratio of protoporphyrin formed to dioxygen consumed is 1:3. Ubiquinone-6, ubiquinone-10 and dicoumarol stimulate protoporphyrinogen oxidase activity at low concentrations (less than 15 microM), whereas coenzyme Q0 and menadione show no activation at these concentrations. Above 30 microM, all five quinones inhibit the enzyme activity. FAD does not significantly affect the activity of the enzyme. Bilirubin, a product of haem catabolism, is shown to be a competitive inhibitor of the penultimate enzyme of the haem-biosynthetic pathway, protoporphyrinogen oxidase, with a calculated Ki of 25 microM. The terminal enzyme of haem-biosynthetic pathway, namely ferrochelatase, is not inhibited by bilirubin at concentrations over double the Ki value for the oxidase. In contrast with other enzymic systems, the toxicity of bilirubin is not reversed by binding to albumin.     

The synthesis of murine ferrochelatase in vitro and in vivo.

Ferrochelatase (protohaem ferro-lyase, EC 4.99.1.1), the terminal enzyme of the haem-biosynthetic pathway, is an integral membrane protein of the mitochondrial inner membrane. When murine erythroleukaemia cells are labelled in vivo with [35S]methionine, lysed, and the extract is immunoprecipitated with rabbit anti-(mouse ferrochelatase) antibody, a protein of Mr 40,000 is isolated. However, when isolated mouse RNA is translated in a cell-free reticulocyte extract, a protein of Mr 43,000 is isolated. Incubation of this Mr 43,000 protein with isolated mitochondria resulted in processing of the Mr 43,000 precursor to the Mr 40,000 mature-sized protein. Addition of carbonyl cyanide m-chlorophenylhydrazone and/or phenanthroline inhibits this processing. These data indicate that ferrochelatase, like most mitochondrial proteins, is synthesized in the cytoplasm as a larger precursor and is then translocated and processed to a mature-sized protein in an energy-required step.     

Rapid modulation of homing receptors (gp90MEL-14) induced by activators of protein kinase C. Receptor shedding due to accelerated proteolytic cleavage at the cell surface

Lymphocyte entry into lymph nodes and Peyer's patches is initiated by the adhesion of the lymphocytes to specialized postcapillary high endothelial venules (HEV). The binding of lymphocytes to lymph node HEV is mediated by the cell surface receptor gp90MEL-14 (gp90). Previous work has shown that gp90 is down-regulated over a period of days after mitogenic or mixed lymphocyte reaction stimulation of T lymphocytes. In our study, it is shown that stimulation of lymphocytes with activators of protein kinase C (PKC), such as PMA or 1-oleoyl 2-acetyl-glycerol, results in the nearly complete loss of surface expression of gp90 within 1 h. Pretreatment of the cells with H-7 or staurosporine, PKC inhibitors, but not HA1004, a general protein kinase inhibitor, prevents the loss of gp90MEL-14. Within 15 min of stimulation of PKC, a novel form of gp90 can be immunoprecipitated from the supernatant of stimulated cells. Upon deglycosylation, this soluble gp90 polypeptide is shown to be 12 kDa smaller than the cell surface protein. Peptide mapping showed identical patterns for surface and soluble receptor, confirming that the soluble Ag is related to the cell membrane protein. Together, these experiments suggest that activation of PKC results in the proteolytic cleavage of gp90MEL-14, resulting in receptor shedding and the inability of the lymphocytes to adhere to HEV endothelium. Furthermore, because supernatant from unstimulated, normal lymphocytes also contains a small amount of the low Mr form of gp90, cell surface proteolysis may be part of the normal turnover of this receptor glycoprotein. These experiments suggest that PKC may play a role in the regulation of lymphocyte traffic to lymphoid tissues.     

A molecular defect in human protoporphyria.

Protoporphyria is generally an autosomal dominant disease that is characterized clinically by photosensitivity and hepatobiliary disease and that is characterized biochemically by elevated protoporphyrin levels. The enzymatic activity of ferrochelatase, which catalyzes the last step in the heme biosynthetic pathway, is deficient in all tissues of patients with protoporphyria. In this study, sequencing of ferrochelatase cDNAs from a patient with protoporphyria revealed a single point mutation in the cDNAs resulting in the conversion of a Phe(TTC) to a Ser(TCC) in the carboxy-terminal end of the protein, F417S. Further, the human ferrochelatase gene was mapped to chromosome 18q21.3 by chromosomal in situ suppression hybridization. Finally, expression of recombinant ferrochelatase in Escherichia coli demonstrated a marked deficiency in activity of the mutant ferrochelatase protein and of mouse-human mutant ferrochelatase chimeric proteins. Therefore, a point mutation in the coding region of the ferrochelatase gene is the genetic defect in some patients with protoporphyria.     

Heme biosynthesis in mammalian systems: evidence of a Schiff base linkage between the pyridoxal 5'-phosphate cofactor and a lysine residue in 5-aminolevulinate synthase.

5-Aminolevulinate synthase is the first enzyme of the heme biosynthetic pathway in nonplant higher eukaryotes. Murine erythroid 5-aminolevulinate synthase has been purified to homogeneity from an Escherichia coli overproducing strain, and the catalytic and spectroscopic properties of this recombinant enzyme were compared with those from nonrecombinant sources (Ferreira, G.C. & Dailey, H.A., 1993, J. Biol. Chem. 268, 584-590). 5-Aminolevulinate synthase is a pyridoxal 5'-phosphate-dependent enzyme and is functional as a homodimer. The recombinant 5-aminolevulinate synthase holoenzyme was reduced with tritiated sodium borohydride and digested with trypsin. A single peptide contained the majority of the label. The tritiated peptide was isolated, and its amino acid sequence was determined; it corresponded to 15 amino acids around lysine 313, to which pyridoxal 5'-phosphate is bound. Significantly, the pyridoxyllysine peptide is conserved in all known cDNA-derived 5-aminolevulinate synthase sequences and is present in the C-terminal (catalytic) domain. Mutagenesis of the 5-aminolevulinate synthase residue, which is involved in the Schiff base linkage with pyridoxal 5'-phosphate, from lysine to alanine or histidine abolished enzyme activity in the expressed protein.     

Amplification and excision of integrated polyoma DNA sequences require a functional origin of replication

Cells transformed by Polyoma virus (Py) can undergo a high rate of excision or amplification of integrated viral DNA sequences, and these phenomena require the presence of homology (i.e., repeats) within the viral insertion as well as a functional viral large T antigen (T-Ag). To determine whether the main role of large T-Ag in excision and amplification was replicative or recombination-promoting, we studied transformed rat cell lines containing tandem insertions of a ts-a Py molecule (encoding a thermolabile large T-Ag) with a deletion of the origin of viral DNA replication. Culturing of these cells at the temperature permissive for large T-Ag function did not result in any detectable excision or amplification of integrated Py sequences. We then introduced into origin-defective lines a recombinant plasmid containing the viral origin of replication and the gene coding for resistance to the antibiotic G418. All G418-resistant clones analyzed readily amplified the integrated plasmid molecules when grown under conditions permissive for large T-Ag function, showing that these cells produced viral large T-Ag capable of promoting amplification in trans of DNA sequences containing the Py origin. These observations strongly suggest that Polyoma large T antigen promotes excision or amplification of viral DNA by initiating replication at the integrated origin, providing a favorable substrate for subsequent recombination.     

Siderophore utilization and iron uptake by Rhodopseudomonas sphaeroides

The growth of Rhodopseudomonas sphaeroides in iron-deficient medium did not result in the production of detectable levels of siderophores of either the catechol or hydroxamate type. Iron-limited cultures of R. sphaeroides were not able to remove iron from ferric transferrin unless supplemented with 2,3-dihydroxybenzoic acid. R. sphaeroides was shown to take up 59Fe+3 when it was supplied as ferric chloride, ferric citrate, or ferric parabactin, but not when supplied as ferric rhodotorulate or ferric Desferal. When iron was supplied as ferric citrate, citrate was not taken up by the cells. The growth rate of R. sphaeroides under iron-limiting conditions was decreased by the addition of either Desferal or rhodotorulic acid, while the addition of citrate or parabactin did not affect growth.