Regulation of biogenesis of liver delta-aminolevulinate synthase: effects of structural modifications of heme on the enzyme's RNA

1. The aim of this study was to determine the effects of several metallo-porphyrins, derived by modifications of heme, on the concentration delta-aminolevulinate (ALA) synthase RNA in hepatocytes. 2. Primary cultures of chick embryo hepatocytes were incubated with allylisopropylacetamide (AIA) for 5 hr in the presence and absence of each metallo-porphyrin (10 microM). At the end of each incubation, total RNA was isolated from the cells and analyzed for ALA synthase-specific RNA by solution hybridization. 3. The concentration of ALA synthase RNA increased 7.3 fold in hepatocytes incubated with AIA alone. The AIA-induced elevations in the enzyme's RNA were blocked partially and equally in cells. incubated with zinc- or with iron-protoporphyrin IX. The block was greater in cells incubated with cobalt-protoporphyrin IX. 4. Modifications of the side chains of the porphyrin ring at positions 2 and 4, giving mesoporphyrin IX and deuteroporphyrin IX, changed the effectiveness of the iron- and the cobalt-porphyrins to limit the AIA-induced increase in ALA synthase RNA. The modifications did not affect the capacities of the zinc-porphyrins to inhibit the rise in RNA. 5. In conclusion, the effect of a given metallo-porphyrin on liver ALA synthase RNA following side chain modification depended on the coordinated metal.     

A rapid method for capture and identification of immunogenic proteins in Bordetella pertussis enriched membranes fractions: a fast-track strategy applicable to other microorganisms

Mass spectrometry (MS) coupled with 1-D and 2-D electrophoresis can be utilized to detect and identify immunogenic proteins, but these methods are laborious and time-consuming. We describe an alternative, simple, rapid gel-free strategy to identify multiple immunogenic proteins from Bordetella pertussis (Bp). It couples immunoprecipitation to nano liquid chromatography- tandem mass spectrometry (IP-nLC-MS/MS) and is significantly both time- and labor-saving. We developed a gel-free magnetic bead-based immunoprecipitation (IP) method using different NP-40/PBS concentrations in which solubilized proteins of Bp Tohama I membrane fractions were precipitated with polyclonal rabbit anti-Bp whole cell immune sera. Immune complexes were analyzed by MS and Scaffold analysis (> 95% protein identification probability). Total immunoproteins identified were 50, 63 and 49 for 0.90%, 0.45% and 0.22% NP-40/PBS buffer concentrations respectively. Known Bp proteins identified included pertactin, serotype 2 fimbrial subunit and filamentous hemagglutinin. As proof of concept that this gel-free protein immunoprecipitation method enabled the capture of multiple immunogenic proteins, IP samples were also analyzed by SDS-PAGE and immunoblotting. Bypassing gels and subjecting immunoprecipitated proteins directly to MS is a simple and rapid antigen identification method with relatively high throughput. IP-nLC-MS/MS provides a novel alternative approach for current methods used for the identification of immunogenic proteins.     

Mitochondrial biogenesis: do liver mitochondria contain glycoproteins and glycosyltransferases?

1. Subcellular fractions isolated from livers of 19-day-old chicken embryos were analyzed in order to assess whether liver mitochondria contained glycosylated proteins or had mannosyl- or sialyl-transferases that could transfer sugars to mitochondrial macromolecules. 2. Proteins in liver mitochondrial membranes and matrix fractions were screened for their affinities for concanavalin A (Con A). 3. After separation by gel electrophoresis under denaturing conditions, a significant number of the proteins bound [125I]Con A, and the binding of the lectin was substantially inhibited by alpha-methyl-D-mannoside. 4. In addition, radio-iodinated matrix proteins were screened for lectin-binding properties by chromatography on Con A covalently linked to agarose. 5. A number of proteins, representing 14% of those loaded onto the column, became tightly bound to the agarose-linked lectin, and the molecular weights of several of those proteins are reported. 6. Mannosyltransferase activities were measured in fractions highly enriched for mitochondria. 7. In the reactions, mannose was transferred from guanosine diphosphomannose to materials insoluble in 0.3% trichloroacetic acid or in chloroform:methanol (2:1). 8. The fractions also catalyzed the transfer of mannose to materials extractable in chloroform:methanol and which migrated with the Rf of dolichol phosphate on Silica Gel H. 9. Dolichol phosphate stimulated the transfer of mannose to those materials extractable in the organic solvents. 10. Marker enzyme analyses indicated that the mannosyl transferase activity in the mitochondrial fraction could not be accounted for entirely by contaminating microsomal membranes. 11. Although sialyltransferase activity was detected also in the mitochondrial fractions, the levels of the activity and the kinetics of the reactions indicated that Golgi membranes were most likely the sources of the enzyme.     

Proteolysis: Adaptor, adaptor, catch me a catch

The ClpXP protease of bacteria can degrade a wide variety of proteins while maintaining remarkable substrate selectivity. New work in Escherichia coli implicates adaptor proteins in enhancing substrate selectivity and regulating the flow of substrates to cellular proteases.     

Analysis of mononuclear cell surfaces with fluoresceinated staphylococcal protein A complexed with IgG antibody of heat-aggregated gamma-globulin.

Fluorescein-conjugated staphylococcal protein A (SPA) was complexed with either: 1) heat-aggregated IgG, 2) B cell specific antibody, or 3) T cell specific antibody and then used for an immunofluorescent analysis of mononuclear cell surfaces. Cellular Fc receptors failed to recognize the Fc region of aggregated IgG that had been blocked by SPA. Moreover, fluoresceinated SPA that had been complexed either with anti-Fab (B-cell specific) or T cell-specific antisera prevented the nonspecific binding of these reagents to the IgG-Fc receptors on mononuclear cells, thereby permitting the latter to be properly identified as B or T lymphocytes. In addition, when unconjugated SPA was added to presensitized target cells in a test for antibody-dependent cell-mediated cytotoxicity, cytolysis was abrogated.     

Characterization of chicken liver L-alanine:4,5-dioxovaleric acid aminotransferase

1. A procedure is described for purifying the enzyme L-alanine:4,5-dioxovaleric acid aminotransferase (DOVA transaminase) from chicken liver. The enzyme catalyzes a transamination reaction between L-alanine and 4,5-dioxovaleric acid (DOVA), yielding delta-aminolevulinic acid (ALA). 2. In cell fractionation studies, DOVA transaminase activities were detected in mitochondria and in the post-mitochondrial supernatant fraction from liver homogenates. 3. For the mitochondrial enzyme, any of most L-amino acids could serve as a source for the amino group transferred to DOVA, but L-alanine appeared the preferred substrate. At pH 7.0, the enzyme had an apparent Km of 60 microM for DOVA and of 400 microM for L-alanine. 4. The enzyme was purified from disrupted mitoplasts in three steps: chromatography on DEAE-Sephacel, gel filtration through Sephadex G-150, and chromatography on hydroxyapatite. The yield was approx. 100 micrograms of enzyme protein per 10 g wet wt of liver. 5. The purified enzyme had a subunit mol. wt of 63,000 as determined by gel electrophoresis under denaturing conditions. 6. The activity of DOVA transaminase was also measured in embryonic chicken liver, and based on activity, the enzyme's capacity to produce ALA was significantly greater than that of ALA synthase. Unlike ALA synthase, however, DOVA transaminase activity did not increase in liver mitochondria of chicken embryos exposed for 18 hr to two potent porphyrogenic agents.