ABSTRACT
The majority of the heme-binding proteins possess a "heme-pocket" that stably binds to heme. Usually known as housekeeping heme-proteins, they participate in a variety of metabolic reactions (e.g., catalase). Heme also binds with lower affinity to the "Heme-Regulatory Motifs" (HRM) in specific regulatory proteins. This type of heme binding is known as exchangeable or regulatory heme (RH). Heme binding to HRM proteins regulates their function (e.g., Bach1). Although there are well-established methods for assaying total cellular heme (e.g., heme-proteins plus RH), currently there is no method available for measuring RH independent of the total heme (TH). The current study describes and validates a new method to measure intracellular RH. This method is based on the reconstitution of apo-horseradish peroxidase (apoHRP) with heme to form holoHRP. The resulting holoHRP activity is then measured with a colorimetric substrate. The results show that apoHRP specifically binds RH but not with heme from housekeeping heme-proteins. The RH assay detects intracellular RH. Furthermore, using conditions that create positive (hemin) or negative (N-methyl protoporphyrin IX) controls for heme in normal human fibroblasts (IMR90), the RH assay shows that RH is dynamic and independent of TH. We also demonstrated that short-term exposure to subcytotoxic concentrations of lead (Pb), mercury (Hg), or amyloid-ß (Aß) significantly alters intracellular RH with little effect on TH. In conclusion the RH assay is an effective assay to investigate intracellular RH concentration and demonstrates that RH represents â¼6% of total heme in IMR90 cells.
Subject(s)
Apoenzymes/metabolism , Enzyme Assays/methods , Heme/metabolism , Horseradish Peroxidase/metabolism , Intracellular Space/metabolism , Amyloid beta-Peptides/metabolism , Cell Line , Heme/biosynthesis , Humans , Lead/metabolism , Mercury/metabolism , Reference Standards , Substrate Specificity , Time FactorsABSTRACT
We have tested the specificity and utility of more than 200 antibodies raised against 57 different histone modifications in Drosophila melanogaster, Caenorhabditis elegans and human cells. Although most antibodies performed well, more than 25% failed specificity tests by dot blot or western blot. Among specific antibodies, more than 20% failed in chromatin immunoprecipitation experiments. We advise rigorous testing of histone-modification antibodies before use, and we provide a website for posting new test results (http://compbio.med.harvard.edu/antibodies/).
Subject(s)
Antibody Specificity , Histones/immunology , Animals , Antibodies/chemistry , Blotting, Western , Caenorhabditis elegans/genetics , Caenorhabditis elegans Proteins/chemistry , Chromatin Immunoprecipitation , Drosophila Proteins/chemistry , Drosophila melanogaster/genetics , Histones/chemistry , Histones/metabolism , Immunoblotting , Protein Processing, Post-Translational , Quality Control , Reproducibility of ResultsABSTRACT
We have identified a novel gene named grappa (gpp) that is the Drosophila ortholog of the Saccharomyces cerevisiae gene Dot1, a histone methyltransferase that modifies the lysine (K)79 residue of histone H3. gpp is an essential gene identified in a genetic screen for dominant suppressors of pairing-dependent silencing, a Polycomb-group (Pc-G)-mediated silencing mechanism necessary for the maintenance phase of Bithorax complex (BX-C) expression. Surprisingly, gpp mutants not only exhibit Pc-G phenotypes, but also display phenotypes characteristic of trithorax-group mutants. Mutations in gpp also disrupt telomeric silencing but do not affect centric heterochromatin. These apparent contradictory phenotypes may result from loss of gpp activity in mutants at sites of both active and inactive chromatin domains. Unlike the early histone H3 K4 and K9 methylation patterns, the appearance of methylated K79 during embryogenesis coincides with the maintenance phase of BX-C expression, suggesting that there is a unique role for this chromatin modification in development.
