A simple spectrophotometric method for uroporphyrinogen I synthetase in erythrocytes is described. Results obtained on intermittent acute porphyria patients and carriers are similar to the results ...obtained with fluorimetric methods. Reproducibility, relationship between enzyme activity and enzyme concentration, and effect of time on enzymatic activity are described.
Limited tryptic digestion of spectrin (Sp) from seven related individuals manifesting hereditary elliptocytosis (HE) or hereditary pyropoikilocytosis (HPP) phenotypes revealed the presence of a novel ...peptide with a molecular weight of 78 Kd and a concomitant decrease in the al domain (80-Kd peptide), which is the domain involved in the dimer self-association process. Sp from the normal members of this white family exhibited a normal peptide pattern, as compared with controls. The abnormal peptide pattern was associated with a decreased ability of Sp dimer to self-associate. In this kindred in which three generations were available for study, the clinical manifestations were quite variable and ranged from the asymptomatic HE carrier state to hemolytic HE or to severe anemia requiring splenectomy. The severity of the disease appeared to be correlated both with the amount of mutant spectrin (31 % to 69%) and with the excess of the Sp dimer found in the membrane (26% to 60%, compared with a normal value of 5.6% ± 2.2%). Partial amino acid sequencing showed that the αl/78-Kd peptide resulted from cleavage at lysine residue 10 of the αl/80-Kd domain. Knowledge of the exon/intron structure of cloned genomic DNA encoding the al domain allowed us to amplify in vitro a DNA fragment containing the third exon of the a-spectrin gene. The amplified fragment was subcloned and sequenced. A G to T transversion was found in the 39th codon (AGT for AGG), which changed the normal arginine to a serine. Hybridization of amplified DNAs with allele-specific oligo-nucleotides corresponding to the normal and mutant sequences confirmed the presence of the mutation in three other HE members of the family (the propositus mother, brother, and sister).
Using the digitonin method and subsequent fractionation of rat liver mitochondria, protoporphyrinogen oxidase (penultimate enzyme in the heme biosynthesis pathway) was found to be closely associated ...with the mitochondrial inner membrane fraction.
Chemical treatment with non‐specific probes (trypsin and diazobenzene sulfonate) of either intact or inverted mitoplasts, indicated that protoporphyrinogen oxidase was anchored within the lipid bilayer of the inner membrane. Protoporphyrinogen had an equal access to the active site of the enzyme from both sides of the inner membrane and its transformation to protoporphyrin did not appear to be energy‐dependent.
Studies of protoporphyrinogen synthesis from exogenously added coproporphyrinogen in either intact or hypoosmotically treated mitochondria underlined the importance of the peculiar submitochondrial location of coproporphyrinogen oxidase and protoporphyrinogen oxidase for the transfer of substrates to the inner membrane.
Uroporphyrinogen decarboxylase was synthesized in a reticulocyte lysate cell-free system under the direction of messenger RNAs isolated from human fetal liver and from human reticulocytes. The enzyme ...was specifically isolated by immuno affinity chromatography. Analysis of the translation products showed that uroporphyrinogen decarboxylase was synthesized in vitro with its mature molecular weight. This enzyme represented 0.04% of the total neosynthesized proteins under the direction of fetal liver mRNA and about ten times less (0.005%) with reticulocyte mRNA.
An assay for coproporphyrinogen oxidase activity is described which uses a 14C-coproporphyrinogen substrate with product isolation by methylation, extraction, and thin layer chromatography. This ...method affords high sensitivity, since a high specific activity of the substrate and good reproducibility due to the incorporation of an internal standard can be obtained. The activity in rat liver and human lymphocytes was found to be 140 nmol protoporphyrin/h/g of liver and 483 pmol protoporphyrin/h/mg of lymphocyte protein, respectively.
DNA amplification by the polymerase chain reaction (PCR) is a method capable of producing a selective and very high enrichment of a specific DNA sequence. Hence it seems to be useful in various ...fields from basic research to clinical applications. In order to automatize PCR we assembled for a very low cost a mechanical system designed to carry a test tube holder successively in three thermal baths set at the required temperatures for the reaction. Two examples of the use of this machine are given: (i) amplification of DNA of a particular subtype of acute intermittent porphyria; (ii) the detection of the chimeric c-abl/bcr message found in chronic myelogenous leukemia cells.
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