Isotopic microassay of histamine, histidine, histidine decarboxylase and histamine methyltransferase in brain tissue

Abstract— Microassays are described for histamine, histidine, and the activities of the enzymes histidine decarboxylase (EC 4.1.1.22) and histamine niethyltransferase (EC 2.1.1.8) in brain tissue. The enzymic-isotopic microassay for histamine is based on the methylation of tissue histamine by added histamine methyl-transferase and [¹⁴C]- or [³H]-labelled S-adenosyl-l -methionine. In a double-isotopic form of the assay, a tracer of [³H]histamine is employed along with [¹⁴C]S-adenosyl-l -methionine, and the ratio [¹⁴C]:[³H] reflects the amount of histamine in the sample. Because the methylation of histamine is uniform in brain samples studied, a single isotopic assay with [³H]S-adenosyl-l -methionine as the methyl donor is possible and increases sensitivity, so that 10 pg of tissue histamine can be estimated reliably. The assay for histidine involves decarboxylation of histidine by a bacterial histidine decarboxylase and measurement of the histamine formed by the enzymicisotopic procedure. In the histidine decarboxylase assay, histamine synthesized from added histidine is measured. The assay for histamine methyltransferase involves measuring the formation of [¹⁴C]methylhistamine with [¹⁴C]S-adenosyl-l -methionine serving as the methyl donor.     

In vivo synthesis of histidine by a cloned histidine ammonia-lyase in Escherichia coli

Histidine ammonia-lyase catalyzes the first step in histidine catabolism, the deamination of histidine to urocanate and ammonia. In vitro experiments have shown that histidine ammonia-lyase also can catalyze the reverse (amination) reaction, histidine synthesis, relatively efficiently under extreme reaction conditions (4 M NH4OH, pH 10). An Escherichia coli hisB deletion strain was transformed with a pBR322 derivative plasmid (pCB101) containing the entire Klebsiella aerogenes histidine utilization (hut) operon to determine whether the catabolic histidine ammonia-lyase could function biosynthetically in vivo to satisfy the histidine auxotrophy. Although the initial construct did not grow on media containing urocanate and ammonia as a source of histidine, spontaneous mutants possessing this ability were isolated. Four mutants characterized grew at doubling times of 4 h compared with 1 h when histidine was present, suggesting that histidine synthesis, although unequivocally present, remained growth limiting. Each mutant contained a plasmid-encoded mutation which eliminated urocanase activity, the second enzyme in the Hut catabolic pathway. This genetic block led to the accumulation of high intracellular levels of urocanate, which was subsequently converted to histidine via histidine ammonia-lyase, thus satisfying the histidine auxotrophic requirement.     

Improved fluorometric assay of histidine and peptides having NH2-terminal histidine using o-Phthalaldehyde

o-Phthalaldehyde (OPT) reacts with many biogenic compounds such as spermidine, histamine, histidine and peptides with NH₂-terminal histidine, yielding intensely fluorescent condensation products. This communication examines the reaction conditions for the OPT-induced fluorescence of histidine and peptides with NH₂-terminal histidine for the purpose of improving the sensitivity as well as the specificity of the assay of these compounds. Reaction with OPT at pH 11.2–11.5 and at 40°C for 10 min was found to be optimal for histidine. After cooling, the fluorescence was read at $\text{360}\text{440}\text{nm}$ (uncorrected instrument values). The method measures as little as 4–5 ng/ml. Peptides with NH₂-terminal histidine were found to interfere with the assay whereas histamine, histidinol and spermidine did not. The optimum reaction and assay conditions for the OPT-induced fluorescence of the histidyl-dipeptides varied markedly from one peptide to another. As a group peptides with NH₂-terminal histidine are best assayed by condensation with OPT at pH 11.8 at room temperature and with a reaction time of 30 min. Fluorescence should be read before as well as after acidification to pH 2.5. Details are given for the assay of individual histidyl-dipeptides.     

Immuno-cross-reactivity of histidine and dopa decarboxylases

Immuno-cross-reactivity between histidine decarboxylase (HDC) and dopa decarboxylase (DDC) was investigated. By comparing the cDNA sequences of rat HDC with rat and guinea-pig DDCs, we found a region that may possibly be related to the cross-reactivity of anti-rat HDC antibody with guinea-pig DDC. The peptide encoded by this region was synthesized and anti-peptide antibody was prepared. We also purified HDC and DDC homogeniously from fetal rat liver and guinea-pig liver, respectively. On immunoblotting, anti-peptide antibody recognized both rat HDC and guinea-pig DDC. Anti-HDC polyclonal antibody which also recognizes both enzymes detected only rat HDC when it was absorbed by the peptide. This result indicates that this region is responsible for the immuno-cross-reactivity of anti-rat HDC antibody with guinea-pig DDC.