Histidine, radioactive

The addition of a radioactive iodine atom to a protein molecule typically has little effect on the resultant protein activity, unless the active center is modified in the process. The size of an iodine atom is relatively small and does not result in many steric problems with large molecules. The sites of potential protein modification are tyrosine and histidine side chains. Tyrosine may be modified with a total of two iodine atoms per phenolate group, whereas histidine can incorporate one iodine. Sulfhydryl modification at cysteine residues is typically unstable. 

Figure 12.5 IODO-GEN is a water-insoluble oxidizing agent that can react with 1251 - to form a highly reactive mixed halogen species, 125IC1. This intermediate can add radioactive iodine atoms to tyrosine or histidine side chain rings.

Figure 265 IODO-BEADS contains immobilized Chloramine-T functional groups that can react with radioactive iodide in aqueous solution to form a highly reactive intermediate. The active species may be an iodosulfonamide derivative, which then can iodinate tyrosine or histidine residues in proteins.

Metabolites of the ergoline type [as (128)], as well as those of type (119), have been isolated from Penicillium roqueforti. The incorporation has been observed of radioactive samples of tryptophan and mevalonate into both series of metabolites, and of histidine into those of type (119).44 Diversion from tryptophan into the two independent biosynthetic pathways is initiated on the one hand by the formation of (122) and on the other by reaction with histidine to give a diketopiperazine precursor for metabolites such as (119). Which route is followed is temperature-dependent. 

The biosynthesis of pilocarpine in Pilocarpus pennatifolius was studied by the administration of radiolabeled precursors. Radioactive sodium acetate, histidine, histidinol, methionine, and threonine were administered by the cut-stem method. Histidine, methionine, and threonine were administered together by a wick inserted through the stem of an intact plant. Sodium acetate and histidine were fed to root cuttings by suspending the roots in aqueous solutions of the precursors. After 64 - 7 5 h, the roots were harvested and total alkaloid extracts made. These extracts were then fed to stem cuttings. 

The most smdied enzyme is histidine decarboxylase from Lactobacillus 30a. There are pyruvate residues at the amino terminals of each of 5 of the 10 subunits in this enzyme. When the organism is grown on [ C] serine, the specific radioactivity of the pymvate is the same as that of serine incorporated into the protein and much greater than that of free lactate or pyruvate in the culture medium This suggests that pyruvate arises by postsynthetic modification of a serine residue. 

The sequence of appearance, relative amounts, and final relationships of cell components that survive lysis are presented diagrammatically in Figure 11. The smallest filaments that have been isolated (35 A thick) have low sulfur content (32). They thicken to 60-90 A with addition of sulfur-rich protein (demonstrated by heavy metal staining) (32, 64) and histidine-rich protein (demonstrated with radioactive labeling) (49). Five to 10 of these thickened filaments aggregate to form fibrils that average 250 A in diameter (70). Meanwhile, KH and ER protein accumulate until the cell is lysed when they are mixed and dispersed (47) to coat the 250-A fibrils (70). The coated fibrils are submerged in a matrix that includes nucleoproteins and nonfibrous proteins these incorporate about 10 times more sulfur than the fibrils (32). The insoluble fibrils and matrix constitute about 65% of the cornified cell (66) other components include 10% soluble keratin, 10% dialyzable substances (amino acids, etc.), 7-9% lipids, and about 5% membrane protein (65, 66). 

The histidine catabolic pathway is discussed under Folate in Chapter 9. The material reveals that histidine is catabolized to produce glutamate. Glutamate in turn, can be converted to a-ketoglutarate and completely oxidized to CO in the Krebs cycle. In the study depicted in Figure 8,26, the dietary histidine was spiked with I Cjhistidine, The term "spiked" means that only a very small proportion of the histidine contained carbon-14. The metabolic behavior of the radioactive histidine, which can be followed, mirrors the metabolic fate of nonradioactive histidine in the diet. All of the CQz exhaled by the rats can be easily collected, The " COj present in the rat s breath can be measured by use of a liquid scintillation counter. The amount of CO2 produced directly mirrors the proportion of histidine, absorbed from the diet that was degraded the rat s body. 

The results in Figure 8,26 reveal that only a tiny proportion of histidine Is catabolized with diets containing up to 0-25% histidine. With diets containing mote than 0,25% histidine, there is a steady increase in the proportion of absorbed amino acid undergoing oxidation. For example about 25% of the absorbed histidine (both radioactive and nonradioactive) is degraded in the rats fed diets containing 0,6% histidine. 

FIGURE R26 Percentage of absorbed radioactive carbon (consumed as histidine) that is exhaled as versus concentrations of histidine in the diet. (Redrawn with peimission from Kang-Lee and Harper, 1977.)... 

The monazo and bisazo derivatives of lysine, tyrosine, and histidine are unstable to acid hydrolysis and the native amino acids are not regenerated. Therefore, the best method to quantitate the extent of derivatization is with radioactive reagent. The characteristic absorp-... 

Chelex-lOO also competes against the added chelators glycine, histidine, and ethylenediaminetetraacetic acid (EDTA) (Table II). In these experiments, various concentrations of chelators or phytoplankton cultures with cells removed were mixed in seawater with Cu, and the radioactivity was counted before and after the water was passed through Chelex columns. Since the only competition observed was with 10" M EDTA, the resin should be able to compete against natural complexes... 

This has been achieved for the production of (146) in D. sphaerica by means of histidine decarboxylase inhibitors. Both a-methylhistidine and a-hydrazinohistidine (inhibitors for mammalian specific histidine decarboxylase) inhibited formation of histamine with the result that more (146) was synthesized at the expense of (144) (based on the radioactivity of the products after feeding [ H]histidine, [ C]isovaleric acid and inactive animomethylimidazole [145]). a-Methyldopa, an inhibitor of mammalian non-specific decarboxylase, was without effect on the proportions of the products formed. 

Fig. 6-25. Analysis of Sr-90 with the aid of a radioactivity monitor. - Separator column IonPac CS2 eluent 0.03 mol/L HC1 + 0.002 mol/L histidine hydrochloride flow rate 1.5 mL/min detection scintillation measurement for Sr-90 injection volume 50 pL solute concentration 76.4 Bq Sr-90 with inactive SrCl2 as the carrier.

The most sensitive method for measuring carbon dioxide production by histidine decarboxylase is that in which histidine labelled with carbon-14 in the carboxyl group is used as substrate. The C02 evolved is trapped in a suitable absorbent medium, and its radioactivity is then determined directly in a scintillation spectrometer. Several variations of this procedure have been described - - - - , and these compare favourably in speed and sensitivity with alternative isotopic methods in which the production of radioactive histamine from ring- C-labelled histidine is measured (see following section). 

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