A Rapid Histidine Decarboxylase Assay1

Home , Glutamate decarboxylase, Histidine

ANALYTICAL BIOCHEMISTRY 87. 293-297 (1978)

Histidine decarboxylase (EC 4.1.1.22) catalyzes the conversion of histidine to histamine. Because current assays for enzyme activity are time consuming and require additional enzymes or large amounts of tissue, a rapid radioisotopic assay was devised. Using commercially available radioactive histidine (without additional purification), the enzyme mediates the formation of histamine. The product is resolved from precursor by paper electrophoresis in a formic acid-acetic acid solution for 15 min. After drying and ninhydrin staining, radioactive histamine is measured by liquid scintillation spectrometry. This assay procedure is sensitive enough to measure decarboxylase activity in milligram quantities of rat brain.

Histidine decarboxylase (EC 4.1.1.22) catalyzes the synthesis of histamine from histidine. In addition to its potent physiologic effects in animals (l), histamine is synthesized in microorganisms and plants (2). The enzyme has been measured manometrically by monitoring CO, evolution (3) and radiometrically by measuring 14C0, liberation (4). Both methods require considerable tissue or enzyme activity. For example, more than 600 mg wet wt of rat stomach-a rather rich mammalian source-is required in the second procedure. Schwartz et al. (5) developed a fluorometric procedure to measure histidine decarboxylase activity in rat brain. In addition to requiring large tissue samples (100 mg), a lengthy histamine purification is required to reduce the fluorescent background. Taylor and Snyder (6) developed a sensitive radioisotopic assay based on the formation of labeled methylhistamine during a second incubation. In addition to requiring partial purification of histamine N-methyltransferase and a second incubation, additional extraction procedures are required to obtain the desired product. Baudry and co- workers (7) also developed a sensitive radioisotopic assay based on the conversion of labeled histidine to histamine. For each determination, histamine was resolved from histidine by ion-exchange column chromatography. Following the methodology developed to measure glutamate decarboxylase in brain (EC 4.1.1.15) (8), an assay based on the conversion of histidine to histamine was also used to measure histidine decarboxylase activity. Histamine is resolved from precursor during a one-step 15min electrophoresis. In addition to rapidity, commercial 14C or 3H-labeled L-histidine can be used without further purification.

1 This work was supported by U. S. Public Health Service Grant NS-11310.

293 0003-2697/78/0871-0293$02.00/O Copyright 0 1978 by Academic Press. Inc. All rights of reproduction in any form reserved 294 SHORT COMMUNICATIONS Furthermore, blank values are fewer than 80 cpm, and the method is sufficiently sensitive to measure activity in 1 mg of brain tissue. Other enzyme sources including rat gastric mucosa and extracts of Cl. wefchii have been assayed by this procedure.

METHODS AND MATERIALS

Histidine decarboxylase assay. The final incubation mixture contained 50 PM L-[14C]histidine (uniformly labeled) (0.2 PCi) or L-[3-3H]histidine (2.0 &i), 0.1 mM EDTA, 0.01 mM pyridoxal phosphate, 0.1% Triton X- 100, and 100 mM potassium phosphate (all adjusted to pH 7.4). The alcohol containing the radioisotope as provided by the supplier was evaporated at 50°C under water aspirator vacuum immediately before use. If the radioisotope was stored with the other components overnight at -20°C, there was a large increase in the blank from 60 cpm to greater than 1000 cpm. The other components were stable at -20°C. To 5 ~1 of a four-fold concentrated solution of the final incubation mixture was added 15 ~1 of homogenate (or 10 ~1 of homogenate and 5 ~1 of reagent where specified), giving a final volume of 20 ~1 in a 1.8-ml stoppered plastic tube. In incubations longer than 90 min, a drop of mineral oil was placed over the reaction mixture to retard evaporation. After incubation at 37°C for the time specified, a 5-yl solution of 10 mM histamine, 10 mM L-histidine, and 0.2 M formic acid was added to stop the reaction. To resolve the labeled histamine from histidine, portions (10 ~1 of the 25-~1 total) were subjected to low voltage (40 V/cm) paper electrophoresis (Whatman No. 1) in 8% acetic acid/2% formic acid for 15 min (ambient temperature). After drying for 5 min at lOO“C, the carrier histamine and histidine were developed with ninhydrin spray. The leading histamine zone was cut out for radioactivity measurement by liquid scintillation spectrometry in toluene-based scintillant (Liquifluor). Cl. welchii decarboxylase was measured as described, except that the pH was adjusted to 5.0. Reagents. L-[“Clhistidine (206 Ci/mol; uniformly labeled), L-[~-~H]- histidine (14 Ci/mmol), and Liquifluor were obtained from New England Nuclear. Other reagents, drugs, and CI. welchii histidine decarboxylase were purchased from Sigma Chemical Co. Hypothalamus and cerebral cortex were dissected from male Sprague-Dawley rats (150-200 g) following decapitation. Histidine decarboxylase activity was stable in tissue stored in liquid nitrogen for 1 month. At the time of assay, the tissue was homogenized in 5 vol (w/v) of ice-cold 0.01 M sodium phosphate (pH 7.9) containing 0.1% (w/v) Triton X-100. Protein was measured by the procedure of Lowry et al. (9). SHORT COMMUNICATIONS 295

10 c

Time, hrs.

FIG. 1. Time course of the histidine decarboxylase reaction. Enzyme activity in portions (15 ~1) of rat hypothalamus homogenates were measured at the specified times as described in Methods and Materials. Background values (about 60 cpm) were obtained from parallel incubations containing extract heated at 100°C for 5 min. The labeled histamine produced per incubation mixture is given. One pmol of histamine is equivalent to 245 cpm (50.8% efficiency). (A) 16 mg of protein/ml (0): 8 mg of protein/ml; and (V) 4 mg of protein/ml; (0) boiled enzyme blank.

RESULTS Using homogenates of rat hypothalamus, the time course of formation of labeled histamine from histidine is linear for 3 hr (Fig. 1). Activity is also proportional to the amount of protein in the extract. There is little contaminant radioactivity associated with the histamine zone in the incubation lacking enzyme or boiled extract (Fig. 2). As described by Schwartz et al. (5) and Taylor and Snyder (6), the K, for L-histidine was found to be about 400 PM (not shown). Also in agreement with these workers, the enzyme exhibits a broad pH optimum of about 7. Commercially available L-[ ‘*C]histidine and L-[3H]histidine can be em- ployed without additional purification. Using cerebral cortex and hypothalamic homogenates, the respective increases in radioactivity comigrating with histamine are 250 and 600-900 cpm greater than the background, 60 to 80 cpm, after a 90-min incubation. With more-active bacterial enzyme, the reaction is still linear after 20,000 cpm (of 280,000 cpm) is converted to product after 10 min. To further distinguish between mammalian histidine decarboxylase and L-aromatic amino acid decarboxylase, specific inhibitors were examined. In agreement with Palacios and co-workers (lo), 1 mM N-methyl histidine inhibits histamine formation by 85% (Table 1). On the other hand, LX- methyldopa, an inhibitor of the aromatic acid decarboxylase, fails to inhibit histamine formation. Dithiothreitol, a thiol reagent, a-aminoisothiurium bromide, used to stabilize glutamate decarboxylase (1 I), and 296 SHORT COMMUNICATIONS

I Hislidine H 66.000 cpm l-l I I

Histamine H I I I --I+& 2 4 6 6 10 Distance, cm FIG. 2. Distribution of radioactivity in electropherograms resolving histamine and histidine. Following a 90-min incubation of rat hypothalamus extract or phosphate buffer in the histidine decarboxylase assay mixture, paper electrophoresis was performed as described in Methods and Materials. Electropherograms were then cut into l.O-cm zones, and radioactivity was measured by liquid scintillation spectrometry. Positions of carrier histamine and histidine are indicated. The cross-hatched area represents the increased radioactivity which occurred when hypothalamic extract was present during the incubation. pyridoxal phosphate, a cofactor for histidine decarboxylase (12), failed to increase or alter histidine decarboxylase activity (Table 1). When experiments were performed with Lj3H]histidine, the labeled histamine zones were counted in Tritosol (12) after extraction overnight. It was unnecessary to purify the commercially available substance further, since the blank values were less than 80 cpm. The appearance of 3H label comigrating with carrier histamine was proportional to the time of incubation and to protein content, exhibited saturation kinetics, and was abolished by prior heating (1OoOC for 5 min) of the homogenate.

TABLE 1

EFFECT OF SEVERAL CHEMICAL SUBSTANCES ON HISTIDINE DECARBOXYLASE ACTIVITY~

Activity Addition (pmoVassay)

None 3.57 N-methylhistidine, I mM 0.51 a-methyldopa, 1 mM 3.47 Dithiothreitol, 1 mM 3.64 Alpha aminoisothiurium bromide, 0.1 mM 3.48 Pyridoxal phosphate, 0.1 mM 3.46

a Histidine decarboxylase activity in lo-/~1 portions of rat hypothalamus homogenates was determined as described in Methods and Materials in the presence of 5 ~1 of the specified compounds to give the final indicated concentrations. Incubations were 90 min, and the initial extracts contained 14.5 mg of protein/ml. SHORT COMMUNICATIONS 297

DISCUSSION A radioisotopic assay for histidine decarboxylase sensitive enough to assay small amounts (1 mg) of brain tissue was developed. The precision among multiple assays is within 5% of the means. The resolution of product from precursor requires only 15 min. Seven samples per electrophoresis chamber can be processed concurrently. Commercially available isotopic compounds do not require additional purification. The procedure is satisfactory for measuring activity in discrete brain regions, in subcellular fractions, or during enzyme purification. The con- centration and specific activity of histidine can be adjusted according to enzyme activity and the experimental requirements. Aminoguanidine and quinidine sulfate, diamine oxidase (12) and histamine N-methyltransferase (14) inhibitors, respectively, did not enhance the formation of histamine using brain homogenates. Triton X-100, how- ever, enhanced activity 25% or more. This suggests that some of the enzyme is sequestered in membranous structures including synapto- somes (7,15).

REFERENCES

1. Kahlson, G., and Rosengren, E. (1968) Physiol. Rev. 48, 155-196. 2. Boeker, E. A., and Snell, E. E. (1972) in The Enzymes (Boyer, P. I)., ed.), 3rd ed., Vol. 6, pp. 217-253, Academic Press, New York. 3. Schayer, R. W. (1971) in Methods of Biochemical Analysis (Glick, D., ed.). pp. 99-117, John Wiley, New York. 4. Kobayashi, Y. (1963) Anal. Biochem. 5, 284-290. 5. Schwartz, J. C., Lampart, C., and Rose, C. (1970) J. Neurochem. 17, 1527-1534. 6. Taylor, K. M., and Snyder, S. H. (1972) J. Neurochem. 19, 1343- 1358. 7. Baudry, M., Martres, M. P., and Schwartz, J. C. (1973) J. Neurochem. 21, 1301-1309. 8. Ryan, L. D., and Roskoski, R., Jr. (1976) Neurochem. Res. 1, 37-45. 9. Lowry, 0.. Rosebrough, N., Farr, A., and Randall, R. J. (1951) J. Biol. Chem. 193, 265-275. 10. Palacios, J. M., Mengod, G.. Picatoste, F., Gran, M., and Blanco, I. (1976) J. Neuro- them. 27, 1455-1460. 11. Susz, J. P., Haber, B., and Roberts, E. (1966) Biochemistry 5, 2870-2877. 12. Schayer, R. W. (1957) Amer. J. Physiol. 189, 533-536. 13. Fricke, U. (1975) Anal. Biochem. 63, 555-558. 14. Cohn, V. H. (1965) Biochem. Pharm. 14, 1686-1688. 15. Snyder, S. H., Brown, B., and Kuhar, M. J. (1974)J. Neurochem. 23, 37-45.

ROBERT ROSKOSKI, JR. LAURA M. ROSKOSKI

Department of Biochemistry The University of Iowa Iowa City, Iowa 52242 Received Ociober 18, 1977; accepted December 27, 1977