ANALYTICAL

BIOCHEMISTRY

A Microassay

66, 194-205 (1975)

for Mammalian

Histidine

Decarboxylase

DAVID G. RITCHIE’ AND DAVID A. LEVY’ Department of Biochemical and Biophysical Sciences, The Johns Hopkins University, School of Hygiene and Public Health, Baltimore, Maryland 21205

Received July 3 1, 1974; accepted January 10, 1975 This report describes a microassay procedure for mammalian histidine decarboxylase (HDC) based on the measurement of [‘YZ]O, formed from L-[lWlhistidine. This assay is particularly useful for quick measurement of HDC activity both in microgram quantities of cell or tissue extract and in tissues that contain significant levels of endogenous histamine. Using this assay, we have shown that the pH optimum, K, and thermolability of HDC are similar for extracts prepared both from normal rat peritoneal mast cells and from the Furth mouse mastocytoma. HDC activity could be detected in homogenates prepared from IO5 rat mast cells, and it was expressed on a per cell basis. Mast cell HDC activity varied with the strain of rat from which the cells were obtained and with the season when they were assayed.

Histamine plays a prominent role in a variety of important physiological responses including gastric acid secretion, allergy, and the control of vascular permeability. Histidine decarboxylase is the major enzyme responsible for the production of histamine in mammalian tissues (1). The action of HCD3 on its substrate, L-histidine, results in the production of equimolar amounts of histamine and COZ. Currently used isotopic assays (2,3), which are based on the measurement of the amount of histamine formed during the reaction, are sufficiently sensitive to measure as little as two pmoles of this product. These assays are, however, very complex and time consuming, usually requiring more than 6 hr to complete. One of them, the method of Taylor and Snyder (3), suffers from the additional drawback of interference by high levels of endogenous histamine. This assay, in which an isotopic derivative of histamine (both endogenous and newly synthesized) is formed, is extremely ’ In partial fulfillment of the requirements of Johns Hopkins University for the degree of Doctor of Philosophy. * Presented in part at the annual meeting of the American Society of Experimental Pharmacology and Therapeutics, Atlantic City, NJ, April 8, 1974. 3 Abbreviations used: Histidine decarboxylase (HDC); mast cell(s) (MC); 4-bromo-3hydroxybenzyloxyamine dihydrogen phosphate (NSD-1055); 25diphenyloxazole (scintillation grade) (PPO); 1,4-his (2,5-phenyloxazolyl) benzene (scintillation grade) (POPOP); phosphate buffered saline (PBS). 194 Copyrisht All rights

Q 1975 by Academic Press, Inc. of reproduction in any form reserved.

HISTIDINE

DECARBOXYLASE

195

ASSAY

useful where the endogenous histamine levels are relatively low, e.g., rat brain contains 0.2-2 pmoles/mg protein (3). However, in some tissues the newly synthesized histamine is only a small fraction of that already present and therefore cannot be detected. For example, the rat mast cell contains about 300 nmoles of histamine/mg protein (4) while its HDC only produces on the order of 1 nmole/mg protein/hr. The development by Kobayashi (5) of an assay for HDC based on the evolution and trapping of radioactive CO, and its subsequent modification by Levine and Watts (6) have provided a means of avoiding these problems. While assays of this type are 2 to loo-fold less sensitive (limit of detection of about 160 pmoles of [14C]02 formed = 2 16 cpm, based on a “blank” value of 108 cpm (7)) than those based on the measurement of histamine (2,3), the procedure is greatly simplified, and the time required is decreased considerably. In this report, we present a modification of the Kobayashi method in which the reaction volume is significantly reduced (to 50 ~1) and the sensitivity increased (limit of detection 45 pmoles [ 14C]02 formed = 160 cpm, based on a “blank” of 80 cpm). We show that the assay is specific for HDC and that the pH optimum, K, and thermolability are similar for HDC from normal rat peritoneal mast cells and from the Furth mouse mastocytoma. HDC activity (pmoles of [14C]02 formed/hi-/lo6 cells) was determined for rat mast cells and was found to vary significantly with the strain of rat from which the cells were obtained. HDC activity in one strain (SpragueDawley) was found to vary with the time of year the cells were collected. MATERIALS

AND

METHODS

Chemicals. L- [ l-14C] Histidine (16.85 mCi/mmole), D-[ 1-14C] histidine ( 16.85 mCi/mmole), L-[4,5-3H]histidine (16.4 Cilmmole) and Na,[14C]03 (4.25 mCi/mmole) were obtained from New England Nuclear Corp. DL-cY-Methylhistidine, DL-a-methyldihydroxyphenylalanine (a-methyl DOPA), and pyridoxal-5’-phosphate were purchased from Sigma Chemical Co. L-Histidine was purchased from Eastman Organic Co., and D-histidine was obtained from K & K Laboratories. 4-Bromo-3hydroxybenzyloxyamine dihydrogen phosphate (NSD- 1055) was provided by Lederle Laboratories. Animals. LAF, mice were obtained from Jackson Laboratories, Bar Harbor, ME. Female Sprague-Dawley rats (200-500 g) were obtained from Charles River, Wilmington, MA, while female MAXX, BN/fMai and BUF/fMai rats were obtained from Microbiological Associates. Walkersville, MD. Preparation

tocytoma

of HDC

was originally

from

murine

mastocytoma

cells. The HC

obtained from Dr. Alan Sartorelli,

mas-

Yale Univer-

196

RITCHIE

AND

LEVY

sity, and has been maintained in the peritoneal cavity of LAF, mice as previously described (8). Seven to eight days after the injection of 5-10 X lo6 HDC mastocytoma cells into an LAF, mouse, the tumor cells were removed as follows: 2 ml of PBS (120 mM NaCl, 2 16 mM KCl, 8.1 mM Na,HPO,, 1.4 mM KH2P04, 5.5 mM glucose, 0.4 mM MgC12*6Hz0, 0.9 mM CaCl,, 70 mM sucrose, 250 mg/liter human serum albumin, pH 7.5, 340 mosM) was injected into the peritoneal cavity. The peritoneum was opened surgically, and a siliconized Pasteur pipet was used to remove the milky fluid containing the mastocytoma cells. Microscopic examination after staining with 0.1% toluidine blue revealed that more than 90% of the cells in the suspension were mastocytoma cells. This fluid was centrifuged 15 min at 1OOg at 5°C. The supernatant portion was discarded, and the remaining cell pellet was then washed with PBS and centrifuged. Then the cells were resuspended in an appropriate volume of ice-cold 50 mM sodium phosphate buffer, pH 6.6, containing 0.1% Triton X-100. This cell suspension was homogenized (10 strokes) in a 5-ml glass homogenizer with a motorized Teflon pestle. Microscopic examination of the resulting homogenate demonstrated that this procedure ruptured more than 95% of the cells, while leaving the nuclei intact. The crude cell homogenate was centrifuged for 10 min at 750g (O’C) to sediment nuclei and membranes. The remaining supernatant fluid was centrifuged for 30 min at 20,OOOg to remove mitochondria and any remaining membrane fragments. This preparation was diluted with 50 mM sodium phosphate buffer, pH 6.6, so that it contained approximately 4 mg/ml protein, as measured by the method of Lowry et al. (9) and it is designated “crude HDC.” Preparation of HDC from rat peritoneal mast cells. Except for the one experiment in which we compared strains and seasonal differences in mast cell HDC activities, the following studies were done with female Sprague-Dawley rats. PBS, 25 ml, was injected into the rat’s peritoneal cavity, and the abdomen then was massaged gently for 30 sec. The peritoneal cavity was then carefully opened and the fluid removed with a siliconiaed Pasteur pipet. For each experiment, the cells from several animals were pooled and centrifuged at room temperature for 15 min at 1OOg. Erythrocytes which contaminated the cell pellet were lysed by brief (30 set) exposure to PBS diluted 1:4 with water and then removed after centrifugation; the cell pellet, devoid of intact erythrocytes, then was resuspended in 1 ml of PBS. Mast cells and total cells were counted in a hemocytometer after suspending the cells in 0.1% toluidine blue. Approximately 1.2 X lo6 mast cells, comprising 4-5% of the total cell population, were obtained regularly from each Sprague-Dawley rat. Their viability, as checked by trypan blue, was greater than 90%. The cells were then centrifuged for 5 min at 350g and resuspended in an

HISTIDINE

OBSERVED

MOLAR

RATIOS

PW,

Mastocytoma HDC

(pmolesk)

10 jd 20 /.d

DECARBOXYLASE

341 696

197

ASSAY

TABLE 1 OF CO,: HISTAMINE

IN

[3H]Histarnine (pmoles/hr)

HDC ASSAY Ratio CO,: histamine

362 714

0.959 0.975

appropriate volume (100 ~l/lO~ mast cells) of homogenizing buffer. Crude HDC was obtained from these rat cells in the same manner as described above for the murine mastocytoma. An alternative method was utilized to obtain the HDC levels shown in Table 1. Peritoneal cells from each rat were centrifuged at 1OOg for 1.5 min. The supernatant fluid was discarded and 100 ~1 of cold 50 mM sodium phosphate buffer, pH 6.6, was added. The cell suspension was then sonicated for 5 set at 0°C using a Bronwill Biosonik apparatus (Bronwill Scientific, Rochester, NY). The homogenate was then centrifuged at 750g for 10 min and the supernatant portion was assayed as usual for HDC activity. Equivalent mast cell HDC activities were obtained with both methods. HDC assay. Each assay tube contained a final concentration of the following components in a total reaction volume of 50 ~1: 50 mM sodium phosphate buffer, pH 6.6, either L- or o-[ l-14C] histidine ( 1 x lob3 M, 1.2 mCi/mmole, 60-70 nCi); pyridoxal-5’-phosphate ( 1 X 10m5M) ; and O-40 ~1 of crude enzyme. The above reactants were combined at 0°C in a 10 X 35-mm glass test tube (Fig. la). The assay tubes were then sealed with rubber stoppers (Arthur H. Thomas Co., Cat. No. 1781-E90), shaken for 30 set by hand to mix the reactants and, except where otherwise stated, incubated routinely in a water bath at 37°C for 60 min. The reaction was stopped by

L4C02Formed la

lb

IC

FIG. 1. Samples of crude HDC are sealed and incubated for 60 min at 37°C (la). The reaction is stopped with 0.1 ml of 1 M citric acid at which time the tubes are sealed in scintillation vials containing fluor (lb). The reaction tubes then are displaced with a wire probe and allowed to fall into the fluor where [‘V]O, trapping occurs (1~).

198

RITCHIE

AND

LEVY

the addition of 0.1 ml of 1 M citric acid which was injected through the rubber cap with a tuberculin syringe fitted with a 25gauge needle. The incubation tubes were then sealed inside low-potassium grade scintillation vials containing 1 ml of ethanol, 0.3 ml of ethanolamine:2-methoxyethanol mixture (1: 2 v/v), and 5 ml of scintillation fluor (4 g of PPO, and 50 mg of POPOP per liter of toluene) as shown in Fig. lb. The assay tubes then were dislodged gently from the stoppers into the scintillation fluor by inserting a sharpened wire probe through the rubber stopper (Fig. Ic). The reaction mixture was never in direct contact with fluor. The [14C]02 that evolved from the acidic reaction mixture was trapped in the fluor at room temperature during the next 60 min. Then the rubber stoppers were removed and the reaction tubes lifted from the scintillation vials. The vials were next filled with an additional 5 ml of scintillation fluor and the radioactivity counted in a Beckman LS 200B liquid scintillation spectrometer. Less than 3 hr, excluding counting time, was required to assay 10 samples in triplicate. The counting efficiency was calculated by using a [14C]toluene standard and found to be 88%. Assay “blanks” containing either the reaction mixture with no enzyme, enzyme plus NSD-1055 (10e4 M), boiled enzyme, or the L-[l14C] histidine replaced by D- [ 1-14C] histidine of identical specific activity and concentration were carried through the entire procedure. Each type of blank yielded 80 +- 6 cpm. Each sample was assayed in triplicate and the resulting radioactivity (cpm) was averaged and the cpm of the blank was subtracted. Enzyme activity was calculated by dividing the mean cpm by the specific activity of the substrate (cpm/nmole of L-[ l-14C] histidine) and expressed either as nmoles or pmoles of [14C]0, formed/hr. One unit (U) of enzyme activity is defined as the amount of enzyme that forms 1 pmole of [ 14C] OJhr at 37°C under our assay conditions. RESULTS

In order to estimate the relationship between [14C]02 produced and [ 14C] 0, recovered, the [ 14C]Oz-trapping efficiency of the scintillation fluor was determined. Na,[14C]0, (18 nCi) was added to a reaction mixture lacking labeled substrate. Following acidification with citric acid, the [14C]0, formed was trapped in the scintillation mixture for 60 min. This resulted in absorption of 91.5 -+ 4.8% (2 * SD, N = 10) of the evolved [14C]02. When the same procedure was extended to 120 min, 92.7 + 5.6% (z? SD, N = 6) of the evolved [‘4C]0, was absorbed Subsequently, 60 min were always allowed for trapping of the [‘“C]O, produced in the enzymatic reaction. In calculating enzyme activity, [ 14C] O2 recoveries were corrected to 100% trapping. The reaction rate and the effect of substrate concentration were studied with the murine enzyme. The results are shown in Fig. 2. It can be

HISTIDINE

DECARBOXYLASE 200

L =

199

ASSAY

b

160

g 140 $ 120 0” 100 P 80 : 2 60 =

40 2.0

lb

io

TIME OF INCUBATION

30

$ 160 0 I_ 0

AT 37’Cthrsl

100 200 300 AMOUNT OF PROTEIN lpg)

400

FIG. 3. (a) Time course for the HDC reaction. Mastocytoma crude HDC (5 ~1) was incubated for varying time intervals over a 3-hr period. At each time-point samples were removed and acidified to stop the reaction. Then the amount of product formed was measured. (b) The effect of enzyme concentration on reaction rate. Varying amounts of mastocytoma crude HDC (5-40 ~1) were incubated for 1 hr at 37°C.

seen that decarboxylation proceeded in a linear fashion for two hours at 37°C (Fig. 2a). Figure 2b shows that, in the presence of increasing amounts of crude enzyme, the reaction remained linear until approximately 10 nmoles of the substrate were consumed. Since the reaction mixture contained 50 nmoles of L-histidine, loss of linearity did not occur until more than 20% of the substrate had been consumed. It is at this point that the substrate concentration (8 X 10e4 M) begins to approach the K, for the enzyme, and therefore the reaction rate decreases. Nevertheless, under these conditions, tissue samples extending over a

l!!!rb z 0 E 0” t6

.

-

120

400

so

H 2 s

200

40

2

2 : a I

I

s 2

600

iI2

6.0 6.2 6.4 6.6 PH 6.6 70 Z2 74

1

FIG. 3. pH optima for mouse mastocytoma cell and rat peritoneal mast cell HDC. Ten microliters of either mastocytoma or rat crude HDC was mixed with 30 ~1 of the appropriate sodium phosphate buffer. The final pH was measured and the reaction was allowed to proceed for I hr at 37°C.

200

FIG. 4. Lineweaver-Burke

RITCHIE

AND

LEVY

plot of initial velocities (v) vs substrate concentrations

PI.

220-fold range in enzyme activity may be assayed without prior dilution. The effect of pH on HDC activity is shown in Fig. 3. The optimum pH for the reaction was found to be 6.6 when the initial histidine concentration was 1 X lop3 M. This contrasts dramatically with the pH optimum for DOPA decarboxylase, a nonspecific histamine-forming enzyme which has previously been detected in these cells (10). DOPA decarboxylase is reported to have a pH optimum close to 9 and, in addition, requires higher histidine concentrations ( 1O-2-1O-1 M) for saturation at pH 6-7 (11). The Michaelis constant (K,) for the mastocytoma HDC (Fig. 4) was found to be 6.7 X 1O-4 M at pH 6.6. Since the K, for DOPA decarboxylase when using histidine as substrate was reported to be 2.2 x lop2 M (1 l), a final L-histidine concentration of 1 X lop3 M was selected for the HDC assay in order to be slightly above the K, for the specific enzyme, yet well below the K, for DOPA decarboxylase. Thus, by taking advantage of differences in substrate affinity and pH optima between the two enzymes, decarboxylation of the substrate could be attributed completely to HDC, with little contribution from the DOPA decarboxylase which the crude extract might also contain. HDC is reported to be stereospecific with respect to its substrate (6). To test this finding, D- [ l-14C] histidine of identical specific radioactivity and concentration was tested as substrate in place of L- [ l-14C] histidine. The amount of [14C]02 evolved from a reaction mixture containing D-[ I-14C]histidine and HDC was similar to that found with L-[ l14C]histidine in the absence of HDC. In order to confirm the theoretical equimolar ratio of CO,: histamine, L-[4,S3H]histidine (152 ncilreaction, 1 X 10e3 M) was used in place of L-[ l-14C]histidine as substrate. Following a 1-hr incubation with two different amounts of HDC, the reaction was stopped by a IO-min incubation at 100°C. r3H]Histamine was separated by paper chromatography (2 propanol:chloroform:ammonium hydroxide:water, 11:5:2:2) and

HISTIDINE

DECARBOXYLASE

201

ASSAY

100 -

g F 5 zz

BO-

s

40-

_

NSD-1055

60-

L-d

Methyl Msfldire

B a f 0

./..

I 10-7

L-0( Methyl DOPA

I I I I U6 IO10-4 lo-’ INHIBITOR CONCENTRATIONIMI

I IO-

FIG. 5. Effect of inhibitors on HDC activity. A constant amount of mastocytoma H DC was incubated under standard conditions in the presence of NSD-1055 or a-methylhistidine, both specific inhibitors of HDC, or a-methyl DOPA, a specific inhibitor of DOPA decarboxylase.

counted in a Triton X-100: toluene (1: 2 v/v) scintillation fluor. As shown in Table 1, the observed CO,: histamine molar ratios approximated the expected ratio. A number of reagents has been shown to inhibit HDC. Figure 5 shows that mastocytoma HDC was inhibited by NSD-1055 (Is,, = I.5 x lo-’ M), a specific competitive inhibitor (12) and by ~-amethylhistidine (I,, = 6.5 x lop4 M), a relatively weak inhibitor of HDC activity (13). In contrast, the addition of up to 9 X 10e3 M L-a-methyl DOPA, a specific inhibitor of DOPA decarboxylase (14) did not inhibit crude mastocytoma HDC. We determined the precision of the assay in two ways. First, crude mastocytoma HDC containing 42 mg of protein/ml was divided into 200~~1 aliquots and frozen at -70°C. On each of the next 5 consecutive days, one of these aliquots was thawed and assayed in triplicate. The mean HDC activity was determined to be 1,900 pmoles of [14C]OZ/hr with a standard deviation of 140 pmoles of [‘“C]O,/hr. Enzyme preparations stored in this manner were found to be stable for as long as 7 months. The second test for precision was performed as follows: Five replicate samples from a single aliquot were assayed and had a mean activity of 2,000 pmoles of [‘“C]O,/hr with a standard deviation of 110 pmoles/hr. Measurement

of HDC from Rat Peritoneal

Mast Cells

Following the isolation of crude HDC from normal rat peritoneal mast cells, the pH optimum and K, of this enzyme were studied. Figures 3 and 4 show that rat mast cell HDC has a pH optimum (6.6) and a K,,, (9.1 X 10e4 M) similar to those for the murine HDC. We therefore judged that the rat HDC could be assayed under the same conditions

202

RITCHIE

AND

LEVY

IO 20 30 40 50 TIME

AT

55-C

(MIN)

6. Heat denaturation kinetics: A comparison between normal rat peritoneal HDC and mouse mastocytoma HDC. Four aliquots each of mouse and rat HDC were incubated in 50 mM sodium phosphate buffer, pH 6.6, for various time intervals at 55°C in a water bath. After all samples had been withdrawn from the bath and placed in ice water, the remaining enzyme activity in each aliquot was assayed. FIG.

used for the murine enzyme. As a further test of similarity, the thermolability of crude preparations of these two enzymes was studied. Denaturation was carried out at 55°C. As shown in Fig. 6, both mouse HDC and rat HDC lost 50% of their activity after 12 min at this temperature. Following isolation and counting of rat mast cells, subsequent assay for HDC activity resulted in a linear correlation between extractable enzyme activity and mast cell number (Fig. 7). In order to substantiate this observation further, rat mast cells were purified, by a modification of a procedure described by Uvnas and Thon (15), to better than 80% with a 30% Ficoll solution, washed twice with PBS and counted, and the crude

2 a Y

600

600 E 0" 5-O 400 B d I: 200 a

1.0 20 3040 NUMBER

OFRAT

50 60 MASTCELLSblO

?

7. Relationship of HDC activity to mast cell number. Peritoneal cells were obtained from three female Sprague-Dawley rats. Crude HDC was prepared and diluted so that 40 ~1 contained the enzyme from 6 X lo5 mast cells. Then 40-, 20-, lo- and S-p1 samples of this preparation were assayed in triplicate. FIG.

HISTIDINE

DECARBOXYLASE

TABLE HISTIDINE

DECARBOXYLASE

LEVELS

203

ASSAY

2 IN

RAT PERITONEAL MAST CELLS HDC

a

Strain

No. ofrats

(units/IO6 MC)

BUF/f Mai female (2174 band S/74)

II

450 t 50’

11

180 2 20d

5

110 ‘- 1.5’

MAXX

female (2/74 and 5/74) BNf Mai female (5/74) a 1 unit = 1 pmole of [‘T]O, * Dates of assay. c Significantly different from d Significantly different from e Significantly different from

formedhr at 37°C. MAXX and BN (p < 0.01). BUF only 0, < 0.01). BUF only (p < 0.01).

HDC was extracted. Enzyme activity from these cells was found to be 400 pmoles of [*“Cl O2 formed/hr/ lo6 mast cells compared to 3 60 & 40 pmoles of [ 14C] O2 formed/hr/ 1O6 mast cells. These values are very similar and indicate further that the nonmast cell fraction in the normal peritoneal cell population probably contains no measureable HDC. Using this technique, we measured HDC levels in mast cells from four inbred strains of rats. Female Sprague-Dawley rats, when assayed over a 7-month period, showed the following enzyme activities: in 10/73, 1,000 ? 80 units/lo6 MC; in 3/74, 360 + 40 units/lo6 MC; and in 5/74, 165 -+ 10 units/lo6 MC. In contrast to these observations, enzyme activities from three other strains were constant over a 3-month span. Within this period, strain differences in enzyme activity were noted. As listed in Table 2, HDC activity in mast cells from BUF rats (450 U/lo6 MC) was significantly greater (p < 0.01) than from MAXX (180 U/10” MC) and BN (110 U/IO6 MC) rats. DlSCUSSlON

Our modification of the HDC assay of Levine and Watts (6) has led to substantial improvements in sensitivity of the assay and economy of the isotope. These improvements have been made possible through changes in both volume (40-fold less) and substrate concentration (fourfold greater) than that used by Levine and Watts (6). Under the conditions described above, the reaction was linear over more than a 200-fold range in enzyme activity, and the synthesis of as little as 45 pmoles of [14C]0, gave radioactivity (cpm) twice that of the blank. We have used this assay to show that HDC from two different sources, viz., the normal rat mast cell and the mouse mastocytoma, have

204

RITCHIE

AND

LEVY

similar pH optima and Michaelis-Menten kinetics. We have also found that HDC from rat and mouse cells have identical inactivation times at 55°C. According to Paigen (16), thermal inactivation provides a sensitive test for changes in primary protein structure. The probability that a random amino acid substitution produces a protein with altered thermolability is more than 50%, as opposed to a 25% probability for a similar substitution to cause a change in the electrophoretic mobility of a protein. While comparative data on the thermolability of HDC from other tissues are not generally available, Leinweber (17) found that the half life of gastric HDC at 53°C was 12 min, a result similar to ours. Thus, the similarity of the thermolabilities suggests that the enzyme from these three sources may have the same structure. HDC activity in mast cells from different inbred strains of rats was investigated. Using female Sprague-Dawley rats, we found that the specific enzyme activities showed significant variations according to the period of the year the cells were harvested for assay. The largest change in mast cell HDC activity occurred between fall and winter, with a smaller yet significant change occurring between winter and spring. Enzyme levels in three other strains, when assayed in March and May, remained constant and were therefore used for interstrain comparison. Mast cells from BUF rats were found to have significantly greater specific activity (450 units/lo6 MC) than cells from either MAXX rats (180 units/lo’ cells) or BN rats (110 units/lo6 cells). These differences deserve further investigation. ACKNOWLEDGMENTS The authors thank Dr. Aileen Ritchie of the Carnegie Institution of Washington and Dr. Elliott Richelson and Dr. P. C. Huang of the Johns Hopkins Medical Institutions for helpful advice. This work was supported in part by U.S.P.H.S. grants, No. AI-08104 and AI-11281.

REFERENCES 1. Aures, D., and Hakanson R. (1971) in Methods in Enzymology (Tabor, H., and Tabor, C. W., eds.), Vol. 17B, pp. 667-677, Academic Press, New York. 2. Schayer, R. W., Rothschild, Z., and Bizony, P. (1959) Amer. J. Physiol. 196, 295-298. 3. Taylor, K. M., and Snyder, S. H. (1972)J. Neurochem. 19, 1343-1358. 4. Levy, D. A., and Ritchie, D. G. (1974) Fed. Proc. 33, 561 (Abstract). 5. Kobayashi, Y. (1963) Anal. Biochem. 5, 284-290. 6. Levine, R. J., and Watts, D. E. (1966) Rio&em. Pharmacol. 15, 841-849. 7. Maudsley, D. V., Radwan, A. G., and West, G. B. (1967) Brit. J. Pharmacol. Chemother.

31, 3 13-3 18.

8. Minard, P., and Levy, D. A. (1972)J. Immunol. 109, 887-890. 9. Lowry, 0. H., Rosenbrough, N. J., Farr, A. L., and Randall, R. J. (1951) J. Biol. Chem.

193, 265-275.

10. Day, M., and Green, J. P. (1962) J. Physiol. 164, 210-226. 11. Ames, D., Davidson, W. D., and Hgkanson, R. (1969) Eur. J. Pharmacol.

8,

100-107.

HISTIDINE

12. 13. 14. 15. 16.

DECARBOXYLASE

ASSAY

Leinweber, F. J. (1968) Mol. PharrnacoL 4, 337-348. Reid, J. D., and Shepherd, D. M. (I 963) Life Sri. 2, 5-8. Kahlson, G., and Rosengren, E. (1968) Physio/. Rev. 48, 155-196. UvnPs, B., and Thon, I. L. (1959) Exp. Cell Res. 18, 5 12-520. Paigen, K. M. (1971) in Enzyme Synthesis and Degradation in Mammalian (Rechcigl. M., Jr., ed.), pp. Z-40. University Park Press, Baltimore, Tokyo.

17. Leinweber,

F. J., and Braun. G. (1969) Mol. Pharmucol. 6. 146-155.

205

Systems London.