Axmuc~~
JOURNAL O? PHY810LocY Vol. 230, No. 5, May 1976. Printed in USA.
Histidine synthesis
decarboxylase-mediated histamine in glomeruli from rat kidneys
JAMES I. HEALD AND THEODORE M. HOLLIS Department of Biology, Pennsylvania State University,
HEALD, JAMES I., AND THEODORE M. Ho~~~s.Histidine decarboxylase-mediated histamine synthesis in glomeruli from mt kidneys. Am. J. Physiol. 230(5): 1349-1353. 1976. -Enzymatic histamine synthesis by renal glomeruli of the rat has been examined. Assays of the partially purified enzyme demonstrated a pH optimum of 6.2, a Michaelis-Menten constant of 2.4 x 10B4 M histidine, and lack of potentiation by benzene. These data thus indicate that renal glomeruli contain the histidine-specific histidine decarboxylase.
PYRIDOXAL PHOSPHATE-DEPENDENT, histidine-decarboxylating enzymes exist in mammals (2, 14, 20). One enzyme, nonspecific aromatic L-amino acid decarboxylase (EC 4.1.1.28), acts on both histidine and aromatic amino acids, notably L-3,4-dihydroxyphenylalanine (L-DOPA), and is commonly referred to as L-DOPA decarboxylase. The other enzyme, i.e., mammalian Lhistidine apodecarboxylase (EC 4.1.1.22), is specific for bhistidine and is commonly referred to as histidine decarboxylase (HD). Histamine formation in rabbit and guinea pig renal homogenates has been generally regarded as being mediated by L-DOPA decarboxylase, since the enzyme shows low affinity for histidine (2,20), is potentiated by addition of benzene (14, 20), and has a relatively high pH optimum (14, 20). Similarly, histamine formation in rat kidney is increased in the presence of benzene (15, 19), leading to the conclusion -that the rat kidney histamine-forming enzyme is likewise LDOPA decarboxylase. However, if L-DOPA decarboxylase is the histamine-forming enzyme in rat kidneys, than administration of alpha-methyl-DOPA should result in decreased urinary histamine levels, since this inhibitor is selective for L-DOPA decarboxylase (2, 20) and since renal decarboxylation of histidine is a major source of urinary histamine (13); this has not been the case in rats (5, 8). Our laboratory has established that endothelial cells isolated from bovine (4) and rabbit (11) aortas demonstrate relatively high histidine decarboxylase activity. Studies of Schayer (13) indicate that rat kidneys form and excrete histamine directly into the urine. Since alpha-methyl-DOPA fails to decrease histamine excretion, and in light of the recent identification of histidine decarboxylase activity in vascular endothelium, the present study was designed to determine if glomeruli of rat kidneys contain specific histidine decarboxylase. TWO
University
MATERIALS
AND
Park, Pennsylvania
16802
METHODS
Male Wistar rats, 300-350 g were used in all studies. Animals were anesthetized by sodium pentobarbital (33 mg/kg body wt, ip), and kidneys were exposed by midline laporotomy. The abdominal aorta was cannulated using PE-90 tubing (Clay-Adams, N. Y .), and kidneys were subsequently perfused with cold N-2-hydroxyethylpiperazine-N-2-ethane-sulfonic acid (HEPES) buffered Earle’s balanced salt solution, pH 7.4. Perfusion pressure never exceeded 100 mmHg. Following perfusion, kidneys were quickly excised and placed in ice-cold Earle’s balanced salt solution. The kidneys were decapsulated and demedullated. Glomeruli were then isolated from the renal cortex by a modification of the methods of Krakower and Greenspon (7) and Spiro (17). Cortical slices were gently forced through a no. 140 stainless steel sieve, 106,mm opening, using the bottom of an ice-filled beaker to apply pressure. Tissue was washed through the sieve and collected in ice-cold phosphate-buffered saline, pH 7.2. This sieved material was then poured through two stainless steel sieves in series; the top sieve had 180.mm openings and the bottom had 90-pm openings. Glomeruli were collected from the final sieve with 0.1 M sodium acetate buffer, pH 5.5, 1 ml/kidney. After homogenization and centrifugation, histidine decarboxylase was partially purified from the supernatant as described by Hakanson (3). This involves heating the supernatant solution in a 55OC water bath for 5 min, followed by ammonium sulfate fractionation at O4°C. Material which precipitated between 25 and 40% saturation was redissolved in a small volume of 0.1 M sodium phosphate buffer, pH 7.0. Following dialysis against redistilled water, a second ammonium sulfate fractionation was conducted, and protein which precipitated between 28 and 42% saturation was resuspended in 0.01 M sodium phosphate buffer, pH 7.0. After dialysis, this material was used for all histidine decarboxylase assays. Protein concentrations were determined by the method of Lowry et al. (10). Histidine decarboxylase activity was determined by a modification of the double isotopic microassay of Taylor and Snyder (18) which involves two incubation media. Initially, a 25-~1 sample is diluted to 45 ~1 with 0.05 M sodium phosphate buffer, pH 6.2, containing 0.025 mM pyridoxal phosphate. After 5 ~1 of 0.1 M L-histidine are added, the medium is incubated at 37°C for 90 min. Blanks are prepared with n-histidine. After stopping
1349
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1350 the reaction bv boiling in a water bath for 10 min. the pH is adjusted to 7.9 with 0.1 M NaOH, and 20-~1 samples are removed to assay for histamine produced. The histamine assay utilizes imidazole-N-methyltransferase (HMT, EC 2.1.1.8) from fresh guinea pig brain which methylates histamine to form l-methyl-(@-aminoethyl)imadazole (methylhistamine). In this incubation, 10 ~1 of HMT solution are added to the sample and incubated for 1 h at 37°C. The HMT solution contains one part SP4Cladenosylmethionine (5.0 @i/ml), one part 13Hlhistamine (0.64 $X/ml), and two parts of guinea pig brain supernatant. HMT transfers of 14Clabeled methyl group from S-adenosylmethionine to
J. I. HEALD
AND
T.
M.
HOLLIS
histamine, forming [14Clmethylhistamine. 13Hlhistamine and S [14C]adenosyImethionine are separated from 14C, [3H]methyhistamine, and [14C]methylhistamine by extracting the methylhistamines from an alkaline, salt-saturated solution into chloroform. The amount of histamine formed per sample is determined by the ratio: 14C:3H. In summary, the following modifications were made: 1) supernatants of crude guinea pig brain homogenates were used as the source of HMT instead of the partially purified enzyme, and 2) pyridoxal phosphate was added to the incubation medium with the buffer instead of with the substrate in order to allow time for the cofactor to react with the enzyme before beginning
FIG. 1. Phase contrast of glomeruli isolated from Magnification is x 553.
photograph rat kidney.
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GLOMERULAR
HISTAMINE
TABLE 1. Purification
of histidine Protein,
S@P
1351
SYNTHESIS
Heat treatmentt
&ml
276
decarboxylase
from rat renal glomeruli
Vol, ml
Units per ml*
3.70
1,942
Sp Act, units 1 mg
Purif. Fold
Total Act
Yield
7,025
1
7,185
100
Ammonium tionation
sulfate IS
frac-
24.5
2.60
2,446
99,857
14.2
6,360
89
Ammonium tionation
sulfate IIt
frac-
7.0
2.30
1,922
274,500
39.1
4,421
62
Rurif. fold-sp
act, Ammonium
sulfate fkaction/sp act, heat treatment. * One unit = 1 pmol histamine formed per hour of incubation. $ Protein precipitated between 25 and 40% saturation in 0.1 M sodium acetate precipitated between 28 and 42% saturation in 0.1 M sodium phosphate buffer, pH 7.0.
t 55OC for 5 min in 0.1 M sodium acetate buffer, pH 5.5. buffer, pH 5.5.
9 Protein
the reaction with addition of L-histidine. Radioactivity was counted in Aquasol (Nuclear Associates, Westbury, N. Y.) using a Unilux II scintillation spectrometer (Nuclear-Chicago Corp., Chicago). RESULTS
s m
Figure 1 is a representative phase contrast photomicrograph of an aliquot of sieved renal tissue prepared as previously described. It is evident that these glomeruli are intact, some surrounded by a tightly adhering membrane which is presumably Bowman’s capsule. No distinct tubular material is present in this photomicrograph. By counting all cellular material showing definitive organization, we observed less than 2% contamination by tubular elements in the least highly purified tissue preparation used for this study. Table 1 presents summary data relative to the influence of ammonium sulfate fractionation on specific activity of histidine decarboxylase, employing techniques by Hakanson (3). As indicated, with respect to the heatH istamine, ng treated supernatant solution, the histidine decarboxylFIG. 2. Linearity of double isotopic microassay. Each point reprease purification procedures employed in the present sents mean (2 SE) of 3 determinations. pH was 6.2 and histidine was study resulted in an approximate 40.fold increase in 10e2 M. Counting efficiency for 14C was 65% and for 3H was 22%. eflzyfne specific activity; the yield was 62%. As indicated in Fig. 2, the double isotopic microassay employed was linear between 0.05 and 5 ng histamine per sample. Counting efficiency for 14C was 65% and for 3H was 22%. 14C overlap into the tritium channel was 8.8%, whereas overlap into the 14C channel was 0.6%. The effect of pH on activity of the partially purified enzyme at a substrate concentration of 10B2 M is illustrated by Fig. 3. From inspection of this figure, it is apparent that the optimal pH is 6.2. A double reciprocal plot was employed for analysis of the results of the enzyme kinetic study (Fig. 4). From this analysis, the apparent K, for histidine is 2.4 x 10B4 M. Benzene, added to the HD incubation medium, failed to stimulate activity of the enzyme (Table 2). DISCUSSION
Kinetic analysis of partially purified enzymes is the most reliable method currently available for identification of histidine decarboxylating enzymes (1). The two histidine decarboxylating enzymes may also be distinguished by the large difference in their pH optima (20). Using such analyses, the purified glomerular enzyme
5.4
5.8
6.2
6.6
7.0
7.4
78
PH 3. Effect of pH on purified histidine decarboxylase activity. Histidine concentration was 10s2 M. Each point represents mean (+ SE) of at least 3 determinations. FIG.
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1352
J. I. HEALD
AND T. M. HOLLIS
TABLE 2. Effect of benzene on glomerular decarboxylase ac’tivity Sample
Without
0.16
Benzene
nglhistuminelmgprotin
?J 012 l
0.08
1
6,479
2 3 4
8,147 7,623 9,576
Mean _+ SE * Five microliters
I I
CHistidinel
1
I
I
I
I
2
4
6
8
10
_-
Ifi
3
‘I”
FIG. 4. Double reciprocal plot for histidine of partially purified histidine decarboxylase from renal glomeruli of rat at pH 6.2. Initial velocity (v) is expressed as nanograms histamine formed per hour. Each point represents mean ( + SE) of at least 3 determinations.
demonstrated an apparent Michaelis-Menten constant of 2.4 x 10e4 M for histidine and a pH optimum of 6.2 (using lo-* M histidine). Hakanson (3) and others (12) have shown that the optimal pH of histidine decarboxylase varies inversely with histidine concentration. Aures and Hakanson (1) summarized reports of pH optima using lOa2 M histidine for the enzyme from whole fetal rat, adult rat stomach, hamster placenta, and murine mastocytoma. They found that the pH optimum had a range of from 5.5 to 6.0. Hakanson (3) has also indicated that the active form of the enzyme protein is more abundant at an acid pH. Renal DOPA decarboxylase, on the other hand, is most active in alkaline solutions, i.e., pH 8.0-9.5, (2, 9, 10) and has an optimum pH independent of histidine concentration (1). The pH optimum of the decarboxylase examined in the present study is thus consistent with that of histidine decarboxylase. The apparent K, of histidine decarboxylase varies inversely with the pH (3, 12). At a pH of 6.2, we found
7,956
histidine With Benzene*
per h
6,384 7,194 6,813 7,813
,+ 645
added to incubation
7,05’1 ,+ 303
medium.
an apparent K, of 2.4 x 10e4 M. Using human gastric carcinoid tumor HD, No11 and Levine (12) obtained values of 2.08 x 10B4 M and 3.85 x 10s4 M at a pH of’6.57 and 3.85, respectively. Hakanson (3) has indicated that the enzyme appears to require the anionic form of histidine as its true substrate. If the K, for anionic histidine is determined, using the Henderson-Hasselbalch equation to calculate the proportion of total histidine which is present as the anionic form (using a pK’ of 1.82, 9.17, and 6.0 for the carboxyl, amino, and imadazole groups, respectively), a value of 1.6 x 10D7 M is obtained. Hakanson (3) reports a “true” K, of 6.7 x 10e7 M for whole fetal rat, whereas No11 and Levine (12) found a true K, of 4 x low7 M for human gastric carcinoid tumor histidine decarboxylase. In contrast, renal DOPA decarboxylase has a much lower affinity for histidine with a K, in the range of 10 -‘-lo-* M (2, 9, 20). From the kinetic and pH determinations and from the failure of benzene potentiation, we conclude that renal glomeruli of the rat contain the specific histidine decarboxylase. This may explain the failure of alpha-methylDOPA to decrease rat urinary histamine levels. Glomerular HD would be capable of excreting histamine directly into the glomerular filtrate, thus contributing to histamine which is excreted directly into the urine, i.e., without first entering the circulation. Such direct excretion of histamine into urine has been found in the mouse, dog, and guinea pig as well as the rat (13,16). Received for publication
10 December 1975.
REFERENCES D., AND R. HAKANSON. Histidine decarboxylase (mammalian). In: Methods in EnzymoZogy,edited by H. Tabor and C. W. Tabor. New York: Academic, 1971, vol. l7B, p. 667-677. GANROT, P. O., A. M. ROSENGREN, AND E. ROSENGREN. On the presence of different histidine decarboxylating enzymes in mammalian tissues. Experientia 17: 263-264, 1961. HAKANEUIN, R. Histidine decarboxylase in the fetal rat. &o&em. Phurmacol. 12: 1289-1296, 1963. Hours, T. M., AND L. A. ROSEN. Histidine decarboxylase activity of bovine aortic endothelium and intima-media. Proc. Sot. Exptl. Biol. Med. 141: 9’78-981, 1972. KAHLSON, G., E. ROSENGREN, AND B. THUNBERG. Observations on the inhibition of histamine formation. J. PhysioZ. London 169:
1. AURES,
2.
3. 4. 5.
467-486,1963. 6. KAHLSON, G., AND
E. ROSENGREN. Histamine: entering physiology. Experientiu 28: 993-1002, 1972. 7. KRAKOWER, C. A., AND S. A. GREENSPON. Localization of the nephrotoxic antigen within the isolated renal glomerulus. A.MA. Arch. Pathol. 51: 629-638, 1951. 8. LEVINE, R. J., T. L. SATO, AND A. SJOERDSMA. Inhibition of histamine synthesis in the rat by cr-hydrazino analog of histidine
and 4-bromo-3-hydroxy 14: 139-149, 9. LOVENBERG,
benzyloxyamine.
Biochem.
Pharmacol.
1965.
W. Aromatic L-amino acid decarboxylase (guinea pig kidney). In: Methods in Enzymology, edited by H. Tabor and C. W. Tabor. New York: Academic, 1971, vol. 17B, p. 652-656. 10. LOWRY, 0. H., N. J. ROSEBROUGH, A. L. FARR, AND R. J. RANDALL. Protein measurement with the Folin phenol reagent. J. BioZ. Chem. 193: 265-275, 1951. 11. MARKLE, R. A., AND T. M. HOLLIS. Rabbit aortic endothelial and medial histamine synthesis following short-term cholesterol feeding. Exptl. Mol. PathoZ. 23: 417-425, 1975. 12. NOLL, W. W., AND R. J. LEVINE. Histidine decarboxylase in gastric tissues of primates. Biochem. PharmacoZ. 19: 1043-1053, 1970. 13. SCHAYER,
R. W., K. Y. TAX WV, AND R. L. SMILEY. Sources of histamine in the rat. Am. J. Physiol. 179: 481-485,1954, 14. SCHAYER, R. W. Histidine decarboxylase of rat stomach and other mammalian tissues. Am. J. Physiol. 189: 533-536, 1957. 15. SCHAYER, R. W. Enzymatic formation of histamine from histidine. In: Handbook of Experimental PharmacoZogy, edited by 0. Eichler and A. Farah. Berlin: Springer, 1966, vol. 18, p. 688-725.
urinary
Downloaded from www.physiology.org/journal/ajplegacy by ${individualUser.givenNames} ${individualUser.surname} (163.015.154.053) on August 22, 2018. Copyright © 1976 American Physiological Society. All rights reserved.
GLOMERULAR
HISTAMINE
R. W., AND M. A. REILLY. Effect of thyroxine on histamine metabolism in mice. Agents Actions 5: 226-230, 1975. 17. SPIBO, R. G. Studies on the glomerular basement membrane. J. Bid. Chem. 242: 1915-1922, 1967. 18. TAYLCIB, K. M., AND S. H. SNYDER. Isotopic microassay of histamine, histidine, hi&dine decarboxylase and histamine methyl16. Sc~ayxm,
1353
SYNTHESIS
transferase in brain tissue. J. Neurochem. 19: 1343~1358,197Z. J. M., AND G. B. WEST. The formation of histamine in the rat. J. Pharm. Pharmacol. 13: 75-82, 1961. 20. WEISSBACH, H., W. LOVENBERG, AND S. UDENFRIEND. Characteristics of mammalian histidine decarboxylating enzymes. B&him. Biophys. Acta 50: 177-179, 1961. 19. T~LFORD,
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JOURNAL O? PHY810LocY Vol. 230, No. 5, May 1976. Printed in USA.
Histidine synthesis
decarboxylase-mediated histamine in glomeruli from rat kidneys
JAMES I. HEALD AND THEODORE M. HOLLIS Department of Biology, Pennsylvania State University,
HEALD, JAMES I., AND THEODORE M. Ho~~~s.Histidine decarboxylase-mediated histamine synthesis in glomeruli from mt kidneys. Am. J. Physiol. 230(5): 1349-1353. 1976. -Enzymatic histamine synthesis by renal glomeruli of the rat has been examined. Assays of the partially purified enzyme demonstrated a pH optimum of 6.2, a Michaelis-Menten constant of 2.4 x 10B4 M histidine, and lack of potentiation by benzene. These data thus indicate that renal glomeruli contain the histidine-specific histidine decarboxylase.
PYRIDOXAL PHOSPHATE-DEPENDENT, histidine-decarboxylating enzymes exist in mammals (2, 14, 20). One enzyme, nonspecific aromatic L-amino acid decarboxylase (EC 4.1.1.28), acts on both histidine and aromatic amino acids, notably L-3,4-dihydroxyphenylalanine (L-DOPA), and is commonly referred to as L-DOPA decarboxylase. The other enzyme, i.e., mammalian Lhistidine apodecarboxylase (EC 4.1.1.22), is specific for bhistidine and is commonly referred to as histidine decarboxylase (HD). Histamine formation in rabbit and guinea pig renal homogenates has been generally regarded as being mediated by L-DOPA decarboxylase, since the enzyme shows low affinity for histidine (2,20), is potentiated by addition of benzene (14, 20), and has a relatively high pH optimum (14, 20). Similarly, histamine formation in rat kidney is increased in the presence of benzene (15, 19), leading to the conclusion -that the rat kidney histamine-forming enzyme is likewise LDOPA decarboxylase. However, if L-DOPA decarboxylase is the histamine-forming enzyme in rat kidneys, than administration of alpha-methyl-DOPA should result in decreased urinary histamine levels, since this inhibitor is selective for L-DOPA decarboxylase (2, 20) and since renal decarboxylation of histidine is a major source of urinary histamine (13); this has not been the case in rats (5, 8). Our laboratory has established that endothelial cells isolated from bovine (4) and rabbit (11) aortas demonstrate relatively high histidine decarboxylase activity. Studies of Schayer (13) indicate that rat kidneys form and excrete histamine directly into the urine. Since alpha-methyl-DOPA fails to decrease histamine excretion, and in light of the recent identification of histidine decarboxylase activity in vascular endothelium, the present study was designed to determine if glomeruli of rat kidneys contain specific histidine decarboxylase. TWO
University
MATERIALS
AND
Park, Pennsylvania
16802
METHODS
Male Wistar rats, 300-350 g were used in all studies. Animals were anesthetized by sodium pentobarbital (33 mg/kg body wt, ip), and kidneys were exposed by midline laporotomy. The abdominal aorta was cannulated using PE-90 tubing (Clay-Adams, N. Y .), and kidneys were subsequently perfused with cold N-2-hydroxyethylpiperazine-N-2-ethane-sulfonic acid (HEPES) buffered Earle’s balanced salt solution, pH 7.4. Perfusion pressure never exceeded 100 mmHg. Following perfusion, kidneys were quickly excised and placed in ice-cold Earle’s balanced salt solution. The kidneys were decapsulated and demedullated. Glomeruli were then isolated from the renal cortex by a modification of the methods of Krakower and Greenspon (7) and Spiro (17). Cortical slices were gently forced through a no. 140 stainless steel sieve, 106,mm opening, using the bottom of an ice-filled beaker to apply pressure. Tissue was washed through the sieve and collected in ice-cold phosphate-buffered saline, pH 7.2. This sieved material was then poured through two stainless steel sieves in series; the top sieve had 180.mm openings and the bottom had 90-pm openings. Glomeruli were collected from the final sieve with 0.1 M sodium acetate buffer, pH 5.5, 1 ml/kidney. After homogenization and centrifugation, histidine decarboxylase was partially purified from the supernatant as described by Hakanson (3). This involves heating the supernatant solution in a 55OC water bath for 5 min, followed by ammonium sulfate fractionation at O4°C. Material which precipitated between 25 and 40% saturation was redissolved in a small volume of 0.1 M sodium phosphate buffer, pH 7.0. Following dialysis against redistilled water, a second ammonium sulfate fractionation was conducted, and protein which precipitated between 28 and 42% saturation was resuspended in 0.01 M sodium phosphate buffer, pH 7.0. After dialysis, this material was used for all histidine decarboxylase assays. Protein concentrations were determined by the method of Lowry et al. (10). Histidine decarboxylase activity was determined by a modification of the double isotopic microassay of Taylor and Snyder (18) which involves two incubation media. Initially, a 25-~1 sample is diluted to 45 ~1 with 0.05 M sodium phosphate buffer, pH 6.2, containing 0.025 mM pyridoxal phosphate. After 5 ~1 of 0.1 M L-histidine are added, the medium is incubated at 37°C for 90 min. Blanks are prepared with n-histidine. After stopping
1349
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1350 the reaction bv boiling in a water bath for 10 min. the pH is adjusted to 7.9 with 0.1 M NaOH, and 20-~1 samples are removed to assay for histamine produced. The histamine assay utilizes imidazole-N-methyltransferase (HMT, EC 2.1.1.8) from fresh guinea pig brain which methylates histamine to form l-methyl-(@-aminoethyl)imadazole (methylhistamine). In this incubation, 10 ~1 of HMT solution are added to the sample and incubated for 1 h at 37°C. The HMT solution contains one part SP4Cladenosylmethionine (5.0 @i/ml), one part 13Hlhistamine (0.64 $X/ml), and two parts of guinea pig brain supernatant. HMT transfers of 14Clabeled methyl group from S-adenosylmethionine to
J. I. HEALD
AND
T.
M.
HOLLIS
histamine, forming [14Clmethylhistamine. 13Hlhistamine and S [14C]adenosyImethionine are separated from 14C, [3H]methyhistamine, and [14C]methylhistamine by extracting the methylhistamines from an alkaline, salt-saturated solution into chloroform. The amount of histamine formed per sample is determined by the ratio: 14C:3H. In summary, the following modifications were made: 1) supernatants of crude guinea pig brain homogenates were used as the source of HMT instead of the partially purified enzyme, and 2) pyridoxal phosphate was added to the incubation medium with the buffer instead of with the substrate in order to allow time for the cofactor to react with the enzyme before beginning
FIG. 1. Phase contrast of glomeruli isolated from Magnification is x 553.
photograph rat kidney.
Downloaded from www.physiology.org/journal/ajplegacy by ${individualUser.givenNames} ${individualUser.surname} (163.015.154.053) on August 22, 2018. Copyright © 1976 American Physiological Society. All rights reserved.
GLOMERULAR
HISTAMINE
TABLE 1. Purification
of histidine Protein,
S@P
1351
SYNTHESIS
Heat treatmentt
&ml
276
decarboxylase
from rat renal glomeruli
Vol, ml
Units per ml*
3.70
1,942
Sp Act, units 1 mg
Purif. Fold
Total Act
Yield
7,025
1
7,185
100
Ammonium tionation
sulfate IS
frac-
24.5
2.60
2,446
99,857
14.2
6,360
89
Ammonium tionation
sulfate IIt
frac-
7.0
2.30
1,922
274,500
39.1
4,421
62
Rurif. fold-sp
act, Ammonium
sulfate fkaction/sp act, heat treatment. * One unit = 1 pmol histamine formed per hour of incubation. $ Protein precipitated between 25 and 40% saturation in 0.1 M sodium acetate precipitated between 28 and 42% saturation in 0.1 M sodium phosphate buffer, pH 7.0.
t 55OC for 5 min in 0.1 M sodium acetate buffer, pH 5.5. buffer, pH 5.5.
9 Protein
the reaction with addition of L-histidine. Radioactivity was counted in Aquasol (Nuclear Associates, Westbury, N. Y.) using a Unilux II scintillation spectrometer (Nuclear-Chicago Corp., Chicago). RESULTS
s m
Figure 1 is a representative phase contrast photomicrograph of an aliquot of sieved renal tissue prepared as previously described. It is evident that these glomeruli are intact, some surrounded by a tightly adhering membrane which is presumably Bowman’s capsule. No distinct tubular material is present in this photomicrograph. By counting all cellular material showing definitive organization, we observed less than 2% contamination by tubular elements in the least highly purified tissue preparation used for this study. Table 1 presents summary data relative to the influence of ammonium sulfate fractionation on specific activity of histidine decarboxylase, employing techniques by Hakanson (3). As indicated, with respect to the heatH istamine, ng treated supernatant solution, the histidine decarboxylFIG. 2. Linearity of double isotopic microassay. Each point reprease purification procedures employed in the present sents mean (2 SE) of 3 determinations. pH was 6.2 and histidine was study resulted in an approximate 40.fold increase in 10e2 M. Counting efficiency for 14C was 65% and for 3H was 22%. eflzyfne specific activity; the yield was 62%. As indicated in Fig. 2, the double isotopic microassay employed was linear between 0.05 and 5 ng histamine per sample. Counting efficiency for 14C was 65% and for 3H was 22%. 14C overlap into the tritium channel was 8.8%, whereas overlap into the 14C channel was 0.6%. The effect of pH on activity of the partially purified enzyme at a substrate concentration of 10B2 M is illustrated by Fig. 3. From inspection of this figure, it is apparent that the optimal pH is 6.2. A double reciprocal plot was employed for analysis of the results of the enzyme kinetic study (Fig. 4). From this analysis, the apparent K, for histidine is 2.4 x 10B4 M. Benzene, added to the HD incubation medium, failed to stimulate activity of the enzyme (Table 2). DISCUSSION
Kinetic analysis of partially purified enzymes is the most reliable method currently available for identification of histidine decarboxylating enzymes (1). The two histidine decarboxylating enzymes may also be distinguished by the large difference in their pH optima (20). Using such analyses, the purified glomerular enzyme
5.4
5.8
6.2
6.6
7.0
7.4
78
PH 3. Effect of pH on purified histidine decarboxylase activity. Histidine concentration was 10s2 M. Each point represents mean (+ SE) of at least 3 determinations. FIG.
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1352
J. I. HEALD
AND T. M. HOLLIS
TABLE 2. Effect of benzene on glomerular decarboxylase ac’tivity Sample
Without
0.16
Benzene
nglhistuminelmgprotin
?J 012 l
0.08
1
6,479
2 3 4
8,147 7,623 9,576
Mean _+ SE * Five microliters
I I
CHistidinel
1
I
I
I
I
2
4
6
8
10
_-
Ifi
3
‘I”
FIG. 4. Double reciprocal plot for histidine of partially purified histidine decarboxylase from renal glomeruli of rat at pH 6.2. Initial velocity (v) is expressed as nanograms histamine formed per hour. Each point represents mean ( + SE) of at least 3 determinations.
demonstrated an apparent Michaelis-Menten constant of 2.4 x 10e4 M for histidine and a pH optimum of 6.2 (using lo-* M histidine). Hakanson (3) and others (12) have shown that the optimal pH of histidine decarboxylase varies inversely with histidine concentration. Aures and Hakanson (1) summarized reports of pH optima using lOa2 M histidine for the enzyme from whole fetal rat, adult rat stomach, hamster placenta, and murine mastocytoma. They found that the pH optimum had a range of from 5.5 to 6.0. Hakanson (3) has also indicated that the active form of the enzyme protein is more abundant at an acid pH. Renal DOPA decarboxylase, on the other hand, is most active in alkaline solutions, i.e., pH 8.0-9.5, (2, 9, 10) and has an optimum pH independent of histidine concentration (1). The pH optimum of the decarboxylase examined in the present study is thus consistent with that of histidine decarboxylase. The apparent K, of histidine decarboxylase varies inversely with the pH (3, 12). At a pH of 6.2, we found
7,956
histidine With Benzene*
per h
6,384 7,194 6,813 7,813
,+ 645
added to incubation
7,05’1 ,+ 303
medium.
an apparent K, of 2.4 x 10e4 M. Using human gastric carcinoid tumor HD, No11 and Levine (12) obtained values of 2.08 x 10B4 M and 3.85 x 10s4 M at a pH of’6.57 and 3.85, respectively. Hakanson (3) has indicated that the enzyme appears to require the anionic form of histidine as its true substrate. If the K, for anionic histidine is determined, using the Henderson-Hasselbalch equation to calculate the proportion of total histidine which is present as the anionic form (using a pK’ of 1.82, 9.17, and 6.0 for the carboxyl, amino, and imadazole groups, respectively), a value of 1.6 x 10D7 M is obtained. Hakanson (3) reports a “true” K, of 6.7 x 10e7 M for whole fetal rat, whereas No11 and Levine (12) found a true K, of 4 x low7 M for human gastric carcinoid tumor histidine decarboxylase. In contrast, renal DOPA decarboxylase has a much lower affinity for histidine with a K, in the range of 10 -‘-lo-* M (2, 9, 20). From the kinetic and pH determinations and from the failure of benzene potentiation, we conclude that renal glomeruli of the rat contain the specific histidine decarboxylase. This may explain the failure of alpha-methylDOPA to decrease rat urinary histamine levels. Glomerular HD would be capable of excreting histamine directly into the glomerular filtrate, thus contributing to histamine which is excreted directly into the urine, i.e., without first entering the circulation. Such direct excretion of histamine into urine has been found in the mouse, dog, and guinea pig as well as the rat (13,16). Received for publication
10 December 1975.
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urinary
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GLOMERULAR
HISTAMINE
R. W., AND M. A. REILLY. Effect of thyroxine on histamine metabolism in mice. Agents Actions 5: 226-230, 1975. 17. SPIBO, R. G. Studies on the glomerular basement membrane. J. Bid. Chem. 242: 1915-1922, 1967. 18. TAYLCIB, K. M., AND S. H. SNYDER. Isotopic microassay of histamine, histidine, hi&dine decarboxylase and histamine methyl16. Sc~ayxm,
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transferase in brain tissue. J. Neurochem. 19: 1343~1358,197Z. J. M., AND G. B. WEST. The formation of histamine in the rat. J. Pharm. Pharmacol. 13: 75-82, 1961. 20. WEISSBACH, H., W. LOVENBERG, AND S. UDENFRIEND. Characteristics of mammalian histidine decarboxylating enzymes. B&him. Biophys. Acta 50: 177-179, 1961. 19. T~LFORD,
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