Decarboxylation of 3,4-Dihydroxyphenylalanine (DOPA) by Erythrocytes: a Reaction Promoted-By Methemoglobin and Other Ferriheme P
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Proc. Nat. Acad. SCi. USA Vol. 69, No. 9, pp. 2505-2508, September 1972 Decarboxylation of 3,4-Dihydroxyphenylalanine (DOPA) by Erythrocytes: A Reaction Promoted-by Methemoglobin and Other Ferriheme Proteins (human/dopamine/Parkinson's disease/hemoglobin/myoglobin) SURESH S. TATE, JOSEPH ORLANDO, AND ALTON MEISTER Department of Biochemistry, Cornell University Medical College, New York, N.Y. 10021 Contributed by A1tot Meister, July 3, 1972 ABSTRACT The decarboxylation of DOPA by erythro- 20-24 hr after death. The minced tissue was homogenized cyte hemolysates differs from DOPA decarboxylation with 10 volumes (w/v) of ice-cold phosphate buffer containing catalyzed by aromatic aminoacid decarboxylases that con- tain vitamin B6 in several significant respects. The ability 5 mM 2-mercaptoethanol and 10,gM pyridoxal 5'-phosphate of erythrocyte hemolysates to decarboxylate DOPA is in a Potter-Elvehjem homogenizer equipped with a Teflon associated with interaction between DOPA and methemo- pestle. The homogenate was centrifuged at 12,000 X g for globin; the ferriheme protein is reduced and DOPA is de- 30 min, and the supernatant solution was used as a source of carboxylated, probably after oxidation to a quinone inter- mediate. An analogous reaction takes place between xvaromatic aminoacid decarboxylase. DOPA a-nd other ferriheme proteins, such as metmyo- The decarboxylation of DOPA was measured essentially as globin and cytochrome c. This phenomenon may be of described (1). The assay solution (2 ml) contained 0.05 M po- significance in relation to the side effects observed in tassium phosphate buffer (pH 7.2) containing 1 mM EDTA patients with Parkinson's disease who are treated with and 1 mM -DOPA, to which [1-14C]D-DOPA was added very large doses of DOPA. to give a final specific activity of 1.7 X 105 cpm/jimol of Recent studies in this laboratory showed that DOPA is DOPA. Incubations were usually performed for 15 min at decarboxylated by-hemolysates of human erythrocytes (1). 37°. Controls were simultaneously run containing all of the We have further investigated this phenomenon and have reagents except hemolysate or heme proteins. found that this reaction is different from that catalyzed by The activity of human kidney extracts was measured as the typical aromatic aminoacid decarboxylases found in described above, except that the assay solution also con- liver, kidney, and brain with respect to heat stability, optical tained 5 mM 2-mercaptoethanol and 10 /AM pyridoxal 5'- specificity, inhibition by typical DOPA decarboxylase in- phosphate. Controls contained heat-inactivated extracts. hibitors, and activation by pyridoxal 5'-phosphate. In the The spectra were determined with a Cary model 15 spectro- present work, evidence has been obtained that the ability of photometer equipped with a water-jacketted cuvet com- erythrocyte hemolysates to decarboxylate DOPA is associated partment. Dopamine was determined as described by Hinten- with an interaction between DOPA and methemoglobin in berger (2). Chromatographic separation of DOPA, dopamine, which methemoglobin is reduced and DOPA is decarboxyl- and other products derived from DOPA was on Whatman ated. That the reaction may be of more general significance No. 3 MM paper strips (2.5 X 55 cm) in a solvent consisting is indicated by the occurrence of an analogous reaction be- of n-butanol-acetic acid-water 25:4: 10; DOPA and dopamine tween DOPA and other ferriheme proteins. had Rf values of 0.18 and 0.51, respectively. The strips were scanned for radioactivity with a Nuclear-Chicago Actigraph EXPERIMENTAL III model 1002 strip scanner. Materials. L-DOPA and N1-(D-seryl)-N2-(2,3,4-trihy- droxybenzyl)-hydrazine (Ro 4-4602) were gifts from Hoff- Preparation of [1-14C]D-DOPA. [1-14C]D-DOPA was pre- mann-La Roche and D-a-hydrazinomethyl-3,4-dihydroxy- pared from [1-'4C]D-DOPA by decarboxylation of the phenylalanine (MK 486) was, a gift from Merck. D-DOPA, L-enantiomer with mouse-liver homogenate (1). The homog- dopamine, [3H]-DOPA (generally labeled), sperm-whale enate was centrifuged at 12,000 X g for 30 min, and the myoglobin, horse-heart cytochrome c, and horse-radish supernatant solution was dialyzed for 18 hr against 200 vol- peroxidase were purchased from Schwarz-Mann. Human umes of phosphate buffer containing 5 mM 2-mercapto- methemoglobin, and beef-liver catalase were obtained from ethanol and 10 /AM pyridoxal 5'-phosphate. Decarboxylation Sigma. [1-'4C] and [2-14C]DL-DOPA, [1-14C]-tyrosine, and was performed in a standard Warburg flask. The side- generally labeled [14C]-histidine were purchased from New arm contained 0.2 ml of 1 N HCl, and the center well was England Nuclear Corp. Hog-kidney D-aminoacid oxidase was supplied with 0.1 ml of 1 N KOH. The reaction mixture a gift from Dr. Daniel Wellner. (2 ml) contained 1.8 ml of phosphate buffer containing 5 mM 5'-phosphate, 0.2 mg of human erythrocytes were pre- 2-mercaptoethanol, 10 &IM pyridoxal Methods. Hemolysates of [1-"4C]DL-DOPA, and 0.2 ml of dialyzed extract from pared as described (1), except that 0.05 M potassium phos- The flask was and incubated at 370 for buffer mM EDTA (referred to below as mouse liver. stoppered phate (pH 7.2)-i 90 min. The reaction was terminated by tipping in HCl, and "phosphate buffer") was used. The hemolysate was centri- then on a shaker for for 15 min at and the supernatant the flask was placed reciprocating fuged at 8000 X g 40, 60 min. An aliquot of the KOH solution was mixed with liquid solution was used in the studies described here. scintillation fluid and counted. About 48% of the total Samples of human kidney cortex were obtained at autopsy radioactivity added as [14C]DL-DOPA was found as 14CO2; 2505 Downloaded by guest on October 1, 2021 2506 Biochemistry: Tate et al. Proc. Nat. Acad. Sci. USA 69 (1972) TABLE 1. Decarboxylation of - and i-DOPA by human the [1-14C]iDOPA in the solution thus obtained was 1.45 X erythrocyte hemolysates and kidney extracts 105 cpm/,umol. Decarboxylase activity RESULTS Enzyme source -DOPA D-DOPA Characteristics of the DOPA Decarboxylase Activity of Human Erythrocytes. The ability of hemolysates of human erythro- Human erythrocyte hemolysate 7.1 7.6 cytes to decarboxylate DOPA was relatively resistant to Human kidney extract 41.7 <1 heating at 1000. Thus, although the DOPA decarboxylase activity of human kidney extracts was completely abolished Extracts were prepared and assayed as described in the text. for 1 treat- Activity is expressed as nmol of DOPA decarboxylated per by incubation of the extracts at 1000 min, similar 15 min per ml of hemolysate or per mg of kidney-extract protein. ment of erythrocyte hemolysates destroyed only 40-70% of the initial activity. Addition of pyridoxal 5'-phosphate(10- thus, virtually all of the [14C]i-DOPA was decarboxylated. 100 MM) had no effect on the decarboxylation of DOPA by The reaction mixture was placed in a centrifuge tube, which erythrocyte hemolysates; on the other hand, the aromatic was immersed in a boiling-water bath for 1 min. After the L-aminoacid decarboxylases from other sources, e.g., rat tube was cooled and centrifuged, 2 ml of the clear supernatant liver (3), rat kidney (4), bovine adrenal medulla (5), and solution was withdrawn and D-DOPA was added to yield a hog kidney (6), are significantly stimulated by pyridoxal specific activity of the 5'-phosphate. Prior incubation of human kidney extracts final concentration of 0.01 M. The Ro [1-14C]D-DOPA in the solution thus obtained was 1.08 X 105 with the aromatic L-aminoacid decarboxylase inhibitors 4-4602 and MK 486 (final concentration, 10 MM; see ref. 7 for cpm/MUmol. abolished Preparation of [1-14C]L-DOPA. [1-14C]DL-DOPA (0.3 mg) a review of these and other inhibitors) completely DOPA decarboxylase activity; such treatment, however, had was dissolved in 0.4 ml of 0.01 N HCl; 0.3 ml of 0.1 M sodium hemol- pyrophosphate buffer (pH 7.2, containing 1 Mumol of 2- no significant effect on the activity of erythrocyte ysates. mercaptoethanol) was added and the pH was adjusted to decar- A mixture (0.3 ml) containing 0.5 mg of hog-kidney D- As indicated in Table 1, erythrocyte hemolysates 7.2. i- at about the same rate, but aminoacid oxidase, 10 ug of FAD, and 3400 units of catalase boxylated and D-DOPA human kidney extract exhibited activity only toward L- was incubated at 370 for 3 hr. The pH of the solution was the de- and after DOPA. Erythrocyte hemolysates did not promote then adjusted to 5.5 by addition of acetic acid, or addition of 5 Mmol of 2-mercaptoethanol, the solution was carboxylation of histidine, tyrosine, 5-hydroxytryptophan. immersed in a boiling-water bath for 1 min. The denatured Decarboxylation of DOPA by Hemoproteins. The experi- protein was centrifuged off, and the supernatant solution was ments described above indicate that the decarboxylation of then applied to a Dowex-1 (acetate) column (0.5 X 2 cm); DOPA by erythrocyte hemolysates differs markedly from L-DOPA was eluted with 3 ml of water. Sufficient L-DOPA that catalyzed by tissue preparations containing aromatic was added to the eluate to yield a final concentration of aminoacid decarboxylase activity. When erythrocyte hemol- 0.01 M. The eluate contained about 54%O of the radioactivity ysates were fractionated by addition of increasing concentra- originally added as [1-14C]DiDOPA. The specific activity of tions of ammonium sulfate, it was found that the fractions that promoted the decarboxylation of DOPA were the same TABLE 2.