Regulation of Dopamine P-Hydroxylase Synthesis and Degradation ASCORBIC ACID STABILIZATION OF THE ENZYME AGAINST TRYPTIC PROTEOLYSIS*

94305-Regulation of dopamine p-hydroxylase degradation may be studied using in vitro model systems in which the purified enzyme is subjected to proteolysis by trypsin. Tryptic proteolysis of dopamine 8-hydroxylase fol- lows a biphasic time course, with a 3-min half-life for “fast” phase degradation and a 15-min half-life for “slow” phase degradation. Ascorbic acid, the co-factor in the dopamine 8-hydroxylase reaction, markedly sta- bilizes the enzyme against both phases of tryptic proteolysis. In the presence of ascorbate, the half-lives for “fast” and “slow” phase degradation increase by 43- fold and 8.9-fold, respectively. At high concentrations of ascorbate, tryptic proteolysis of dopamine P-hydrox- ylase shifts to a monophasic time course. Phenylethylamine, a substrate of dopamine P-hydroxylase, only min- imally protects the enzyme against tryptic proteolysis. However, phenylethylamine markedly augments ascorbate stabilization, suggesting that, in vivo, sub- strate and co-factor may work synergistically to protect dopamine &hydroxylase against proteolytic breakdown. Immunochemical studies with homologous anti-dopamine /?-hydroxylase IgG suggest that the na- tive enzyme and the enzyme remaining after “fast” phase tryptic proteolysis have similar antigenic config-urations and are

Regulation of dopamine p-hydroxylase degradation may be studied using in vitro model systems in which the purified enzyme is subjected to proteolysis by trypsin. Tryptic proteolysis of dopamine 8-hydroxylase follows a biphasic time course, with a 3-min half-life f o r "fast" phase degradation and a 15-min half-life for "slow" phase degradation. Ascorbic acid, the co-factor in the dopamine 8-hydroxylase reaction, markedly stabilizes the enzyme against both phases of tryptic proteolysis. In the presence of ascorbate, the half-lives for "fast" and "slow" phase degradation increase by 43fold and 8.9-fold, respectively. A t high concentrations of ascorbate, tryptic proteolysis of dopamine P-hydroxylase shifts to a monophasic time course. Phenylethylamine, a substrate of dopamine P-hydroxylase, only minimally protects the enzyme against tryptic proteolysis. However, phenylethylamine markedly augments ascorbate stabilization, suggesting that, in vivo, substrate and co-factor may work synergistically to protect dopamine &hydroxylase against proteolytic breakdown. Immunochemical studies with homologous anti-dopamine /?-hydroxylase IgG suggest that the native enzyme and the enzyme remaining after "fast" phase tryptic proteolysis have similar antigenic configurations and are of a molecular species distinct from that remaining after "slow" phase proteolysis. Brief exposure to tryptic proteolysis reduces dopamine phydroxylase catalytic activity, while native antigenic configuration is preserved. More extensive tryptic breakdown reduces both catalytic activity and antigenic structure. In the presence of ascorbate, only a single immunochemically definable molecular species of dopamine &hydroxylase is observed. The importance of these findings in relation to the glucocorticoid regulation of dopamine /3-hydroxylase proteolysis in vivo is discussed.
Dopamine P-hydroxylase is the terminal enzyme in norepinephrine synthesis. In rat adrenal medulla, this enzyme is regulated by both neuronal and hormonal stimuli. Splanchnic neuronal stimulation induces de novo enzyme synthesis, while glucocorticoids inhibit enzyme proteolysis (1).
Phenylethanolamine N-methyltransferase, the terminal enzyme in epinephrine synthesis, is similarly controlled by neu-* This work was supported by grants from the National Science Foundation (PCM 78-14183) and the National Institute of Mental Health (MH 25998). This paper is the third in a series; the preceding articles are Refs. 1 and 2. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
$Recipient of Research Career Development Award MH 00219 from the National Institute of Mental Health. ronal and hormonal stimuli (2)(3)(4)(5). Glucocorticoid control of this enzyme is accomplished via the cofactor S-adenosylmethionine (6, 7). S-Adenosylmethionine confers in vitro protection against thermal and tryptic degradation of phenylethanolamine N-methyltransferase. S-Adenosylmethionine levels decrease following hypophysectomy and are restored with glucocorticoid administration. Norepinephrine, the substrate for phenylethanolamine N-methyltransferase, is ineffectual by itself in stabilizing the enzyme, but acts synergistically to enhance the protection S-adenosylmethionine confers against proteolysis (6-8).
The precedence for small molecule protection of enzymes against proteolysis is well established. In Neurospora crassa, biosynthesis of aromatic amino acids occurs via five enzymatic steps clustered on a single polypeptide chain. The first substrate, 3-deoxy-~-arabino-heptulosonate 7-phosphate protects the enzymic cluster against degradation (9). The enzymes serine dehydratase and tyrosine transaminase are protected by their cofactor, pyridoxal phosphate, against inactivation by trypsin and chymotrypsin (10). Similarly, the apo-forms of several other pyridoxal-dependent enzymes, ornithine aminotransferase, cystathionase, and 6-aminolevulinic acid synthetase, are less susceptible to serine proteinases than their holo-forms (11). In vivo studies of ornithine aminotransferase show faster degradation of the enzyme in vitamin B6-deficient animals as compared to controls (12). Likewise, in vivo studies show that tryptophan prevents the proteolytic breakdown of tryptophan pyrrolase (13).
The similarities in glucocorticoid regulation of the degradation of dopamine ,&hydroxylase and phenylethanolamine N-methyltransferase and the importance of S-adenosylmethionine in regulating phenylethanolamine N-methyltransferase proteolysis raise the possibility that glucocorticoids control dopamine P-hydroxylase proteolysis by an analogous mechanism, i.e. by modulating the levels of ascorbic acid, the cofactor in the dopamine ,&hydroxylase reaction. In this report, we describe our studies showing that dopamine /3-hydroxylase proteolysis by trypsin follows a biphasic time course with "fast" and "slow" phases of degradation. Ascorbic acid stabilizes the enzyme markedly against proteolysis. Phenylethylamine, a dopamine ,B-hydroxylase substrate, is of limited efficacy by itself but enhances protection by ascorbate. These findings provide further support to the hypothesis that small molecule cofactors may be important regulatory agents in controlling the intracellular turnover of enzymes.

MATERIALS AND METHODS
Assay of Dopamine /3-Hydroxylase--Dopamine /3-hydroxylase was assayed by a modification of the method of Molinoff et al. (14). as previously described (1). This assay is a coupled radioenzymatic assay in which phenylethanolamine N-methyltransferase and S-adenosyl-~-[methyl-~H]methionine are used. Phenylethanolamine N-methyl-

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Ascorbic Acid Stabilization of Dopamine P-Hydroxylase transferase was purified by the method of Ciaranello and Axelrod (15). Purification of Dopamine P-Hydroxylase-Bovine adrenal medullary dopamine P-hydroxylase was prepared as previously described (1). Bovine adrenals were obtained from the local slaughterhouse and stored on cracked ice in transit. The adrenal medulla was dissected from the cortex and stored overnight a t 4OC. The tissue was homogenized in an equal volume of ice-cold 0.32 M sucrose in a Waring Blendor for 30 s and diluted to 4 volumes with sucrose. The homogenate was centrifuged at 1000 X g for 15 min at 0-4"C, and the pellet was discarded. The resulting supernatant was centrifuged again for 30 min at I0,OOO X g . The pellet was resuspended in 0.32 M sucrose to a final volume of 1 m1/1.25 g of starting material. This solution was layered onto 2 volumes of 1.6 M sucrose and centrifuged for 90 min at 78,000 X g and 0°C. The pellet from the high speed centrifugation is a nearly pure fraction of chromaffin granules, the soluble contents of which are enriched with dopamine P-hydroxylase. The enzyme is liberated from the granules by placing them in 5 mM Tris-HC1, pH 7.4, and freezing the material overnight. The enzyme solution is then centrifuged at 78,000 X g for 0°C for 90 min. The supernatant was pooled and concentrated by ultrafiltration using an Amicon pressure dialysis apparatus fitted with a PM-10 membrane. This sample was then further purified by Sephadex G-200 chromatography using 50 mM Tris-HC1, pH 7.4, as the eluting buffer. Fractions containing maximum dopamine P-hydroxylase activity were pooled, concentrated by ultrafiltration, and further purified on a Sepharose 4B column equilibrated in 50 mM potassium phosphate buffer, pH 7.4. Fractions were pooled, concentrated as above, and stored at -2O'C until use. Such a preparation yielded a single band when subjected to polyacrylamide-sodium dodecyl sulfate electrophoresis (1). The specific activity of the purified enzyme was 114.5 nmol of phenylethanolamine formed/h/mg of protein.
Preparation of Dopamine P-Hydroxylase Antiserum-Male goats were immunized with 0.5 to 1.0 mg of purified bovine adrenal dopamine P-hydroxylase. Purified enzyme initially was administered intramuscularly in complete Freund's adjuvant. Subsequent injections were administered in incomplete Freund's adjuvant a t intervals of 6 weeks. The animals were bled by subclavian venipuncture one week after the third injection. Serum was isolated by centrifugation of the whole blood a t 5000 X g for 30 min, and stored at -20°C.
The IgG fraction was purified from antiserum by ammonium sulfate precipitation at 0-4°C. Sixty milliliters of a saturated solution of ammonium sulfate was added dropwise at 0°C with stirring to a 100ml portion of immune serum. The mixture was stirred for an additional 15 min and then allowed to equilibrate for 15 min on ice. The suspension was centrifuged a t 20,000 X g for 30 min. The pellet was resuspended in 100 ml of 150 mM NaCl in 20 mM Tris-HC1, pH 7.4. The ammonium sulfate precipitation and washing procedure was repeated twice, and the final pellet was dissolved in 50 ml of NaC1/ Tris buffer. Particulate matter was removed by centrifugation, and the supernatant dialyzed for 24 h against isotonic Tris/saline buffer. Purified IgG was stored a t -20°C until used.
Tryptic Degradation of Dopamine P-Hydroxylase-Tryptic digestion was carried out as follows: 25 pl of purified dopamine P-hydroxylase, containing 4 pg of protein, was incubated for varying times at 37°C with 25 pg of trypsin (1 mg/ml in 50 mM Tris-HC1, pH 8.2) and 150 p1 of the same buffer. Incubation was carried out from 0 to 40 min. Each digest mixture contained enough enzyme and trypsin for 12 duplicate time points. Proteolysis was terminated by transferring 200 pl from the tryptic mix to ice-cold tubes containing 50 pg of soybean trypsin inhibitor (STI, Sigma) in 100 pi of distilled water. Samples were stored on ice for assay of residual dopamine P-hydroxylase. Tryptic half-life was determined from a plot of the natural logarithm of dopamine P-hydroxylase activity uersus the time of digestion (Fig. 1). The linear portions of the curve were analyzed by regression analysis. The slope for each linear phase gives k , the apparent first order rate constant of tryptic degradation. The tryptic half-life is calculated using the equation tl L = In 2/17. Stabilization Studies-Ascorbic acid, the oxidation-reduction cofactor, and phenylethylamine, the enzyme substrate, were tested for their ability to protect dopamine [$hydroxylase from tryptic proteolysis. Each compound was tested for stabilization efficacy separately and together over a range of concentrations.
Ascorbic acid was studied at concentrations from 0 to 3.58 mM. Catalase, an essential constituent in the enzymatic assay for dopamine fi-hydroxylase, was included routinely in the digestion mixture at a concentration of 2200 units/Gmol of ascorbate. Failure to include catalase results in the autooxidation of ascorbate during the tryptic digestion and formation of peroxides which totally inhibit dopamine P-hydroxylase activity. Presence of these peroxides prevents measurement of residual enzymatic activity.
Phenylethylamine, the enzyme substrate, was tested for stabilization of dopamine P-hydroxylase at concentrations from 0 to 0.90 mM. When ascorbate and phenylethylamine both were present, the phenylethylamine concentration was 0.90 mM while the ascorbate concentration was varied. Tryptic half-lives were determined as previously described.
Zmmunotitration Studies-Dopamine P-hydroxylase was subjected to the action of trypsin for 0, 3, or 15 min. Three minutes and five minutes represent the half-lives for "fast" and "slow" phase tryptic degradation as determined from earlier studies. After termination of the tryptic digestion, 300-pI aliquots of the reaction mixture were transferred to glass test tubes (10 X 75 mm) which contained varying volumes of diluted goat anti-dopamine P-hydroxylase IgG (1:20 in isotonic saline). The tubes were held at 37°C for 1 h and then a t 4°C overnight. The immune complex was precipitated by centrifugation at 30,000 X g for 30 min. Residual dopamine /?-hydroxylase activity was assayed in the usual manner. Titration curves were generated by plotting residual enzyme activity uersus volume of antibody for each time point of tryptic proteolysis. The slopes were determined for the linear portion of each curve by linear regression analysis. Extrapolation of the line to the abscissa gave an estimate of the equivalence point, that amount of antibody required for complete removal of antigen (Fig. 2).

RESULTS
Tryptic Proteolysis of Dopamine P-Hydroxylase- Fig.   1 represents a typical curve for the tryptic degradation of dopamine /3-hydroxylase. The time course of enzyme proteolysis is biphasic. The first ("fast") phase of tryptic proteolysis is linear from 0 to 4 min, while the second ("slow") phase is linear from 6 to 40 min. Regression analysis of each linear phase and calculation of half-lives from their respective slopes yields a "fast" phase half-life of 3 min and a "slow" phase halflife of 15 min. This biphasic degradation curve is not due to subsaturating amounts of trypsin, since it is seen over a wide range of trypsin concentrations, including those which saturate the dopamine P-hydroxylase present.
Stabilization of Dopamine P-Hydroxylase Proteolysis by Ascorbic Acid-Because glucocorticoids regulate the in vivo degradation of both dopamine @-hydroxylase and phenylethanolamine N-methyltransferase, we examined the possibility that glucocorticoids controlled dopamine @-hydroxylase degradation by acting through ascorbic acid, the cofactor in the reaction catalyzed by this enzyme. To test this, tryptic proteolysis of the enzyme was carried out in the presence of varying amounts of ascorbate. Although ascorbate stabilized both phases of tryptic proteolysis, the effect of the cofactor was most pronounced on "fast" phase proteolysis ( Table I). The half-life for "fast" phase degradation maximally increased by 43-fold while the half-life for the "slow" phase increased by 8.9-fold. Maximal stabilization was observed at an ascorbate concentration of 2.89 mM.
At this concentration, the "slow" and "fast" phase components of dopamine /I-hydroxylase degradation merge to yield a monophasic degradation curve.

TABLE I
Ascorbate protection of dopamine /%hydroxylase against tryptic proteolysis Tryptic proteolysis of dopamine a-hydroxylase was measured as described previously in the presence of ascorbic acid at the concentrations indicated. The half-life of dopamine ,&hydroxylase a t each ascorbate concentration was determined from the "fast" and "slow" phases of proteolysis. Statistical analyses were performed to determine f i s t order rate constants and their standard deviations, but for brevity, only half-life is tabulated below. In the "fast" phase of proteolysis, all half-lives are significantly different from control (no ascorbate) at 0.01 > p < lo-'. In the "slow" phase, all half-lives are also significantly different from control samples a t 0. OOO1 > p < 10.'.
The fast and slow phase half-lives differ significantly ( 1 0 -' > p < 10.") at ascorbate concentrations below 2.39 mM. Above this concentration the "fast" and "slow" phases merge into a single curve.

TABLE I1
Phenylethylamineprotection of dopamine P-hydroxylase against tryptic proteolysis Tryptic proteolysis of dopamine a-hydroxylase was measured as described at the concentrations of phenylethylamine shown below. In the "fast" phase of proteolysis, phenylethylamine stabilized dopamine a-hydroxylase at concentrations of 0.36 mM and above with 0.01 > p < Phenylethylamine significantly stabilized the "slow" phase of proteolysis at 0.54 mM and above with 0.001 > p < lo-'. Below 0.90 mM, the "fast" and "slow" phases were distinguishable (0.01 > p < Above this concentration, the "fast" and "slow" phases merge into a single curve. it conferred significant protection only to the "fast" phase component of degradation (Table 11). The maximum stabilization seen with phenylethylamine was 2.8-fold. Thus, phenylethylamine is considerably less effective in stabilizing dopamine @-hydroxylase than ascorbic acid.
Synergistic Effects of Ascorbate a n d Phenylethylamine-To test whether phenylethylamine facilitated stabilization of dopamine @-hydroxylase by ascorbate, we conducted a series of studies in which both compounds were present during tryptic proteolysis. Table I11 shows the results of these studies. When phenylethylamine is present by itself, it only minimally protects dopamine P-hydroxylase against tryptic proteolysis (1.6-fold stabilization for both phases). When ascorbate is present alone, it stabilizes the "fast" phase component of degradation by 68-fold and the "slow" phase by 10-fold. Phenylethylamine markedly facilitates ascorbate stabilization. In the presence of a fixed concentration of phenylethylarnine, ascorbate stabilizes "fast" phase breakdown by .?&fold and "slow" phase degradation by 15-fold.
Zmmunotitration Studies-Dopamine @-hydroxylase was subjected to tryptic proteolysis for 3 or 15 min, and the immunotitration profiles of the enzyme at these times com-Stabilization of Dopamine /3-Hydroxylase Significantly different from control withp < lo-'.
Significantly different from control with p < < 0.0001. Significantly different from control with p c: lo-!'. pared to that of native enzyme. Fig. 3 represents a typical immunotitration of control dopamine /?-hydroxylase, a 3-min tryptic digest, and a 15-min tryptic digest against diluted goat anti-dopamine /?-hydroxylase antisera. The slopes for control and 3-min digests are parallel while that for the 15-min digest is considerably more shallow. This suggests that after 3 min of tryptic proteolysis, dopamine /?-hydroxylase has undergone a loss of enzyme activity (lower y intercept), but that the affinity of the enzyme for the antibody is similar to that of the native enzyme (parallel slopes). One possible interpretation of this is that during "fast" phase proteolysis, dopamine P-hydroxylase is cleaved at the catalytic site, but the antigenic determinants of the molecule remain intact.
In contrast, after 15 min of proteolysis, both the activity and the antigenic configuration of the enzyme are altered.
This suggests that as proteolysis proceeds, the dopamine phydroxylase molecule is progressively altered. The change in the slope of the immunotitration curve after 15 min of trypsin action suggests that enzyme activity is lost more rapidly than antigenic structure.
When ascorbate is present, the slope of the immunotitration curve at 15 min of tryptic digestion becomes parallel to that of the 3-min and control curves (Fig. 3). This suggests that by stabilizing the enzyme in the fist phase of tryptic proteolysis, ascorbate prevents the second phase of proteolysis.

DISCUSSION
These studies were undertaken to more fully understand the mechanism by which glucocorticoids regulate adrenal dopamine /?-hydroxylase degradation in vivo. Earlier findings on this enzyme and phenylethanolamine N-methyltransferase suggested that glucocorticoids regulate dopamine /?-hydroxylase turnover by maintaining levels of ascorbic acid, the enzyme cofactor. Ascorbate, in turn, stabilizes the enzyme against in vivo proteolysis. Proof of this hypothesis requires satisfaction of three criteria: ( a ) ascorbic acid must stabilize dopamine p-hydroxylase against proteolytic degradation in vitro, ( b ) glucocorticoids must control the levels of adrenal ascorbic acid, and ( c ) ascorbic acid administered to hypophysectomized rats must inhibit proteolysis of dopamine /?-hydroxylase in vivo.
Using tryptic degradation as an in vitro model system to study the enzymatic degradation of dopamine /?-hydroxylase, we have shown that proteolysis of the purified enzyme follows a biphasic course, consisting of "fast" and "slow" phases. Ascorbic acid markedly stabilizes the "fast" phase of degradation, has a lesser effect on "slow" phase degradation, and converts the biphasic tryptic degradation pattern into a monophasic one. Phenylethylamine, a substrate for dopamine /?hydroxylase, is a relatively ineffective stabilizer against dopamine /?-hydroxylase proteolysis. However, phenylethylamine markedly facilitates stabilization of the enzyme by ascorbate.
The kinetics of tryptic breakdown suggest that the degradation of dopamine /?-hydroxylase proceeds by a multi-step process. In the fist phase, cleavage most likely occurs at the active site of the enzyme, forming a molecular species in which catalytic activity is reduced, but antigenic configuration is preserved. In the second phase of proteolysis, the antigenic configuration of the enzyme is destroyed, but at a rate slower than the loss of catalytic activity. Ascorbate prevents the second phase of this process from occurring by stabilizing the enzyme in the fist phase of proteolysis.
The relative ineffectiveness of phenylethylamine in stabilizing the enzyme against tryptic degradation is consistent with our previously observed results with phenylethanolamine Nmethyltransferase and hydroxyindole O-methyltransferase. The substrates of these enzymes, norepinephrine and N-acetylserotonin, are only weakly effective as stabilizers against tryptic degradation. However, they markedly facilitate cofactor stabilization against proteolysis (8, 16).
The real significance of these studies lies in the clues they provide as to the in vivo regulation of dopamine P-hydroxylase proteolysis. Our data show that trypsin degrades dopamine /?-hydroxylase by a multistep mechanism. Does intracellular proteolysis occur in the same way? Nagatsu et al. (17) have demonstrated the existence of a catalytically inactive, fully antigenic form of dopamine P-hydroxylase in sympathetic neurons which seems to exist as a normal intermediate in the enzyme turnover cycle. Our unpublished findings include the observation that two genetically distinct mouse strains with different levels of adrenal dopamine P-hydroxylase catalytic activity have the same total number of antigenic enzyme molecules. However, the proportion of active to inactive enzyme differs. Thus, existing data are not incompatible with a multistep breakdown for dopamine P-hydroxylase in vivo.
In order to generalize our studies to the in vivo situation, however, additional requirements beside in vitro stabilization must be met. These include demonstrating that adrenal medullary ascorbic acid levels are under glucocorticoid control and that administration of ascorbic acid to hypophysectomized rats elevates dopamine /?-hydroxylase levels by inhibiting intracellular proteolysis of the enzyme.
What is the evidence that adrenal ascorbate levels are regulated by glucocorticoids? Currently, relatively little is known about regulation of ascorbic acid levels in the adrenal. The total adrenal ascorbic acid content is about 0.9 pmol/pair in the rat. Nearly all of this is localized in the cortex (18). Administration of adrenocorticotrophic hormone results in ascorbic acid depletion, possibly due to utilization of the compound in reactions of glucocorticoid biosynt.hesis (19). Ascorbic acid does not seem to be incorporated into adrenal medullary cells; rather the reduced form of the cofactor, dehydroascorbate, is preferentially incorporated and then converted to ascorbate. Administration of dehydroascorbate increases tissue levels of ascorbic acid (20). It is unclear what role ascorbic acid metabolites, such as dehydroascorbate and semidehydroascorbate play in the regulation of dopamine p- hydroxylase (21-25). However, their importance may lie in their effects on intracellular ascorbic acid concentration.
Evidence accumulated from other enzyme systems suggests that cofactor regulation of enzyme proteolysis is an important intracellular regulatory mechanism. Katunuma et al. (26) have shown that the proteolytic vulnerability of the pyridoxaldependent rat intestinal mucosal enzymes is carried out by a single proteolytic enzyme. This protease rapidly degrades the apoenzyme, but the pyridoxal phosphate-containing holoenzyme is resistant to proteolytic attack. Recently, Dunaway et al. (27) have demonstrated the existence of a peptide-stabilizing factor that is regulated by insulin and that controls the degradation of phosphofructokinase (PFK-L,). Although the mechanism is unknown, this stabilizing factor is thought to alter the susceptibility of PFK-L, by controlling the susceptibility of the enzyme to degradation. Similarly, Schimke has proposed a model whereby the presence of substrate alters the tertiary conformation of an enzyme, making it more resistant to proteolytic destruction (28). Matsuzawa (29) has proposed a reversible conversion between the holoenzyme and apoenzyme forms of ornithine &aminotransferase. When pyridoxal phosphate is bound, the holoenzyme assumes a stable "tight-state" configuration, whereas the apoenzyme is in a "relaxed" conformation that is labile to proteolytic degradation. There seems to be a general agreement that good correlation exists between data derived from in vitro systems using model proteases such as trypsin and pronase and the degradation of enzymes by intracellular proteases (30). The type of model we have proposed for the regulation of phenylethanolamine N-methyltransferase and hydroxyindole 0-methyltransferase, and are now proposing for the regulation of dopamine /I-hydroxylase, seems to be one that has already gained acceptance among other workers studying different enzyme systems. Verification of this model system would also advance our understanding of the regulation of the adrenal catecholamine metabolic enzymes. The steady state levels of tyrosine hydroxylase, dopamine ,&hydroxylase, phenylethanolamine Nmethyltransferase, monoamine oxidase, and catechol O-methyltransferase seem to be modulated by glucocorticoids. For two of the enzymes, the mechanism of this control is on intracellular proteolysis. The possibility exists, of course, that this is a control mechanism for all the enzymes. If glucocorticoids regulate the proteolysis of these enzymes, what is the mechanism by which this occurs? The enzymes have little in common biochemically. They differ vastly in molecular weight and in substrate specificity. Except for the two methyltransferases, they do not share cofactors. There is some evidence that three of them are rich in glutamic acid (15, 31), but it is not known whether this is true for all. It is tempting to speculate that glucocorticoid control night be exerted at the level of cofactor production. This, of course, requires detailed inquiry into the controls on the production of each of the cofactors before the model can be accepted with confidence.